The Role of Natural and Synthetic Flavonoids in the Prevention of Marine Biofouling

Marine biofouling is a major concern for the maritime industry, environment, and human health. Biocides which are currently used in marine coatings to prevent this phenomenon are toxic to the marine environment, and therefore a search for antifoulants with environmentally safe properties is needed. A large number of scientific papers have been published showing natural and synthetic compounds with potential to prevent the attachment of macro- and microfouling marine organisms on submerged surfaces. Flavonoids are a class of compounds which are highly present in nature, including in marine organisms, and have been found in a wide range of biological activities. Some natural and synthetic flavonoids have been evaluated over the last few years for their potential to prevent the settlement and/or the growth of marine organisms on submerged structures, thereby preventing marine biofouling. This review compiles, for the first-time, natural flavonoids as well as their synthetic analogues with attributed antifouling activity against macrofouling and microfouling marine organisms.


Introduction
Marine biofouling is a consequence of the settlement and accumulation of adhesive marine organisms' forms on submerged surfaces, namely on ships, causing huge concerns for the maritime industry, the marine environment, and human health.The process of marine biofouling involves different stages, starting with the adsorption of proteins, glycoproteins, or polysaccharides, constituting the conditioning film, which is followed by the deposition of marine bacteria, leading to the formation of a bacterial biofilm.Consequently, extracellular polymeric substances are secreted by the bacterial biofilms, leading to the settlement and accumulation of diatoms and macroalgal spores on the surface of the biofilm, and subsequently to the settlement of macrofouling organisms, including mussels, barnacles, and algae [1,2].This phenomenon causes huge economic impacts for maritime industries, since the increase in a ship's roughness leads to an increase in fuel consumption and consequently to extra fuel costs.Moreover, the damage to ships due to discoloration and corrosion are also notorious, and a longer drydock period is necessary to repair the ship [3].Marine biofouling is also responsible for environmental and health concerns, as it promotes an increase in the emission of polluting gases and the spread of invasive species to other geographical locations, threatening the native species, reducing the biodiversity, and causing the introduction of diseases [3][4][5].Considering the huge impact of marine biofouling, some antifouling strategies have been adopted to prevent this phenomenon.Since the 1960s, the incorporation of tributyltin (TBT) in marine coatings were used by the maritime industries worldwide, as TBT was able to control a broad spectrum of fouling organisms.However, TBT has been demonstrated to cause impairments in the growth, development, reproduction, and survival of many marine species, and therefore, the use of this biocide was completely banned by the International Maritime Organization in 2008 [6].As an alternative to the use of TBT and other organotins, some old-fashioned techniques, namely using copper and zinc, and booster biocides, including Irgarol 1051, Zinc pyrithione, Chlorothalonil, and Sea Nine 211, have been used in marine coatings.However, although some of these molecules were described as environmentally friendly biocides, several toxic effects have been attributed to these alternative biocides [7].
Since the antifouling compounds used have proven to be harmful to the marine environment, the search for new more sustainable alternatives is urgent.One of these strategies can be the use of natural and nature-inspired compounds, namely flavonoids, which have shown interesting antifouling activity when tested against some biofouling marine organisms [8].Flavonoids are natural polyphenols divided into several subclasses, including chalcones, flavonols, flavones, flavanones, isoflavones, flavans, flavanols, and flavanonols (Figure 1), which are not only commonly found in terrestrial plants, but also found in marine sources [9].These compounds have been widely described over the last decades for having a wide range of biological properties, namely anticancer, antimicrobial, anti-inflammatory, antidiabetic, antioxidant, cardioprotective, and neuroprotective [9][10][11][12].Despite the wide range of biological activities attributed to flavonoids over the last few decades, few studies reported their potential as antifoulants.To the best of our knowledge, the first paper reporting an antifouling assay using a flavonoid was carried out in 1989 [13].Until now, 21 research papers described the antifouling potential of flavonoids obtained from marine and terrestrial sources, as well as synthetic ones, through the inhibition of the settlement of macrofouling species, namely mussels, barnacles, and algae, as well as their activity against microfouling organisms, including marine bacteria and diatoms.This article reviews, for the first time, the flavonoids with antifouling activity reported until now.The presentation of all flavonoids is organized by chemical class, and, within each chemical class, in chronological order of the year of the first report of their antifouling activity.The effect of flavonoids against the settlement/growth of some marine species involved in marine biofouling is presented, as well as the main structure-activity relationship (SAR) which is highlighted to support the design of new and optimized antifouling flavonoids.
Mar. Drugs 2024, 22, x FOR PEER REVIEW were used by the maritime industries worldwide, as TBT was able to control a broad trum of fouling organisms.However, TBT has been demonstrated to cause impairm in the growth, development, reproduction, and survival of many marine species, therefore, the use of this biocide was completely banned by the International Mar Organization in 2008 [6].As an alternative to the use of TBT and other organotins, s old-fashioned techniques, namely using copper and zinc, and booster biocides, inclu Irgarol 1051, Zinc pyrithione, Chlorothalonil, and Sea Nine 211, have been used in m coatings.However, although some of these molecules were described as environmen friendly biocides, several toxic effects have been attributed to these alternative bio [7].
Since the antifouling compounds used have proven to be harmful to the marin vironment, the search for new more sustainable alternatives is urgent.One of these s egies can be the use of natural and nature-inspired compounds, namely flavonoids, w have shown interesting antifouling activity when tested against some biofouling m organisms [8].Flavonoids are natural polyphenols divided into several subclasses, in ing chalcones, flavonols, flavones, flavanones, isoflavones, flavans, flavanols, and vanonols (Figure 1), which are not only commonly found in terrestrial plants, but found in marine sources [9].These compounds have been widely described over the decades for having a wide range of biological properties, namely anticancer, antim bial, anti-inflammatory, antidiabetic, antioxidant, cardioprotective, and neuroprote [9][10][11][12].Despite the wide range of biological activities attributed to flavonoids over th few decades, few studies reported their potential as antifoulants.To the best of knowledge, the first paper reporting an antifouling assay using a flavonoid was ca out in 1989 [13].Until now, 21 research papers described the antifouling potential o vonoids obtained from marine and terrestrial sources, as well as synthetic ones, thro the inhibition of the settlement of macrofouling species, namely mussels, barnacles algae, as well as their activity against microfouling organisms, including marine bac and diatoms.This article reviews, for the first time, the flavonoids with antifouling act reported until now.The presentation of all flavonoids is organized by chemical class, within each chemical class, in chronological order of the year of the first report of antifouling activity.The effect of flavonoids against the settlement/growth of some m species involved in marine biofouling is presented, as well as the main structure-act relationship (SAR) which is highlighted to support the design of new and optimized tifouling flavonoids.

Antifouling Flavonoids
A total of 106 flavonoids with antifouling activity against macrofouling (mussels, barnacles, algae) and microfouling (diatoms, bacteria, protozoans) species have been reported, including mainly chalcones, flavonols, and flavones.Other classes of flavonoids with antifouling potential were also reported but less represented, namely flavanone, isoflavone, flavan, flavanol, and flavanonol derivatives (Figure 2A).Most of the studies (60%) were performed using microfouling species (namely marine bacteria and diatoms), and only 24% of the compounds were evaluated against macrofouling organisms, mainly mussel and barnacle species.Nonetheless, 16% of the tested compounds were evaluated against both macrofouling and microfouling organisms (Figure 2B).Considering the origin of the flavonoids reported in this review, as shown in Figure 2C, most of the compounds were obtained through synthesis in a laboratory (55%), followed by natural flavonoids (26%) and commercial compounds (19%).Among natural flavonoids, the percentage of antifouling flavonoids obtained from marine sources is almost as high as those from terrestrial sources, as shown in Figure 2D.The structure, origin, and antifouling effects of flavonoids are presented in Table S1 (Supplementary Material), which is organized in chronological order of the year of the first report of the antifouling activity.
A total of 106 flavonoids with antifouling activity against macrofouling (mussels, barnacles, algae) and microfouling (diatoms, bacteria, protozoans) species have been reported, including mainly chalcones, flavonols, and flavones.Other classes of flavonoids with antifouling potential were also reported but less represented, namely flavanone, isoflavone, flavan, flavanol, and flavanonol derivatives (Figure 2A).Most of the studies (60%) were performed using microfouling species (namely marine bacteria and diatoms), and only 24% of the compounds were evaluated against macrofouling organisms, mainly mussel and barnacle species.Nonetheless, 16% of the tested compounds were evaluated against both macrofouling and microfouling organisms (Figure 2B).Considering the origin of the flavonoids reported in this review, as shown in Figure 2C, most of the compounds were obtained through synthesis in a laboratory (55%), followed by natural flavonoids (26%) and commercial compounds (19%).Among natural flavonoids, the percentage of antifouling flavonoids obtained from marine sources is almost as high as those from terrestrial sources, as shown in Figure 2D.The structure, origin, and antifouling effects of flavonoids are presented in Table S1 (Supplementary Material), which is organized in chronological order of the year of the first report of the antifouling activity.

Chalcones
A total of 61 chalcones with antifouling potential have been reported.Although this class of flavonoids is common in nature, all chalcones reported to have antifouling activity were obtained by chemical synthesis.The structures of chalcones with antifouling potential are shown in Figure 3.
Sivakumar and colleagues evaluated the antifouling activity of 47 synthetic chalcone derivatives (1-47) against three marine bacteria (Bacillus flexus, Pseudomonas fluorescens, and Vibrio natriegens) obtained from biofilms formed on polymer and metal surfaces immersed in ocean water [14].All compounds were shown to be highly active against B. flexus bacterial growth, with minimum inhibitory concentration (MIC) values between

Chalcones
A total of 61 chalcones with antifouling potential have been reported.Although this class of flavonoids is common in nature, all chalcones reported to have antifouling activity were obtained by chemical synthesis.The structures of chalcones with antifouling potential are shown in Figure 3.
Sivakumar and colleagues evaluated the antifouling activity of 47 synthetic chalcone derivatives (1-47) against three marine bacteria (Bacillus flexus, Pseudomonas fluorescens, and Vibrio natriegens) obtained from biofilms formed on polymer and metal surfaces immersed in ocean water [14].All compounds were shown to be highly active against B. flexus bacterial growth, with minimum inhibitory concentration (MIC) values between 0.002 and 0.466 µM, P. fluorescens (MIC values between 0.004 and 0.133 µM), and V. natriegens (MIC values between 0.024 and 0.249 µM) [14].Among tested compounds, chalcones 5, 7, 16, 23, and 36 were the most active against B. flexus, with MIC values of 0.002-0.031µM.All these chalcones presented a hydroxylated ring on their A ring, which seems to be an important substituent for their antibacterial activity against B. flexus.For V. natriegens, chalcones 31 and 32 presented the highest activity, with MIC values of 0.024 µM.All compounds showed potent activity regarding P. fluorescens.

Flavonols
Flavonols comprise the second most reported class of flavonoids with an effect in the prevention of marine biofouling.The structures of flavonols with antifouling potential are presented in Figure 4.
The Kaempferol glucoside (kaempferol 3-O-(2",6"-di-O-(E)-p-coumaroyl-β-D-glucopyranoside, 62), isolated for the first time from the leaves of Quercus dentata, was the first The same research group showed that 2-methoxy-2 ,4 -dichlorochalcone (45), mixed with a marine paint and coated on polycarbonate (PC), polymethylmethacrylate (PMMA), and a glass-fiber-reinforced plastic (GFRP) surface, inhibited the marine bacterium V. natriegens biofilm [15].This coated surface was also compared with surfaces coated with marine paint without any additive and the one mixed with copper (1%), and it was found that surfaces coated with the paint containing the 0.423 µM of 2-methoxy-2 ,4dichlorochalcone were the most effective amongst these three in reducing the amount of biofilm as well as its thickness.The biofilm formation can be attributed to the presence of fimbria structures in the V. natriegens bacteria, which are used for attachment on surfaces, and these structures are highly enriched with proteins, which is one possible reason for its hydrophobicity.Since the coating with dichlorochalcone 45 increases the hydrophilicity of the polymeric surfaces, it decreases the formation of biofilm, which can explain the activity of this coating.Another assay showed that only about 15% of chalcone 45 was lost from the surface after 28 days, maintaining its activity in a sustained manner [15].
Sathicq et al. reported the synthesis and antifouling evaluation of a series of seven chalcones, including the non-substituted chalcone (4) and six furylchalcones (51-56) [17].These compounds were incorporated into a rosin-based antifouling coating, and acrylic panels were painted with these paints and further immersed in the sea for 45 days, and then the results were compared with a control paint (without the incorporation of chalcones).Concerning the evaluation of their antifouling activity against macrofouling species, paints containing non-substituted chalcone 4 did not show significant differences compared with the control paint, with only a reduction of approximately 20% in the presence of macrofouling organisms compared to the control paint, whereas furylchalcones 51-56 displayed a strong anti-macrofouling effect, showing a high reduction in the settlement of algae (filamentous red alga Polysiphonia sp., green alga Ulva sp., filamentous brown alga Ectocarpus sp.) as well as calcareous tubeworms (Hydroides sp. and Spirorbis sp.).In fact, paints with furylchalcones 53 and 55 showed an inhibition of almost 100% of the settlement of macrofouling species, and compounds 51, 52, 54, and 56 showed only about 10%-25% of settlement of these organisms compared to the control paint.Regarding their anti-microfouling effect, all paints containing chalcones 4 and 51-56 were able to inhibit the settlement and growth of diatoms, namely Achnanthes sp., Coscinodiscus sp., Grammatophora sp., Licmophora sp., Navicula sp., Nitzschia longissima, Pinnularia sp., Pleurosigma sp., Synedra sp., and Skeletonema costatum, and protozoans such as Vorticella sp. and Zoothamnium sp.Compared to the control paint, which was 100% covered with diatoms and protozoans, for paints with unsubstituted chalcone and furylchalcones only from about 20% to 40% of the painted plate was covered with these fouling organisms, with furylchalcone 51 being the most active with less than 20% of the plate being covered with microfouling organisms.SAR studies showed that, in general, compounds 55 and 56, with the presence of a 2 -hydroxy group on their A ring, showed higher activity than compounds 51-54 in the inhibition of the settlement of macrofouling species.Compounds 53-54, with a methyl group on their A ring, also showed higher activity than compounds 51-52 against the settlement of macrofouling species, suggesting that the presence of methyl and hydroxy groups is beneficial to the antimacrofouling activity of this series of compounds [17].
More recently, our group reported a synthesis and antifouling activity evaluation for a series of eight triazole-linked flavonoid glycosides, as well as intermediate compounds.
Among them, chalcone 57, as well as structure-related non-glycosylated chalcones 58 and 59 and the 4 -propargylated chalcones obtained as synthetic intermediates (60 and 61), displayed antifouling activity against macro-and/or microfouling species [18].The hybridization of intermediate chalcone with a glycosyl moiety was performed through the click chemistry reaction, obtaining a 1,2,3-triazole ring as a linker, which has been much highlighted as a promising strategy in medicinal chemistry, giving rise to hybrids with a wide range of biological activities [19].Moreover, as the triazole ring was proven to be an effective scaffold for marine biofouling prevention, the use of flavonoids with a triazole ring could result in promising antifouling agents [20].To assess their antifouling activity, the compounds were studied concerning their effect on the settlement of the macrofouling species M. galloprovincialis mussel larvae and on the growth of five biofilm-forming marine bacteria, namely Vibrio harveyi, Cobetia marina, H. aquamarina, Pseudoalteromonas atlantica, and R. litoralis.Among the tested compounds, chalcones 57 (EC 50 = 3.28 µM; 2.43 µg/mL), 58 (EC 50 = 18.10 µM; 5.44 µg/mL), and 59 (EC 50 = 9.64 µM; 3.18 µg/mL) displayed effective anti-settlement activity (EC 50 values < 25 µg/mL), with triazolyl glycosylated chalcone 57 being the most potent and having the highest therapeutic ratio (>60.98).Concerning the antimicrofouling activity, only propargylated chalcones 60 and 61 presented some activity towards the growth of R. litoralis (EC 30 values of 135 and 83.5 µM, respectively).Later, chalcone 57 was shown to also be able to inhibit the biofilm formation of two strains of bacteria S. aureus [21].The compounds showing the highest anti-settlement effect towards the mussel larvae (57-59) were further evaluated against the biofilm-forming marine diatom Navicula sp.Only chalcone 57 was able to display some activity against this microalga's growth (EC 50 = 41.76 µM; 30.94 µg/mL).An ecotoxicity assay performed against the marine non-target organism Artemia salina showed no toxicity for the chalcones 57-59 (less than 10% mortality at 50 µM) [18].In this study, it was highlighted that the presence of a 3,4,5-trimethoxy group on the B ring of the chalcone scaffold seems to be beneficial for antifouling activity.Following the promising results obtained for compound 57, the toxicity of compound 57 to humans was preliminarily assessed with an immortalized human retinal pigment epithelial cell line (hTERT-RPE-1) and compared with the commercial biocide Econea®.While the commercial biocide caused a 50% decrease in the cell viability at the lowest concentration tested (1 µM), chalcone 57 was only able to decrease the cell viability by 50% at the highest concentration tested (100 µM), which allows us to hypothesize that chalcone 57 may present a more safe profile [22].Overall, the results obtained for triazolelinked chalcone glycoside 57 suggest that the conjugation of flavonoids with a glycosyl moiety through a click chemistry approach might be a potential tool for the development of new antifouling agents.

Flavonols
Flavonols comprise the second most reported class of flavonoids with an effect in the prevention of marine biofouling.The structures of flavonols with antifouling potential are presented in Figure 4.
The Kaempferol glucoside (kaempferol 3-O-(2 ,6 -di-O-(E)-p-coumaroyl-β-D-glucopyranoside, 62), isolated for the first time from the leaves of Quercus dentata, was the first flavonol reported for antifouling evaluation [13].This flavonol showed high repellent activity against the settlement of blue mussel Mytilus edulis at 0.22 µmol/cm 2 , whereas the positive control, copper sulfate, showed similar activity at higher concentration (0.50 µmol/cm 2 ), indicating a twofold more potent effect of this flavonol than copper sulfate.Authors also compared the activity of compound 62 with kaempferol (63) (concentration of 6.0 µmol/cm 2 to exert the same activity) and p-coumaric acid (7.3 µmol/cm 2 to exert the same activity), as well as a mixture of kaempferol and p-coumaric acid (1:2) (2.2 µmol/cm 2 to exert the same activity), concluding that compound 62 presented higher activity than both kaempferol (63) and p-coumaric acid, thus suggesting that the combination of the kaempferol and p-coumaric acid moieties is associated with an improvement in antifouling activity [13].

Flavones
Thirteen flavones were also found to exert some influence in the prevention of marine biofouling, being the structures of compounds presented in Figure 5.
Besides the evaluation of a series of flavonols, already reported in Section 2.2, Singh et al. also tested the activity of 7,8-dihydroxyflavone (83), known as tropoflavin, against  62), previously reported by Yamashita et al. [13], were tested by Singh and collaborators for their antifouling potential against the settlement of blue mussel M. edulis, and their activity was compared with the standard antifouling compound copper sulfate [23].Although these flavonols were obtained commercially for this study, these compounds are found in nature, namely in terrestrial and marine sources, and are associated with a wide range of biological activities [9,[24][25][26][27].Flavonoid 62 presented the highest activity in this study, with a more than twofold higher activity than the positive control copper sulfate.On the other hand, the other flavonoids presented only moderate activity, with 5% to 20% of the activity of copper sulfate.By comparing the activity of the tested flavonols, it appeared that flavonoid glycosides with a rhamnosyl moiety were more active than analogues with glycosyl moiety, and the presence of two hydroxy groups on the B ring allied to the two hydroxy groups on the A ring appeared to enhance the anti-settlement activity [23].
In a study performed by Kong et al., the flavonols kaempferol (63) and quercetin (64), isolated from the methanol extract of the leaves of the marine halophyte Apocynum venetum, were tested for their antifouling activity against the marine fouling bacteria Bacillus thuringiensis, Pseudoalteromonas elyakovii and Pseudomonas aeruginosa [29].At a concentration of 100 µg/disc, quercetin (64) (inhibition zone: 9.3-9.5 mm) and kaempferol (63) (inhibition zone: 9.5-17.4mm) displayed moderate antibacterial activities against the three tested bacteria, compared to the positive control chloramphenicol at the same concentration (inhibition zone 29.2-33.7 mm) [29].
Our group also reported the synthesis and antifouling evaluation of a series of natureinspired sulfated compounds, including flavonoids [31].Among them, rutin persulfate (75) showed moderate anti-settlement activity towards plantigrades of the mussel Mytilus galloprovincialis, with an EC 50 value of 22.59 µM and a LC 50 /EC 50 ratio >22.13.Moreover, this flavonoid also displayed significant inhibitory activity against the growth of the marine bacteria Vibrio harveyi (EC 50 value of 7.69 µM) and was shown to be non-toxic in Artemia salina and Luminescent Vibrio fischeri ecotoxicity assays.In this study, another sulfated flavonoid glycoside (compound 76) was shown to moderately inhibit the growth of the marine bacteria Halomonas aquamarina, with an EC 50 value of 42.3 µM [31].Haider and colleagues tested the antifouling activity of a series of flavonoids against diatoms Chaetoceros socialis, Navicula leavissima, and Navicula parva, including seven flavonols, namely casticin (71) and flavonols 77-82 [32].Flavonols 77-79 were isolated from the marine-derived fungus Aspergillus candidus, whereas the natural flavonoids, 80, 81 (morin) and 82, tested in this study were commercially obtained.All tested flavonols displayed antidiatom activity against C. socialis (EC 50 11.62-44.73µM, 4.0-13.52µg/mL).Regarding their activity against N. leavissima, compounds 71, 80, and 81 displayed activities with EC 50 values ranging between 20.32 and 54.33 µM (6.14-16.42µg/mL).Only flavonols 80-81 showed activity against N. parva (EC 50 18.76-20.38µM, 5.67-6.16µg/mL), whereas the commercial biocide Sea-Nine 211 showed activity against all strains of diatoms, with EC 50 values ranging between 3.79 and 5.21 µM (1.07-1.47µg/mL).Interestingly, these two flavonols with hydroxy groups as substituents on the B ring displayed activity against the three strains of diatoms tested, being the most promising compounds in the study, whereas an unsubstituted B ring leads to compound 82, which has lower antidiatom activity [32].

Flavones
Thirteen flavones were also found to exert some influence in the prevention of marine biofouling, being the structures of compounds presented in Figure 5.  [32].The presence of hydroxy groups on the B ring seems to be favorable for antidiatom activity, as well as the substituents on the A ring, but the presence of adjacent hydroxy groups on the A ring is not beneficial.Flavone tricetin 3',4',5'-trimethyl ether (95), synthesized by us, showed promising activity against the settlement of the mussel M. galloprovincialis larvae, with an EC50 value of 8.34 µ M (2.87 µ g/mL), and with low toxicity against the non-target marine organism A. salina [18].In an attempt to study the mechanism of action of antifouling compounds, and based on the role of tyrosinase enzyme on the production of byssal threads by fouling organisms, an evaluation of the inhibition of this enzyme was performed, with flavone 95 able being to decrease tyrosinase activity, reaching 23.5% of inhibition at 100 µ M [18].

Flavanones
As far as we know, only four flavanones with antifouling activity have been reported so far (Figure 6).
Yoshioka et al. showed that the flavanone naringenin (96), isolated from the methanol extracts of the bark of Prunus jamasakura showed some repellent activity against the blue mussel M. edulis at a concentration of 1.2 mg/cm 2 , although the activity of this flavonoid is lower than the positive control, copper sulfate, at a concentration of 0.08 mg/cm 2 [39].
Naringenin (96), as well as the flavanones naringin (97) (obtained commercially) and pinocembrin (98) (isolated from Eucalyptus signata), were evaluated against the settlement of blue mussel M. edulis by Singh et al. and the activity was compared with the standard antifouling copper sulfate [23].In comparison with positive control, compounds 96, 97 and 98 presented lower activity, having 11%, 24%, and 25% of the activity of the positive Besides the evaluation of a series of flavonols, already reported in Section 2.2, Singh et al. also tested the activity of 7,8-dihydroxyflavone (83), known as tropoflavin, against the settlement of the blue mussel M. edulis.This compound showed 25% of the activity of the positive control copper sulfate [23].Although this compound was commercially obtained for this study, tropoflavin occurs in nature, namely in terrestrial plants, and has been proven to exert therapeutic potential [33].
Jensen et al. observed that a lower number of thraustochytrid protists were observed to be associated with healthy leaf tissue in the marine angiosperm Thalassia testudinum than were found in sterilized samples, suggesting that T. testudinum may biosynthesize some metabolites which could be responsible for the low attachment of the thraustochytrid Schizochytrium aggregatum [34].Therefore, the antifouling potential of T. testudinum extracts was evaluated and studies were performed aiming to identify the possible natural products responsible for the anti-settlement activity.It was demonstrated that extracts of T. testudinum leaf tissues inhibited the growth of the co-occurring thraustochytrid S. aggregatum and deterred the attachment of S. aggregatum motile zoospores to an extract-impregnated substrate.Bioassay-guided chemical fractionation of this extract allowed us to identify the flavone glycoside luteolin 7-O-β-D-glucopyranosyl-2 -sulfate (84), which was able to reduce the attachment of S. aggregatum zoospores, with an IC 50 of 511.9 µM (270 µg/mL) [34].These results support the hypothesis that luteolin 7-O-β-D-glucopyranosyl-2 -sulfate (84) could be responsible for the anti-settlement activity observed in the Thalassia testudinum extracts.
Later, luteolin (85), obtained from the terrestrial plant Arachis hypogata, as well as apigenin (87), obtained from terrestrial plant Apium graveolens, and the commercially obtained primuletin (88) were evaluated against the larval settlement of the barnacle A. amphitrite by Zhou et al., along with the flavonols reported in Section 2.2.[28].Although primuletin was commercially obtained for this study, this flavone is found in nature, namely in terrestrial plants [36].These compounds were shown to have a high activity, with EC 50 values ranging from 11.47 to 13.28 µM (2.8-3.8 µg/mL).Considering that these compounds presented with similar activity, it seems that the presence of the hydroxy group at C5 is important for antifouling activity against barnacle larval settlement, whereas the presence of the other substituents is not essential for this activity [28].
Considering the potential of the surface of the eelgrass Zostera marina to inhibit the colonization of microfouler organisms, Guan et al. performed an extraction of the Z. marina leaves in propanol and tested the extracted fractions against marine bacterial strains [38].The authors identified the presence of the flavones luteolin (85) and luteolin-7sulfate (90) in the extract which might be responsible for the activity.Furthermore, both flavones, luteolin (85) and luteolin-7-sulfate (90) were tested against the marine bacteria Vibrio cyclitrophicus and Marivita litorea.At concentration of 14.5 µg/mL, both luteolin (85) and luteolin-7-sulfate (90) exhibited comparable activity against both marine bacteria [38].
Besides the evaluation of flavonols 71 and 77-82 against diatoms C. socialis, N. leavissima, and N. parva, Haider et al. also studied the activity of the flavones luteolin (85) and flavones 91-94 against these biofouling organisms [32].Luteolin (85) was the most promising flavone in this study, being active against all strains of diatoms tested (N.leavissima: EC 50 = [32].The presence of hydroxy groups on the B ring seems to be favorable for antidiatom activity, as well as the substituents on the A ring, but the presence of adjacent hydroxy groups on the A ring is not beneficial. Flavone tricetin 3 ,4 ,5 -trimethyl ether (95), synthesized by us, showed promising activity against the settlement of the mussel M. galloprovincialis larvae, with an EC 50 value of 8.34 µM (2.87 µg/mL), and with low toxicity against the non-target marine organism A. salina [18].In an attempt to study the mechanism of action of antifouling compounds, and based on the role of tyrosinase enzyme on the production of byssal threads by fouling organisms, an evaluation of the inhibition of this enzyme was performed, with flavone 95 able being to decrease tyrosinase activity, reaching 23.5% of inhibition at 100 µM [18].

Flavanones
As far as we know, only four flavanones with antifouling activity have been reported so far (Figure 6).control copper sulfate, respectively.Naringin 97 showed to be more active than naringenin 96, inferring the importance of glycosyl linkage for this activity [23].
The flavanone 2'-methoxykurarinone (99), isolated from the ethyl acetate extract of the Chinese herb Sophora flavescens by Feng and coworkers, was shown to strongly inhibit the settlement of cyprid larvae of Balanus albicostatus (EC50 of 4.46 µ M, 2.02 µ g/mL) by a non-toxic mechanism, since the LC50 value was higher than 25 µ g/mL [40].

Isoflavones
A total of three isoflavones with antifouling activity were described (Figure 7).Besides the activity evaluation of the flavanone naringenin (96), Yoshioka et al. showed that the isoflavone genistein (100), also isolated from the methanolic extracts of the bark of Prunus jamasakura, exhibited some anti-settlement activity against the blue mussel M. edulis.Nevertheless, the activity of this isoflavone (9% of mussel settlement inhibition at 1.6 mg/cm 2 concentration) was substantially lower when compared with the positive control copper sulfate (100% of mussel settlement inhibition at 0.08 mg/cm 2 concentration) and also lower than the flavanone naringenin [39].
In addition to the evaluation of seven flavonols (Section 2.2), one flavone (Section 2.3) and three flavanones (Section 2.4), Singh and collaborators also tested the activity of isoflavone genistein (100) against the settlement of blue mussel M. edulis.This compound was found to display activity in the inhibition of the settlement of mussels, with 9% of the activity of the positive control copper sulfate, having a similar activity compared to the other flavonoids tested in the same study [23].
The antifouling activity of genistein (100), as well as the isoflavone 8-O-methylretusin (101), was also assessed by Zhou et al.The authors studied the effect of some flavonoids, including isoflavones genistein and 8-O-methylretusin, which were obtained by the purification of extracts of terrestrial plants Genista tinctoria and Pueraria alopecuroides, respectively, against the larval settlement of the barnacle A. amphitrite [28].Genistein (100) presented strong activity (EC50: 11.10 µ M, 3.0 µ g/mL; LC50/EC50: >16.7), while isoflavone 8-Omethylretusin (101) presented with an EC50 value of 44.59 µ M (13.3 µ g/mL).Complementary assays in the field with the incorporation of the genistein in a resin-based paint showed that, after one month of the test panels being submerged, the density of barnacles was significantly lower when compared to the control panel tests.Moreover, some panels were coated with a mixture of genistein and a copper sulfate formulation, which showed lower densities of barnacles than the panels coated with the single compound after 3 months of submergence.Considering that the fate of the environment is a concern for the study of potential antifoulants, to evaluate the genistein behavior in seawater, hydrolysis assays were performed.Genistein seemed to be readily hydrolyzed in the marine environment, with the degradation speed being significantly affected by pH, namely in an alkaline environment, which should counteract the accumulation of high levels of this Yoshioka et al. showed that the flavanone naringenin (96), isolated from the methanol extracts of the bark of Prunus jamasakura showed some repellent activity against the blue mussel M. edulis at a concentration of 1.2 mg/cm 2 , although the activity of this flavonoid is lower than the positive control, copper sulfate, at a concentration of 0.08 mg/cm 2 [39].
Naringenin (96), as well as the flavanones naringin (97) (obtained commercially) and pinocembrin (98) (isolated from Eucalyptus signata), were evaluated against the settlement of blue mussel M. edulis by Singh et al. and the activity was compared with the standard antifouling copper sulfate [23].In comparison with positive control, compounds 96, 97 and 98 presented lower activity, having 11%, 24%, and 25% of the activity of the positive control copper sulfate, respectively.Naringin 97 showed to be more active than naringenin 96, inferring the importance of glycosyl linkage for this activity [23].
The flavanone 2 -methoxykurarinone (99), isolated from the ethyl acetate extract of the Chinese herb Sophora flavescens by Feng and coworkers, was shown to strongly inhibit the settlement of cyprid larvae of Balanus albicostatus (EC 50 of 4.46 µM, 2.02 µg/mL) by a non-toxic mechanism, since the LC 50 value was higher than 25 µg/mL [40].

Isoflavones
A total of three isoflavones with antifouling activity were described (Figure 7).Besides the activity evaluation of the flavanone naringenin (96), Yoshioka et al. showed that the isoflavone genistein (100), also isolated from the methanolic extracts of the bark of Prunus jamasakura, exhibited some anti-settlement activity against the blue mussel M. edulis.Nevertheless, the activity of this isoflavone (9% of mussel settlement inhibition at 1.6 mg/cm 2 concentration) was substantially lower when compared with the positive control copper sulfate (100% of mussel settlement inhibition at 0.08 mg/cm 2 concentration) and also lower than the flavanone naringenin [39].
in nature, particularly in terrestrial plants, and display a wide range of biological activities [41].This compound was able to inhibit the growth of diatom C. socialis, with an EC50 value of 28.60 µ M (8.13 µ g/mL), whereas the positive control Sea-Nine 211 ® presented an EC50 value of 3.93 µ M (1.11 µ g/mL).Moreover, compared to the other flavonoids tested in the same study, biochanin A (102) was only active against one strain of diatom, as well as flavonoids 77-79, 82, and 93-94, whereas the remaining flavonoids were active against two or three strains of diatoms [32].

Other Flavonoids
Other flavonoids, including two flavans, one flavanol, and one flavanonol (Figure 8), also showed some potential in marine biofouling prevention and are reported below.
Sideroxylonal A (103), a flavan isolated from the leaves of Eucalyptus grandis by Singh et al., showed high repellent activity against blue mussel M. edulis at a concentration of 0.032 µ mol/cm 2 [42].Another flavan, grandinal (104), isolated from the methanolic extract of the leaves of E. grandis by the same research group, also showed anti-settlement activity against the blue mussel M. edulis, comparable with the standard antifouling copper sulfate at the same concentrations tested [43].Interestingly, the weaker activity of grandinal (104) compared with sideroxylonal A (103) may be due to the presence of tautomeric forms of grandinal.Grandinal was also found to exert some antibacterial activity against Staphylococcus aureus and B. subtilis, with an MIC of 50 and 100 µ g/mL, respectively, which leads us to infer the dual activity of this molecule against both macrofouling and microfouling species [43].
In addition to the evaluation of flavonols 63 and 64, and isoflavone 101 as referred to in Sections 2.2 and 2.5, respectively, Kong et al. also studied the activity of the flavanol epicatechin (105), isolated from the methanolic extract of the leaves of the marine halophyte Apocynum venetum, against the marine fouling bacteria B. thuringiensis, P. elyakovii, and P. aeruginosa.At a concentration of 100 μg/disc, this compound showed some antibacterial activity against all the tested bacteria, with an inhibition zone diameter of 9.2-10.9mm, whereas the positive control chloramphenicol presented an inhibition zone diameter of 29.2-33.7 mm at the same concentration.Moreover, compound 105 was shown to display a similar activity to flavonols 63 and 64, whereas isoflavone 101 was only active against two strains of marine bacteria [29].
Gopikrishnan et al. reported the production and isolation of the flavanonol taxifolin (106) from the mangrove-derived actinobacterium Streptomyces sampsonii (PM33) [44].After the isolation and characterization of this flavonoid, antifouling assays were performed.In addition to the evaluation of seven flavonols (Section 2.2), one flavone (Section 2.3) and three flavanones (Section 2.4), Singh and collaborators also tested the activity of isoflavone genistein (100) against the settlement of blue mussel M. edulis.This was found to display activity in the inhibition of the settlement of mussels, with 9% of the activity of the positive control copper sulfate, having a similar activity compared to the other flavonoids tested in the same study [23].
The antifouling activity of genistein (100), as well as the isoflavone 8-O-methylretusin (101), was also assessed by Zhou et al.The authors studied the effect of some flavonoids, including isoflavones genistein and 8-O-methylretusin, which were obtained by the purification of extracts of terrestrial plants Genista tinctoria and Pueraria alopecuroides, respectively, against the larval settlement of the barnacle A. amphitrite [28].Genistein (100) presented strong activity (EC 50 : 11.10 µM, 3.0 µg/mL; LC 50 /EC 50 : >16.7), while isoflavone 8-Omethylretusin (101) presented with an EC 50 value of 44.59 µM (13.3 µg/mL).Complementary assays in the field with the incorporation of the genistein in a resin-based paint showed that, after one month of the test panels being submerged, the density of barnacles was significantly lower when compared to the control panel tests.Moreover, some panels were coated with a mixture of genistein and a copper sulfate formulation, which showed lower densities of barnacles than the panels coated with the single compound after 3 months of submergence.Considering that the fate of the environment is a concern for the study of potential antifoulants, to evaluate the genistein behavior in seawater, hydrolysis assays were performed.Genistein seemed to be readily hydrolyzed in the marine environment, with the degradation speed being significantly affected by pH, namely in an alkaline environment, which should counteract the accumulation of high levels of this compound in marine environments [28].
In other study, it was reported that O-methylretusin (101), isolated from the methanolic extract of the leaves of the marine halophyte Apocynum venetum, presented activity against the marine fouling bacteria B. thuringiensis (inhibition zone 9.4 mm) and P. aeruginosa (inhibition zone 10.5 mm) at a concentration of 100 µg/disc, whereas the positive control chloramphenicol presented an inhibition zone of 33.7 and 30.3 mm at the same concentration [29].These results suggest the potential of O-methylretusin (101) to prevent biofouling promoted by macro and microfouling agents.
In addition to the evaluation of flavonols 71 and 77-82, and flavones 81 and 91-94 described in Sections 2.2 and 2.3, respectively, Haider and collaborators also tested the antifouling activity of the isoflavone biochanin A (102) against diatoms C. socialis, N. leavissima, and N. parva [32].Although a commercial stock was used, this compound is found in nature, particularly in terrestrial plants, and display a wide range of biological activities [41].This compound was able to inhibit the growth of diatom C. socialis, with an EC 50 value of 28.60 µM (8.13 µg/mL), whereas the positive control Sea-Nine 211 ® presented an EC 50 value of 3.93 µM (1.11 µg/mL).Moreover, compared to the other flavonoids tested in the same study, biochanin A (102) was only active against one strain of diatom, as well as flavonoids 77-79, 82, and 93-94, whereas the remaining flavonoids were active against two or three strains of diatoms [32].

Other Flavonoids
Other flavonoids, including two flavans, one flavanol, and one flavanonol (Figure 8), also showed some potential in marine biofouling prevention and are reported below.
Pseudomonas sp.P1, Pseudomonas sp.N9, and Kurthia sp.P3, with MIC values ranging from 5.32 to 82.17 µ M; 1.62-25 μg/mL.Moreover, at a concentration of 100 μg/mL, this compound was shown to reduce the Anabaena sp. and Nostoc sp.spore germination by more than 70%.Taxifolin (106) was also shown to reduce the adherence of the mussel Perna indica.Furthermore, field experiments revealed the antifouling activity of taxifolin when tested on wooden surface and PVC panels.A toxicity assay using zebra fish model showed that in a concentration of 0.5 μg/mL and 1 μg/mL, taxifolin (106) caused pericardial edema in 0.6% of zebrafish embryos, while with an increase in the concentration to 1.5 μg/mL and 2.0 μg/mL, 60% of zebrafish embryos had pericardial edema.The cytotoxicity of taxifolin (106) was also assayed in vitro against the normal lung Bronchial Epithelial BEAS -2B cell line, with LC50 values ranging between 150 μg/mL and 200 μg/mL [44].

Conclusions
The phenomenon of marine biofouling is very complex, involving a wide diversity of marine organisms and depending on multiple variables, and, generally, the screening studies performed englobe a few or even just one biofouling species, as described in this study.
In this review, a total of 106 flavonoids with antifouling activity against macro-and microfouling organisms were compiled by chemical classes.Although some flavonoids were only evaluated for their activity against one single species of fouling organisms, namely the mussel M. galloprovincialis or the barnacle A. amphitrite, some compounds were tested against several marine organisms, showing their potential to be effective in real scenarios of complex biofouling communities.For example, flavonol kaempferol (63) was found to display antibacterial activity against several marine bacteria and antifouling activity against two species of macrofouling organisms (mussel and barnacles) whereas quercetin (64) showed antibacterial activity and antifouling activity against a species of mussel.The flavone luteolin (85) was found to be active against marine bacteria, diatoms, and a macrofouling barnacle.Some flavonoids, namely the isoflavone genistein (100) and chalcones 4, 45, and 51-56, were incorporated into marine coatings, showing interesting antifouling potential in field assays.
Several mechanisms of action have been associated with antifouling activity.However, only few studies reported the evaluation of the mechanism of action for antifouling flavonoids.Therefore, future assays should include the assessment of the mechanism involved in the antifouling activity, which could be beneficial in the design of more effective agents.
Considering the need of obtaining environmentally safe antifoulants, ecotoxicological assessments are needed.In fact, only a few flavonoids were assessed for their ecotoxicological behavior against marine non-target species, namely chalcones 48-50 and 57-59 and flavones 75 and 95, which proved to be non-toxic against A. salina.In addition, studies Sideroxylonal A (103), a flavan isolated from the leaves of Eucalyptus grandis by Singh et al., showed high repellent activity against blue mussel M. edulis at a concentration of 0.032 µmol/cm 2 [42].Another flavan, grandinal (104), isolated from the methanolic extract of the leaves of E. grandis by the same research group, also showed anti-settlement activity against the blue mussel M. edulis, comparable with the standard antifouling copper sulfate at the same concentrations tested [43].Interestingly, the weaker activity of grandinal (104) compared with sideroxylonal A (103) may be due to the presence of tautomeric forms of grandinal.Grandinal was also found to exert some antibacterial activity against Staphylococcus aureus and B. subtilis, with an MIC of 50 and 100 µg/mL, respectively, which leads us to infer the dual activity of this molecule against both macrofouling and microfouling species [43].
In addition to the evaluation of flavonols 63 and 64, and isoflavone 101 as referred to in Sections 2.2 and 2.5, respectively, Kong et al. also studied the activity of the flavanol epicatechin (105), isolated from the methanolic extract of the leaves of the marine halophyte Apocynum venetum, against the marine fouling bacteria B. thuringiensis, P. elyakovii, and P. aeruginosa.At a concentration of 100 µg/disc, this compound showed some antibacterial activity against all the tested bacteria, with an inhibition zone diameter of 9.2-10.9mm, whereas the positive control chloramphenicol presented an inhibition zone diameter of 29.2-33.7 mm at the same concentration.Moreover, compound 105 was shown to display a similar activity to flavonols 63 and 64, whereas isoflavone 101 was only active against two strains of marine bacteria [29].
Gopikrishnan et al. reported the production and isolation of the flavanonol taxifolin (106) from the mangrove-derived actinobacterium Streptomyces sampsonii (PM33) [44].After the isolation and characterization of this flavonoid, antifouling assays were performed.It was found that the taxifolin (106) is active against several biofouling bacterial strains, namely Staphylococcus sp.M1, Micrococcus sp.M50, Lactobacillus sp.M6, Bacillus sp.N16, Aeromonas sp.N8, Alcaligenes sp.P2, Alcaligenes sp.N22, Alcaligenes sp.E4, Vibrio sp.M25, Pseudomonas sp.P1, Pseudomonas sp.N9, and Kurthia sp.P3, with MIC values ranging from 5.32 to 82.17 µM; 1.62-25 µg/mL.Moreover, at a concentration of 100 µg/mL, this compound was shown to reduce the Anabaena sp. and Nostoc sp.spore germination by more than 70%.Taxifolin (106) was also shown to reduce the adherence of the mussel Perna indica.Furthermore, field experiments revealed the antifouling activity of taxifolin when tested on wooden surface and PVC panels.A toxicity assay using zebra fish model showed that in a concentration of 0.5 µg/mL and 1 µg/mL, taxifolin (106) caused pericardial edema in 0.6% of zebrafish embryos, while with an increase in the concentration to 1.5 µg/mL and 2.0 µg/mL, 60% of zebrafish embryos had pericardial edema.The cytotoxicity of taxifolin (106) was also assayed in vitro against the normal lung Bronchial Epithelial BEAS -2B cell line, with LC 50 values ranging between 150 µg/mL and 200 µg/mL [44].

Figure 2 .
Figure 2. (A) Classes of flavonoids with antifouling activity reported in this review.(B) Bioactivity evaluated for antifouling flavonoids against macrofouler, microfouler, or both types of fouling organisms.(C) Basis of flavonoids studied for antifouling activity.(D) Source of natural flavonoids.

Figure 2 .
Figure 2. (A) Classes of flavonoids with antifouling activity reported in this review.(B) Bioactivity evaluated for antifouling flavonoids against macrofouler, microfouler, or both types of fouling organisms.(C) Basis of flavonoids studied for antifouling activity.(D) Source of natural flavonoids.