Seaweed-Derived Phlorotannins: A Review of Multiple Biological Roles and Action Mechanisms

Phlorotannins are a group of phenolic secondary metabolites isolated from a variety of brown algal species belonging to the Fucaceae, Sargassaceae, and Alariaceae families. The isolation of phlorotannins from various algal species has received a lot of interest owing to the fact that they have a range of biological features and are very biocompatible in their applications. Phlorotannins have a wide range of therapeutic biological actions, including antimicrobial, antidiabetic, antioxidant, anticancer, anti-inflammatory, anti-adipogenesis, and numerous other biomedical applications. The current review has extensively addressed the application of phlorotannins, which have been extensively investigated for the above-mentioned biological action and the underlying mechanism of action. Furthermore, the current review offers many ways to use phlorotannins to avoid certain downsides, such as low stability. This review article will assist the scientific community in investigating the greater biological significance of phlorotannins and developing innovative techniques for treating both infectious and non-infectious diseases in humans.


Introduction
Marine organisms, particularly brown seaweeds, have garnered a lot of interest across the world for their potential use in treating a variety of infectious and non-infectious diseases [1,2]. Several compounds have been isolated from brown seaweeds, including phlorotannins, fucoxanthin, fucoidan, alginate, fucoidan, and laminarin [3]. Almost all secondary metabolites have been found to possess various biological activities. Among these, phlorotannins, which are members of the polyphenolic group of secondary metabolites, has been found in abundance in brown algae belonging to the Fucaceae, Sargassaceae, and Alariaceae families [4]. Some red and green algae also contain phlorotannins, albeit to a lesser extent than brown algae [5]. Despite problems in isolating and purifying phlorotannins from brown seaweeds, more than 150 phlorotannins have been identified [6]. The different sources from which phlorotannins have been isolated and identified are given in Tables 1 and 2. Figure 1 depicts the chemical structures of these phlorotannins. The use of traditional antibiotics and other pharmaceuticals to treat infectious diseases caused by bacterial, viral, and fungal pathogens [7][8][9], as well as non-infectious diseases such as cancer, diabetes, inflammation, obesity, and adipogenesis, has been shown to be detrimental [10][11][12]. Microbial infections caused by viruses, bacteria, and fungus have become one of the most serious threats to human health across the world [13][14][15]. detrimental [10][11][12]. Microbial infections caused by viruses, bacteria, and fungus have become one of the most serious threats to human health across the world [13][14][15].  Furthermore, microbial pathogens such as bacteria and fungi evolved many resistance mechanisms against commonly used antibiotics and antifungals [16,17]. Multi-drug resistant strains of microorganisms from various hospital and clinical environments are also rapidly growing [18][19][20]. With rising antimicrobial resistance, the characterization of microbial pathogens and innovative strategies to combat microbial diseases are in great demand. Recent and previous research has found that phlorotannins have antibacterial, antifungal, and antiviral effects against a wide variety of microbial pathogens [21][22][23]. Non-infectious disorders, including cancer, diabetes, inflammation, and obesity, are also on the rise and creating a strong need for newer therapeutic drugs [24][25][26]. The current review addresses the biological properties of several types of phlorotannins in treating both infectious and non-infectious diseases. Furthermore, the underlying mechanism involved in treating diseases using phlorotannins have also been explored. Additionally, the current limitations and future perspectives associated with the isolation and application of phlorotannins have been thoroughly discussed.

Phlorotannin against Pathogenic Bacterial and Fungal Species
Antibiotics are used to treat the majority of bacterial pathogens, but the development of antibiotic resistance in pathogens has recently grown substantially, directly affecting human mortality. Antibiotic resistance is mostly caused by the indiscriminate use of antibiotics for pathogen treatment, long-term antibiotic therapy for patients, and patient discontinuation of antibiotics before finishing treatment [27]. According to reports, Acinetobacter baumannii, Enterococcus faecium, Enterobacter spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus spp. are the major bacterial pathogens involved in the development of antibiotic resistance [28]. Antibiotic resistance can be developed or acquired through processes such as (1) lower intracellular concentration of antibiotics owing to pathogen efflux, (2) genetic change of the antibiotic target, and (3) antibiotic deactivation by hydrolysis or modification [29]. This is comparable to the situation in fungal pathogens, where studies have shown that antifungal drugs are successful in the treatment of common fungal pathogens such as Candida spp., Cryptococcus spp., and Aspergillus spp. However, fungi develop resistance as a result of the indiscriminate use of antifungal drugs, resulting in increased fungal disease mortality [30]. Furthermore, the majority of antifungal agents utilized in the treatment of human diseases include polyenes, triazoles, and echinocandins, but they are toxic to patients [31]. Aside from intrinsic and acquired resistance mechanisms, bacterial and fungal species have evolved phenotypic adaptive resistance mechanisms known as biofilm formation, which is an assembly of cells surrounded by self-produced polymeric substances [32]. Biofilms act as a structural and functional barrier to the entry of antibacterial and antifungal drugs into the microbial cell. Furthermore, these biofilms contained a small population of cells (persister cells) that are metabolically inactive and have been linked to persistent and recurring infection. As a result, there is an urgent need to develop a strategy or find novel molecules to battle antibiotic-resistant bacteria and fungi. Several studies have proven the antibacterial action of phlorotannins isolated from various seaweeds. Phlorotannins have an antibacterial effect by suppressing oxidative phosphorylation, altering target microorganism cell permeability, and triggering death by interacting with target microbial proteins [33]. The phenolic aromatic ring and hydroxyl groups of various phlorotannins have been shown to bind to microbial proteins and induce cell lysis [34]. The susceptibility of Gram-positive and Gram-negative bacteria to phlorotannins varies owing to structural differences in the cell membrane and cell wall. Gram-negative bacteria are often less susceptible to phlorotannin than Gram-positive bacteria [35]. Nagayama et al. [21] exhibited the bactericidal action of phlorotannins derived from Ecklonia kurome for the first time, where phlorotannins showed excellent activity against MRSA. Several types of phlorotannins isolated from Eisenia bicyclis showed antibacterial activity against Listeria monocytogenes at 16-256 µg/mL [36]. Furthermore, the combination of phlorotannins and streptomycin demonstrated strong synergistic activity against aminoglycoside antibiotic-resistant clinical strains of L. monocytogenes (fractional Mar. Drugs 2022, 20, 384 4 of 31 inhibitory concentration index, ∑FIC < 0.5). Cox et al. [37] discovered that extracts prepared from several seaweeds inhibited E. faecalis, L. monocytogenes, P. aeruginosa, and Salmonella abony. Brown seaweed, for instance, outperformed red and green seaweed in antibacterial activity. Another research group found that phlorotannins inhibited quorum sensing (QS) activity in Chromobacterium violaceum 12472 by preventing the synthesis of purple pigment [38]. Similarly, phlorotannins reduced P. aeruginosa virulence by inhibiting motility and decreasing the production of virulence factors such as extracellular protease, hemolysis, and pyocyanin. Eom et al. [39] conducted the first investigation on the anti-MRSA (methicillin-resistant S. aureus) activity of phlorotannins from E. bicyclis. The anti-MRSA activity of phlorofucofuroeckol-A was associated with a MIC value of 32 µg/mL. Furthermore, the combinations of phlorofucofuroeckol-A and β-lactam antibiotics such as ampicillin, penicillin, and oxacillin have shown strong synergistic effects. Choi et al. [40] discovered phlorotannin activity against the acne-causing bacteria Propionibacterium acnes. Dieckol and phlorofucofuroeckol-A, in particular, were found to be very active against acne-causing bacteria when isolated from E. cava. Lee et al. [41] also discovered that the compounds isolated from E. bicyclis have strong antibacterial action against acne-causing bacteria. Furthermore, fucofuroeckol-A combined with erythromycin and lincomycin had a synergistic impact on P. acnes. Ethanolic extracts of E. cava were found to have effective antibacterial activity towards S. aureus, MRSA, and Salmonella spp. [42]. Eckol from E. cava substantially reduced the MIC value of ampicillin against MRSA and Salmonella spp. Mittal et al. [43] discovered that phloroglucinol and related compounds exhibited antibacterial action against various Gram-positive bacteria. Furthermore, the combination of phlorodipropanophenone and oxycycline had a strong synergistic bactericidal effect. Kim et al. assessed the antibacterial activity of E. cava phlorotannin against fish infectious diseases caused by marine bacterial pathogens [44]. The methanol extract of E. cava was shown to have potent antibacterial action against several pathogens such as Streptococcus iniae, Edwardsiella tarda, S. parauberis, V. harveyi, V. scophthalm, and Vibrio anguillarum. Phlorotannin isolated from E. arborea has shown antibacterial action against V. parahaemolyticus, indicating its potential as an antibiotic to combat acute hepatopancreatic necrosis disease caused by this pathogen [45]. Furthermore, dieckol derived from E. stolonifera has shown antibacterial efficacy against MRSA and methicillin-susceptible S. aureus (MSSA) [46]. In addition, the application of dieckol reversed the resistance properties developed against ampicillin and penicillin by MRSA. Wei et al. [47] studied the mechanism of phlorotannins derived from Sargassum thunbergii against V. parahaemolyticus. Their findings revealed that phlorotannin was able to suppress bacterial growth and damaged the cytoplasmic membrane. Some studies have also found that phlorotannins damage the cell membranes of anaerobic bacteria, resulting in extracellular and intracellular repercussions [48]. According to Lopes et al. [22] phlorotannin isolated from seaweed has an inhibitory effect against yeast and dermatophytes. Dieckol extracted from E. cava showed fungicidal activity towards Trichophyton rubrum (200 µM) [49], resulting in the loss of cytoplasmic membrane integrity. According to Kim et al. [50] fucofuroeckol-A has significant antifungal action towards C. albicans with the MIC value of 512 µg/mL. Fucofuroeckol-A enhanced fluconazole's antifungal activity against fluconazole-resistant C. albicans. Figure 2 depicts a diagrammatic explanation of the mechanism involved in the antimicrobial activity of several forms of phlorotannins against bacterial and fungal pathogens. Table 1 shows several other examples of phlorotannins with antibacterial and antifungal activities. Table 1

Phlorotannins as a Natural Adjuvant of Antiviral Therapy
Viral diseases are one of the major issues caused by several viruses, including the human immunodeficiency virus [51], hepatitis B and C virus, and coronavirus . All of these have a severe negative impact on human life. Antiviral agents exhibit a variety of action mechanisms, including inhibition of viral fusion and mRNA entry, antagonism of chemokine co-receptors and cyclophilins, and inhibition of proteases and reverse transcriptase within the host cell ( Figure 3). Viruses are acellular and have a simple structure with a protein coat, nucleic acid, viral enzyme, and a lipid envelope, where they replicate after infecting host cells [52]. The viral life cycle inside host cells is extremely complicated, requiring several phases from invasion through maturity. As a result, one of the key problems in designing antiviral agents with selective toxicity is the lack of these characteristics [53]. Although various antiviral medications have been identified, the existing antiviral agents have significant drawbacks, such as the development of resistance, limited bioavailability, and high toxicity [54]. Besednova et al. [55] demonstrated that compounds with antiviral activities obtained from marine species do not cause resistance owing to a variety of mechanisms, including antiviral, immunostimulatory, anti-inflammatory, and antioxidant capabilities. One of the compounds obtained from marine organisms, phlorotannin, has high antioxidant capabilities, particularly due to the presence of the phenol group. These properties have been shown to prevent viral infection as well as interfere with virus adherence, penetration, and replication [56]. According to Artan et al. [57], 6,6′-bieckol derived from E. cava prevented HIV-1 induced fusion formation (EC50 1.72 µM), viral p24 antigen production (EC50 1.26 µM), and lytic effect (EC50 1.23 µM), thereby exhibiting antiviral activities. Moreover, at concentrations that suppressed HIV-1 replication, 6,6′-bieckol did not exhibit cytotoxicity. At non-cytotoxic doses, 8, 4′′′-dieckol derived from E. cava also inhibited HIV-1 lytic effect and viral p24 antigen production

Phlorotannins as a Natural Adjuvant of Antiviral Therapy
Viral diseases are one of the major issues caused by several viruses, including the human immunodeficiency virus [51], hepatitis B and C virus, and coronavirus (COVID-19). All of these have a severe negative impact on human life. Antiviral agents exhibit a variety of action mechanisms, including inhibition of viral fusion and mRNA entry, antagonism of chemokine co-receptors and cyclophilins, and inhibition of proteases and reverse transcriptase within the host cell ( Figure 3). Viruses are acellular and have a simple structure with a protein coat, nucleic acid, viral enzyme, and a lipid envelope, where they replicate after infecting host cells [52]. The viral life cycle inside host cells is extremely complicated, requiring several phases from invasion through maturity. As a result, one of the key problems in designing antiviral agents with selective toxicity is the lack of these characteristics [53]. Although various antiviral medications have been identified, the existing antiviral agents have significant drawbacks, such as the development of resistance, limited bioavailability, and high toxicity [54]. Besednova et al. [55] demonstrated that compounds with antiviral activities obtained from marine species do not cause resistance owing to a variety of mechanisms, including antiviral, immunostimulatory, anti-inflammatory, and antioxidant capabilities. One of the compounds obtained from marine organisms, phlorotannin, has high antioxidant capabilities, particularly due to the presence of the phenol group. These properties have been shown to prevent viral infection as well as interfere with virus adherence, penetration, and replication [56]. According to Artan et al. [57], 6,6 -bieckol derived from E. cava prevented HIV-1 induced fusion formation (EC 50 1.72 µM), viral p24 antigen production (EC 50 1.26 µM), and lytic effect (EC 50 1.23 µM), thereby exhibiting antiviral activities. Moreover, at concentrations that suppressed HIV-1 replication, 6,6 -bieckol did not exhibit cytotoxicity. At non-cytotoxic doses, 8, 4 -dieckol derived from E. cava also inhibited HIV-1 lytic effect and viral p24 antigen production [57]. According to the findings, 8,4 -dieckol preferentially inhibited the reverse transcriptase activity of HIV-1 ( Figure 3). Ahn et al. [58] discovered that 8,4"-dieckol and 8,8 -bieckol inhibited HIV-1 protease and reverse transcriptase. Furthermore, these drugs inhibited HIV-1 reverse transcriptase's RNA-dependent DNA synthesis activity against dUTP/dTTP in a non-competitive manner. In a cell-free-based system, phlorotannins from E. cava competitively suppressed SARS-CoV-2 3CLpro [23]. These compounds showed an inhibitory effect on 3CLpro hydrolysis (IC 50 2.7~164.7 µM). Ryu et al. [59] showed that the ethanol extract of E. cava exhibited inhibitory activity against neuraminidase action, thereby inhibiting the influenza virus. In particular, the ethanol extract effectively inhibited neuraminidase by synergizing with oseltamivir.

Anti-Inflammatory and Immunomodulatory Properties of Phlorotannins
Inflammation is the body's reaction to harmful stimuli, such as an infection or injury, which leads to the development of symptoms such as seizure, pain, fever, and swelling. The inflammatory response is an immunological reaction to exogenous and endogenous signals that serve as the host's defensive systems against potentially harmful stimuli [32]. An inflammatory response is necessary for survival, and it plays an important role in cellular physiology; nevertheless, uncontrolled and excessive inflammation leads to a variety of chronic diseases. [60]. Inflammatory drugs include both steroidal and nonsteroidal, which are synthetic drugs used to treat inflammatory disorders that have potentially dangerous side effects and are not safe for humans [61]. In recent years, many investigations on the isolation and identification of natural anti-inflammatory drugs have been carried out. As a result, naturally occurring organisms have components that chemically resemble steroid structures and have been reported to exhibit a high potential for anti-inflammatory action [62]. Natural compounds with anti-inflammatory potential have antioxidant and radical scavenging characteristics, as well as the capacity to control the activity of proinflammatory enzymes such as phospholipases A2 (PLA2s), cyclooxygenase, and lipoxygenases (LOX) [37]. They also control the production of pro-inflammatory factors and

Anti-Inflammatory and Immunomodulatory Properties of Phlorotannins
Inflammation is the body's reaction to harmful stimuli, such as an infection or injury, which leads to the development of symptoms such as seizure, pain, fever, and swelling. The inflammatory response is an immunological reaction to exogenous and endogenous signals that serve as the host's defensive systems against potentially harmful stimuli [32]. An inflammatory response is necessary for survival, and it plays an important role in cellular physiology; nevertheless, uncontrolled and excessive inflammation leads to a variety of chronic diseases. [60]. Inflammatory drugs include both steroidal and nonsteroidal, which are synthetic drugs used to treat inflammatory disorders that have potentially dangerous side effects and are not safe for humans [61]. In recent years, many investigations on the isolation and identification of natural anti-inflammatory drugs have been carried out. As a result, naturally occurring organisms have components that chemically resemble steroid structures and have been reported to exhibit a high potential for anti-inflammatory action [62]. Natural compounds with anti-inflammatory potential have antioxidant and radical scavenging characteristics, as well as the capacity to control the activity of proinflammatory enzymes such as phospholipases A2 (PLA2s), cyclooxygenase, and lipoxygenases (LOX) [37]. They also control the production of pro-inflammatory factors and genes [63]. As a result, these discoveries may open the path for the discovery of natural anti-inflammatory drugs. Fucosterol isolated from E. bicyclis reduced LPS-induced nitric oxide (NO) generation at non-toxic doses [64]. Furthermore, fucosterol inhibited tert-butyl hydroperoxide-induced reactive oxygen species (ROS) production and the expression of COX-2i and NOS. Several researchers used RAW 264.7 cells activated by lipopolysaccharide (LPS) to assess anti-inflammatory activity. Phlorotannins isolated from Fucales were found to be non-toxic at concentrations ranging from 31.25-500 µg/mL [65]. In RAW 264.7 cells activated by LPS, phloroglucinol reduced the generation of inflammatory mediators such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and prostaglandin E 2 (PGE 2 ) in RAW 264.7 cells stimulated by LPS [66]. Furthermore, phloroglucinol inhibited the production of metalloproteinase, which is involved in chronic inflammation in HT 1080 cells. In macrophages activated by LPS, phlorofucofuroeckol-B isolated from Myagropsis myagroides blocked the NF-κB pathway via suppression of the Akt-ERK pathway ( Figure 4) [67]. Gonçalves-Fernández et al. [68] confirmed the cytotoxic activity against murine cell lines by identifying fractions of low molecular size and medium polarity among fractionated phlorotannins. At a concentration of 100 µM, the fraction reduced ATDC-5 cell proliferation by 50%. In another study, phlorotannins isolated from the brown macroalga Padina tetrastromatica were shown to have strong anti-MRSA, anti-inflammatory, and antioxidant activities [69]. It was discovered, in particular, that the content and structure of phlorotannins differed depending on environmental and geographic conditions. Yu et al. [70] discovered that phlorofucofuroeckol-B isolated from E. stolonifera suppressed IL-1β, IL-6, and TNF-α ( Figure 4). Furthermore, phlorofucofuroeckol-B suppressed the generation of NO and prostaglandins and downregulated the LPS-induced expression of inducible cyclooxygenase-2 and NO synthase in LPS-stimulated BV-2. The compound 6,6 -Bieckol decreased nitric oxide and prostaglandin production and suppressed LPS-induced expression of inducible cyclooxygenase-2 and NO synthase ( Figure 4) [71]. Importantly, 6,6 -bieckol inhibited the expression of IL-6 and TNF-α. genes [63]. As a result, these discoveries may open the path for the discovery of natural anti-inflammatory drugs. Fucosterol isolated from E. bicyclis reduced LPS-induced nitric oxide (NO) generation at non-toxic doses [64]. Furthermore, fucosterol inhibited tert-butyl hydroperoxide-induced reactive oxygen species (ROS) production and the expression of COX-2i and NOS. Several researchers used RAW 264.7 cells activated by lipopolysaccharide (LPS) to assess anti-inflammatory activity. Phlorotannins isolated from Fucales were found to be non-toxic at concentrations ranging from 31.25-500 µg/mL [65]. In RAW 264.7 cells activated by LPS, phloroglucinol reduced the generation of inflammatory mediators such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) in RAW 264.7 cells stimulated by LPS [66]. Furthermore, phloroglucinol inhibited the production of metalloproteinase, which is involved in chronic inflammation in HT 1080 cells. In macrophages activated by LPS, phlorofucofuroeckol-B isolated from Myagropsis myagroides blocked the NF-κB pathway via suppression of the Akt-ERK pathway ( Figure 4) [67]. Gonçalves-Fernández et al. [68] confirmed the cytotoxic activity against murine cell lines by identifying fractions of low molecular size and medium polarity among fractionated phlorotannins. At a concentration of 100 µM, the fraction reduced ATDC-5 cell proliferation by 50%. In another study, phlorotannins isolated from the brown macroalga Padina tetrastromatica were shown to have strong anti-MRSA, anti-inflammatory, and antioxidant activities [69]. It was discovered, in particular, that the content and structure of phlorotannins differed depending on environmental and geographic conditions. Yu et al. [70] discovered that phlorofucofuroeckol-B isolated from E. stolonifera suppressed IL-1β, IL-6, and TNF-α ( Figure 4). Furthermore, phlorofucofuroeckol-B suppressed the generation of NO and prostaglandins and downregulated the LPS-induced expression of inducible cyclooxygenase-2 and NO synthase in LPS-stimulated BV-2. The compound 6,6′-Bieckol decreased nitric oxide and prostaglandin production and suppressed LPS-induced expression of inducible cyclooxygenase-2 and NO synthase ( Figure 4) [71]. Importantly, 6,6′-bieckol inhibited the expression of IL-6 and TNF-α.

Phlorotaninns Having Anticancer Potential
Cancer is a non-infectious disease caused by uncontrolled cell division or malignant cell growth. Normal cells are still being attacked and destroyed by abnormal cells. Death can occur if these systems are not managed [72]. Therapeutics for many forms of cancer have been developed over the years. However, because normal and cancer cells are so similar, anticancer drugs are likely to have negative effects on normal cells. Furthermore, because anticancer drugs are very toxic, cancer patients usually have side effects after therapeutic procedures [73,74]. These problems explain why research is being conducted to discover new therapeutic agents to replace existing ones. Many metabolites isolated from organisms are being studied for anticancer properties. According to studies, the majority of natural anticancer compounds have no side effects and control the growth of cancer cells [74]. As a result, one of the most significant strategies for cancer control is the quest for effective natural anticancer drugs. The anticancer mechanism of phlorotannins encompasses several pathways that are linked to the development of cancer and the triggering of cell death [75][76][77]. Phlorotannins prevent cancer via increasing cytotoxic T lymphocytes, dendritic cells, the epithelial-mesenchymal transition process, matrix metalloproteinase, phagocyte release, and decreasing SLUG and VEGF expression. Furthermore, phlorotannins modulate apoptosis induction by increasing the expression of apoptosis antigen 1 (APO-1), B-cell lymphoma 2 (Bcl-2) protein, caspase-3, -7, -9, cysteinyl aspartate specific proteinase (casp), and down-regulating the expression of the protein kinase B (AKT) pathway, B-cell lymphoma-extra-large (Bcl-xL) protein, extracellular signal-related kinase (ERK) pathway, FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (FLIP), nuclear factor-kB (NF-κB), phosphoinositide 3-kinase (PI3K) pathway, and X-linked inhibitor of apoptosis (XIAP) ( Figure 5). Dieckol extracted from E. cava was shown to be cytotoxic to A2780 and SKOV3 ovarian cancer cells, as reported by Ahn et al. [78]. Dieckol promoted apoptosis in SKOV3 cells and reduced tumor growth in SKOV3-bearing mouse models without causing severe toxicity. Furthermore, phloroglucinol from E. cava inhibited multiplication and triggered apoptosis in MCF-7 human cancer cells [79]. Phloroglucinol induced an increase in apoptosis-related gene expression while decreasing anti-apoptotic gene expression. Another study found that phloroglucinol effectively suppressed breast cancer cells by downregulating SLUG without generating cytotoxicity [80]. Kang et al. [81] discovered that phloroglucinol decreased cell viability and promoted apoptosis in HT-29 colon cancer cells. Phloroglucinol induced apoptosis-related alterations such as Bcl-2 family protein modification, cytochrome c release, and caspase-3 and -8 activation ( Figure 5). Phlorotannins isolated from Cystoseira sedoides induced apoptosis in more than half of MCF-7 breast cancer cells, with the IC 50 value of 78 µg/mL [82]. Furthermore, in Nnitrosodiethylamine-induced hepatocarcinogenesis, dieckol inhibited lipid peroxidation, hepatic cell damage, and increased antioxidant defense mechanisms ( Figure 5) [83]. Mar. Drugs 2022, 20, x 9 of 32

Antioxidant Abilities of Phlorotannins in Modulating Oxidative Stress
Oxidation is a key metabolic process in which electrons or hydrogen are transferred from a material to an oxidizing agent. Free radicals are produced during oxidative processes, which damage or destroy cells. Excess-free radical generation destroys enzymes like superoxide, dismutase, catalase, and peroxidase, resulting in a variety of human disorders, including diabetes and cancer [84,85]. The consequences of excessive oxidation highlight the need for a wide range of antioxidants obtained from natural sources. By preventing oxidation, antioxidants serve as a defensive mechanism against the consequences of excessive oxidation. These antioxidants found in the body have been linked to protection from various diseases, including cancer, aging, and Alzheimer's [51,86]. Various in-vitro methods are applied to determine the antioxidant activity present in natural sources. Scavenging assays utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical are used to evaluate the radical scavenging activity of antioxidants. The β-carotene-linoleate model systems and the thiobarbituric acid reactive substances tests are widely used to assess the antioxidant activity of foods and organic compounds containing lipids. The reducing power of antioxidants is measured using the ferric antioxidant power reduction and cupric ion reducing antioxidant capacity assays. These assays are commonly used to identify prospective antioxidant sources [87,88]. Sathya et al. [89] found that the phlorotannins from C. trinodis had significant radical-scavenging activity against superoxide and DPPH radicals and were as effective as ascorbic acid and α-tocopherol. Previously, it was attempted to determine the relationship between the number of hydroxyl groups in phlorotannins and antioxidant activity. The results showed the antioxidant activity of phlorotannins to be dependent on the structure (substitution pattern) of phlorotannins [90]. Moreover, Ahn et al. [91] demonstrated that phlorotannins isolated from E. cava have better free radical scavenging and DNA damage inhibition properties. Nakai et al. [92] conducted a study to enhance

Antioxidant Abilities of Phlorotannins in Modulating Oxidative Stress
Oxidation is a key metabolic process in which electrons or hydrogen are transferred from a material to an oxidizing agent. Free radicals are produced during oxidative processes, which damage or destroy cells. Excess-free radical generation destroys enzymes like superoxide, dismutase, catalase, and peroxidase, resulting in a variety of human disorders, including diabetes and cancer [84,85]. The consequences of excessive oxidation highlight the need for a wide range of antioxidants obtained from natural sources. By preventing oxidation, antioxidants serve as a defensive mechanism against the consequences of excessive oxidation. These antioxidants found in the body have been linked to protection from various diseases, including cancer, aging, and Alzheimer's [51,86]. Various in-vitro methods are applied to determine the antioxidant activity present in natural sources. Scavenging assays utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and 2,2 -azino-bis-3ethylbenzthiazoline-6-sulphonic acid radical are used to evaluate the radical scavenging activity of antioxidants. The β-carotene-linoleate model systems and the thiobarbituric acid reactive substances tests are widely used to assess the antioxidant activity of foods and organic compounds containing lipids. The reducing power of antioxidants is measured using the ferric antioxidant power reduction and cupric ion reducing antioxidant capacity assays. These assays are commonly used to identify prospective antioxidant sources [87,88]. Sathya et al. [89] found that the phlorotannins from C. trinodis had significant radical-scavenging activity against superoxide and DPPH radicals and were as effective as ascorbic acid and α-tocopherol. Previously, it was attempted to determine the relationship between the number of hydroxyl groups in phlorotannins and antioxidant activity. The results showed the antioxidant activity of phlorotannins to be dependent on the structure (substitution pattern) of phlorotannins [90]. Moreover, Ahn et al. [91] demonstrated that phlorotannins isolated from E. cava have better free radical scavenging and DNA damage inhibition properties. Nakai et al. [92] conducted a study to enhance the radi-cal scavenging activity of S. ringgoldianum. As a result, it was shown that oligomerization of phloroglucinol was required for the improvement of radical scavenging activity. A recent study found a relationship between phlorotannin content and total antioxidant activity, indicating that phlorotannins play a role in determining total antioxidant activity [93]. Lee et al. [94] discovered that phlorofucofuroeckol-A exhibited excellent antioxidant properties against AAPH-induced ROS generation and lipid peroxidation in both in vitro and in vivo systems. Similarly, it was demonstrated that phlorotannins isolated from E. cava protected neurons against H 2 O 2 -induced neurotoxicity and significantly reduced intracellular ROS production, lipid peroxidation, and apoptosis generated by AAPH [95,96].

Phlorotannins with Anti-Adipogenesis, Antidiabetic, and Anti-Obesity Activities
Obesity, diabetes, dyslipidemia, myocardial infarction, stroke, and hypertension are all symptoms of metabolic syndrome [97]. Metabolic syndrome is becoming more common in modern life and is regarded as a major health issue. According to Blüher [98], metabolic syndrome and obesity are closely associated. Obesity is defined by a rise in adipose tissue mass and an increase in fat cell size and number. Adipogenic transcription factors that control enzymes involved in lipid metabolism induce adipogenesis [99]. Transcription factors such as CCAAT/enhancer-binding protein (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), in particular, have a direct influence on adipogenesis [100,101]. Adipocyte hypertrophy and hyperplasia can be induced during adipogenesis by expressing adipogenesis-specific genes [102]. The model utilized to assess adipogenesis and adipocyte differentiation are 3T3-L1 cells [103]. Despite the fact that several anti-obesity drugs have been developed to treat obesity, current anti-obesity treatments have substantial adverse effects. Existing anti-obesity drugs have been linked to major psychological and cardiovascular problems [104]. Given the negative effects of anti-obesity drugs, there is a need for research on natural compounds having anti-obesity efficacy. Hu et al. [105] discovered that phlorotannins had an anti-obesity action by inhibiting pancreatic lipase and protein tyrosine phosphatase 1B and inducing adipocyte differentiation and apoptosis. Dioxynodehydroeckol and fucofuroeckol A isolated from E. bicyclis were shown to have substantial antidiabetic action against α-glucosidase (IC 50 value of 131.34, 93.33 nmol/L) and α-amylase (IC 50 value of 42.91, 472.7 µmol/L) [106]. Furthermore, fucofuroeckol A and dioxinodehydroeckol inhibited α-glucosidase in a noncompetitive manner. Another investigation found that among the E. cava phlorotannins, dieckol had the greatest inhibitory effect against α-glucosidase (IC 50 value of 10.8 µmol/L) and α-amylase (IC 50 value of 124.9 µmol/L) [107]. Gheda et al. [108] found that providing phlorotannins from C. compressa to diabetic rats lowered their serum glucose levels, total serum cholesterol, total triglycerides, liver malondialdehyde, α-amylase, and glucosidase activity. Moreover, the histological investigation revealed that the diabetic group's islets of Langerhans and certain necrotic regions were substantially damaged. In contrast, sections of diabetic rats treated with phlorotannin exhibited a considerable improvement and recovery in the size of the islets of Langerhans. Fucus vesiculosus phlorotannin extract inhibited pancreatic lipase, α-amylase, and notably, µ-glucosidase [109]. Ryu et al. [110] discovered that isophloroglucin A isolated from Ishige okamurae had an IC 50 value of 54.97 µM in α-glucosidase inhibition. It showed the greatest σ-glucosidase inhibitory activity among the phlorotannin groups isolated from I. okamurae. The IC 50 values of phlorofucofuroeckol A for α-glucosidase and α-amylase from E. cava were 19.52 and 6.34 µM, respectively [111]. The enzyme activity was inhibited by the hydroxyl group of phlorofucofuroeckol A, which is bound to it. Kang et al. [112] administered dieckol (10 and 20 mg/kg body weight) to diabetic mice for 14 days and observed that the blood sugar levels, serum insulin levels, body weight, and thiobarbituric acid reactive substrates in the dieckol-administered group were significantly decreased compared to the saline-administered group. Furthermore, the dieckol-administered group showed enhanced activity of several antioxidant enzymes e.g., catalase, glutathione peroxidase, and superoxide dismutase. Kim et al. [113] reported that dioxynodehydroeckol reduced lipid accumulation by inhibiting PPARγ, CEBP/α, and sterol regulatory element-binding protein 1 (SREBP). In addition, dioxynodehydroeckol suppressed fatty acid synthase, fatty acid-binding protein, lipoprotein lipase, acyl-CoA synthetase 1, and fatty acid transport protein. In a tyrosinase inhibitor experiment, dieckol from E. cava outperformed a commercial tyrosinase inhibitor (kojic acid) [114]. Interestingly, E. stolonifera eckol, phlorofucofuroeckol A, and phloroglucinol, inhibited lipid accumulation in 3T3-L1 cells, as well as the expression of key adipocyte marker genes such as C/EBPα and PPARγ [100]. Liu et al. [115] administered dieckol extract (50 mg/kg/day) to nonalcoholic fatty liver induced rats for 4 weeks. Significant improvement was observed in the plasma lipid profile, visceral fat index, liver index, and liver fat accumulation in the dieckol extract administration group. Ko et al. [116] reported that dieckol of E. cava exhibited an adipogenesis inhibitory activity that downregulates the expression of SREBP1, PPARγ, and C/EBPα. Figure 6 depicts the diagrammatic explanation of phlorotannin's activity as an antidiabetic, anti-obesity, and anti-adipogenesis agent via the activation and inhibition of various enzymes and signaling molecules. Table 2 lists a number of other phlorotannins with various biological functions.
Mar. Drugs 2022, 20, x 11 of 32 CEBP/α, and sterol regulatory element-binding protein 1 (SREBP). In addition, dioxynodehydroeckol suppressed fatty acid synthase, fatty acid-binding protein, lipoprotein lipase, acyl-CoA synthetase 1, and fatty acid transport protein. In a tyrosinase inhibitor experiment, dieckol from E. cava outperformed a commercial tyrosinase inhibitor (kojic acid) [114]. Interestingly, E. stolonifera eckol, phlorofucofuroeckol A, and phloroglucinol, inhibited lipid accumulation in 3T3-L1 cells, as well as the expression of key adipocyte marker genes such as C/EBPα and PPARγ [100]. Liu et al. [115] administered dieckol extract (50 mg/kg/day) to nonalcoholic fatty liver induced rats for 4 weeks. Significant improvement was observed in the plasma lipid profile, visceral fat index, liver index, and liver fat accumulation in the dieckol extract administration group. Ko et al. [116] reported that dieckol of E. cava exhibited an adipogenesis inhibitory activity that downregulates the expression of SREBP1, PPARγ, and C/EBPα. Figure 6 depicts the diagrammatic explanation of phlorotannin's activity as an antidiabetic, anti-obesity, and anti-adipogenesis agent via the activation and inhibition of various enzymes and signaling molecules. Table 2 lists a number of other phlorotannins with various biological functions.

Limitations Related to the Phlorotannin Isolation, Purification, Characterization, and Application
Phlorotannins isolated from seaweed are compounds with high biological activity and have been found to be effective as a natural antibacterial, antiviral, antioxidant, anticancer, and anti-obesity agent. However, several constraints must be overcome in order to take full advantage of these properties. Because certain seaweeds contain heavy metals such as Cu, Hg, As, and Cd, unpolished seaweed can be lethal to humans [117][118][119]. To address this issue, a high level purification technique is required. Although there is a rising interest in bioactive compounds found in seaweed, it is difficult to validate comprehensive in-vivo processes due to a lack of bioavailability research [120][121][122]. Furthermore, in vitro investigations make it difficult to identify a compound's genuine potential in for-

Limitations Related to the Phlorotannin Isolation, Purification, Characterization, and Application
Phlorotannins isolated from seaweed are compounds with high biological activity and have been found to be effective as a natural antibacterial, antiviral, antioxidant, anticancer, and anti-obesity agent. However, several constraints must be overcome in order to take full advantage of these properties. Because certain seaweeds contain heavy metals such as Cu, Hg, As, and Cd, unpolished seaweed can be lethal to humans [117][118][119]. To address this issue, a high level purification technique is required. Although there is a rising interest in bioactive compounds found in seaweed, it is difficult to validate comprehensive in-vivo processes due to a lack of bioavailability research [120][121][122]. Furthermore, in vitro investigations make it difficult to identify a compound's genuine potential in formulation development [35]. As a result, there is a need for a study to clarify the mechanism of phlorotannins in vivo and clinically. Methanol, ethyl acetate, ethanol, and acetone are the most common solvents used for phlorotannin extraction. These solvents are not safe for animals or humans, and researchers should be aware of the maximum residual limit of solvents in phlorotannins [123,124]. High-temperature extraction of phlorotannins or microwave extraction would dissolve more compounds and increase extraction yield. The heat generated at high temperatures and the high output of microwaves may reduce antioxidants in phlorotannins, hence it is critical to investigate proper extraction conditions [125,126]. Enzyme-assisted extraction of phlorotannins is a safe and ecologically friendly approach. However, due to the cost of enzymes, lack of substrate-specific enzymes, and limitations in maintaining bioreactor conditions, using an appropriate extraction method is required [127,128]. Several experiments have been carried out to identify the structural characteristics of purified phlorotannins, but the drawback is that there is no available library for comparison with the standard [129,130]. The spectrophotometer-based methods, such as the Folin-Ciocâlteu assay, FRAP assay, and ORAC assay, have a low specificity because non-phenolic substances are prone to overestimate the results [131,132]. Although GC-MS technology is capable of detecting pure compounds, its limits come when the analytical technique is more sensitive than the assay employed to evaluate biological activity [133]. This might lead to the loss of trace quantities of biologically active compounds, emphasizing the need to isolate pure molecules. Furthermore, the solvent employed in the extraction should be evaluated critically at each stage of the cycle process. The technique should be clarified fundamentally, including the rationale of the maximum residual limit of solvent within the pure compound. It might be argued that optimizing extraction processes would result in high-quality phlorotannin from various seaweed sources as nutraceuticals for improving human health. However, because most research on the biological use of phlorotannins have used extracts, efforts must be made to develop a standard purification technique for the active component present in the extracts.          • MTT assay • Aminopeptidase-N enzyme assay • Cell proliferation assay Cell migration was inhibited by lowering the level of protein and gene expression of matrix metalloproteinases such as MMP-2 and -9 [142] Mar. Drugs 2022, 20, 384 25 of 31

Conclusions and Future Perspectives
In conclusion, the present review paper describes the many forms of phlorotannins produced from various marine algae sources. These phlorotannins, which are polyphenolic compounds derived from brown algae, have a wide range of biological characteristics, including antimicrobial, antidiabetic, anticancer, and anti-inflammatory properties (Tables 1 and 2). The vast spectrum of biological activities associated with phlorotannins is projected to increase their favorable health value in the food, pharmaceutical, and cosmeceutical industries. Despite the prospective uses of phlorotannins, the successful development of a series of products produced from brown algal polyphenols as nutraceuticals have remained an unmet goal. Nutritional epidemiology studies demonstrate a strong link between consuming polyphenols and modifying the molecular pathways of carcinogenesis, as well as lowering cancer cell growth. Despite its extensive use as an antimicrobial, anticancer, antidiabetic, and anti-inflammatory agent, phlorotannin's low stability and solubility restrict its application. In recent trends, nanotechnology, particularly nanocarriers and nanoformulation, are evolving to overcome the limits associated with natural products and allow targeted and controlled drug delivery. To treat microbial infections, the phlorotannin must be loaded onto the nanocarrier (metallic or nonmetallic) in such a way that the nanocarriers are biocompatible and have inherent antimicrobial capabilities allowing for the synergistic killing of the microbial pathogens [143].
Furthermore, combination therapy has emerged as a viable strategy for revitalizing antibiotics or antifungals in order to fight microbial pathogens in a synergistic manner. The reported chemical structures for some of the phlorotannins (Figure 1) can be employed as scaffolds for the synthesis of the derivatives by grafting some of the active molecules on the phlorotannin to produce broad-spectrum and effective biological effects. Studying the structure-activity relationships of phlorotannins is essential in order to explain the molecular action mechanisms in various biological roles, since various antimicrobial resistance signaling proteins and transcription factors, such as efflux pumps, biofilms, virulence, reverse transcriptase, protease, integrase, and so on, have been thoroughly described in microbial pathogens. As a result, elucidating the molecular docking-interaction of phlorotannins with known microbial proteins relevant to pathogenesis and resistance mechanisms will be more intriguing in future studies. Aside from gene expression studies of microbial virulence, biofilms, antimicrobial resistance genes, and genes implicated in cancer signaling pathways, inflammation, diabetes, and obesity would provide more supporting research to validate the results of the in vitro studies. In addition, to confirm the in vitro antimicrobial activity of phlorotannins, an in vivo investigation employing an animal model organism is required.