Abstract
Medicinal chemists and pharmacognosists have relied on terrestrial sources for bioactive phytochemicals to manage and treat disease conditions. However, minimal interest is given to sea life, especially macroalgae and their inherent phytochemical reserves. Phlorotannins are a special class of phytochemicals mainly predominant in brown algae of marine and estuarine habitats. Phlorotannins are formed through the polymerization of phloroglucinol residues and derivatives via the polyketide (acetate–malonate) pathway. Studies over the past decades have implicated phlorotannins with several bioactivities, including anti-herbivory, antioxidants, anti-inflammatory, anti-microbial, anti-proliferative, anti-diabetic, radio-protective, adipogenic, anti-allergic, and anti-human immunodeficiency virus (anti-HIV) properties. All these activities are reflected in their applications as nutraceuticals and cosmeceutical agents. This article reviews the chemical composition of phlorotannins, their biological roles, and their applications. Moreover, very few studies on phlorotannin bioavailability, safety, and toxicity have been thoroughly reviewed. The paper concludes by suggesting exciting research questions for further studies.
Introduction
One day the tables will turn, and the wealth of every country will be defined by the nature they hold. When that day comes, we shall tap into the ubiquitous architecture of nature's reservoir of remedies. According to a notable quote by Hippocrates, “Let food be your medicine and medicine be your food”. 1 Nature's architecture is so designed that numerous plants and animals from both terrestrial and marine sources have become the primary source of food for humans, since time immemorial, with the marine/aquatic ecosystem serving as the principal and best reservoirs. A large body of growing evidence shows that plants and animals from marine sources are potential food sources for man's daily life. In recent years, marine macroalgae have received much interest globally as a source of animal feed and healthy food supplements. Minerals, soluble dietary fibers, peptides, proteins, carotenoids, polysaccharides, potential metabolites, and other bioactive compounds are abundant in these macroalgae.2,3 Previous studies have also reported that macroalgal metabolites have anti-adipogenic, antioxidant, antibacterial, anti-inflammatory, antidiabetic, anti-cancer, and anti-human immunodeficiency virus (anti-HIV) properties. 3 Seaweeds are not photo-dynamically damaged despite being subjected to severe environmental conditions such as high oxygen concentrations and light, which contribute to the formation of free radicals, as well as other potent oxidizing agents. 4 As a result, marine algae can synthesize bioactive substances to protect themselves from external threats, including stress, herbivores, and ultraviolet (UV) radiation. 5 Brown seaweeds have also been found to lower methane emissions when fed to ruminants, which is critical given the increasingly stringent emission standards imposed on this intensive farming sector. 6 Animal feeds supplemented with seaweed are nutritionally rich renewable feedstocks with several attributes vital for livestock health.6–9 Such attributes include reduced fecal shedding and improved gut health.10,11 A large body of evidence from previous studies has shown that a pig diet supplemented with seaweed leads to decreased need for antibiotics, which could avert the antibiotic resistance crisis currently facing the livestock industry. 12 These observed antibacterial and antimicrobial activities are linked to complex polyphenolic structures known as phlorotannins. 13
Phlorotannins are phloroglucinol-based (13,5-trihydroxy benzene) polyphenolic compounds that are accumulated by marine brown algae (Phaeophyta), consisting of 5 to 12% of their total dry mass. 14 Phlorotannins are formed through the phloroglucinol oligomer polymerization via the polyketide (acetate–malonate) pathway.14,15 Various marine brown algae, including Hizikia fusiformis, Ecklonia cava, Laminaria japonica, E kurome, Ishige okamurae, Sargassum thunbergii, Eisenia bicyclis, Undaria pinnatifida, and E stolonifera have been reported to contain phlorotannins with health-promoting biological activity. E cava, E kurome and E bicyclis are predominant in Korean and Japanese coastal regions. A report from previous studies showed that the content of phlorotannins in E bicyclis was 3% of its total dry mass, and high-performance liquid chromatography (HPLC) results showed that it contains 0.9% of phloroglucinol, 4.4% of a tetramer of phloroglucinol, 21.9% of phlorofuroeckol, 7.5% of eckol, 23.4% of dieckol, and 24.6% of 8,8′-bieckol, as well as other unknown phenolic compounds (7.5%). 16 However, the amount of phlorotannins in seaweeds varies depending on the species, geographic region, and extraction method adopted. 17 Although they are structurally distinct, phlorotannins have many similarities to tannins produced by terrestrial plants. 18 Fucodiphlorethol G was isolated from E cava and acetylated to produce several derivatives of the compound. 19 The presence of numerous phenolic -OH groups in the structure makes them highly hydrophobic, allowing them to be easily absorbed into the biological system upon digestion. Phlorotannins are concentrated and predominant in the epidermal cortex of brown seaweeds, but are also known to be bound to marine microalgal cell walls. 20 Phlorotannins are thought to help the plant defend itself by acting as a herbivore deterrent and potentially as an appetite suppressant. 21 This review focuses on the sources of phlorotannins, their chemical compositions and properties, analytical techniques for their extraction, characterization, and quantification, biological roles and other prospects, with their huge potentials as pharmaceuticals, nutraceuticals, cosmeceuticals (skin-protective effects) as well as metal sequestration agents.
Chemical Composition and Properties of Phlorotannins
The production pathways of phlorotannins in brown seaweed are still unclear since they have been poorly elucidated. Despite this, a school of thought suggests that they are biosynthesized via a malonate and acetate condensation reaction seen in the phenylpropanoid or shikimate pathway. This condensation is usually with two molecules of acetyl-CoA with carbon dioxide to yield malonyl-CoA, 22 a reaction catalyzed by polyketide type III synthase. This compound undergoes further chemical transformations, including a Claisen cyclization of 3 molecules of malonyl CoA, which leads to the formation of structural isomers of phloroglucinol. The tautomerization of these compounds produces a stable thermal form of the compound. 23 The carbon to carbon and carbon to oxygen linkages observed due to tautomerization of phloroglucinol have led to various types of phlorotannins. 24
Structural Classification of Phlorotannins
Phlorotannins are polymers of phloroglucinol (1, 3, 5 trihydrobenzene). This compound has been classified into linear and branched phloroglucinol. It has also been given six different classes based on monomeric bindings found in the glucinol ring. The presence of ether bonds gives rise to phloroethols and fuhalols (and its isoform isofuhalols). Those with the presence of both ether and phenyl bonds are categorized as fucophloroethols, and those with phenyl bonds as fucols. The dibenzodioxin linkage gives rise to phloroeckols (Figure 1). 18
Structure of phlorotannins. (A) Phloroglucinol (the monomeric unit of phlorotannins). (B1) and (B2) show dibenzodioxin linkages in Phloroeckol and Carmalol; (C1) and (C2) show ether linkages in Phloroethol and Fuhalol, respectively. (D) Shows phenyl linkage in fucols. (E) Shows both ether and phenyl linkages in Fucophlorethol.
Phloroethols are formed from the ether bonds of phloroglucinol rings in different positions such as the 1 and 2 positions (ortho), 1 and 3 positions (meta), and 1 and 4 positions (para), giving rise to triphloroethol A, tetraphloroethol C and tetraphloroethol B. 22 The ethols also contain fucophloroethols, which are obtained from the condensation of a phenyl bond and an ether bond, as observed in fucophlorethol A, fucophlorethol B, and fucodiphlorethol B. 13 Fuhalols, such as trifuhalol B, bifuhalol, and trifuhalol A, possess an additional -OH group in their monomer unit. Eckols also have this type of -OH group found in fuhalols. These eckols include phloroeckols, eckols and dieckols, fucophloroeckols (eckmaxol, 6, 6′ -biekol and 8, 8′ -bieckol), fucofuroeckols (phlorofucofuroeckol-A, phlorofucofuroeckol B, and phlorofucofuroeckol) and dioxinodehydroeckol. The interlinkage of phloroglucinol in the 1, 3 (meta) positions of phlorotannins give rise to the likes of fucols such as tetrafucols A and B, and heptafucols. 22
Extraction of Phlorotannins
Several methods have been adopted to extract phlorotannins. However, it is essential to note that the extraction methods significantly impact the possible elucidation and quantification of phlorotannins. It is usually advised to follow methods that will limit the oxidation of these phlorotannins. One interesting approach to achieve limitation of phlorotannin oxidation is by acetylating them 25 or possibly adding antioxidants while extracting, such as ascorbic acid and K2S2O5, to halt the oxidation process caused by phenols on phlorotannins, since they are extracted together. 4
Generally, the solvent extraction method has been widely applied for the extraction of phlorotannins. However, before maceration with polar organic solvents, the lipid contents and other pigments are first extracted from the dried seaweeds. The dried form of seaweed is treated or washed with n-hexane 3 times and centrifuged at 3200 g for 3 min after each wash. 25 The mixture concentration or ratio of the dried seaweed (solid) to n-hexane is 1:5 w/v, 22 although some other studies have tried other ratios. 25 This protocol is based on the principle that nonpolar solvents such as n-hexane are suitable for extracting lipids and highly hydrophobic secondary metabolites. Treatment with n-hexane was made to remove the lipid content and other pigments present. Going forward, the marc, which has been freed of lipids and pigments, is re-macerated either with polar or organic solvent, or a mixture of both to extract phlorotannins based on their high solubility. Some of the solvents used for phlorotannins extraction include methanol, ethanol, acetone, water, water/methanol 50:50, n-hexane/ethanol 88:12, ethanol/water 25:75 or 80:20, ethyl acetate/water 50:50, water/acetone 20:80 or 30:70, and methanol/chloroform 66:33% (v/v). 22 Koiviko et al 25 showed that aqueous acetone (water/acetone 20:80 or 30:70) yielded more phlorotannin (70%) in comparison with other extraction solvents.
Similarly, another study reported an optimum yield of phlorotannins from Fucus vesiculosus with 67% acetone. 26 Conversely, Yoon et al 27 reported the best yield of phlorotannins from the edible brown seaweed E cava with 95% ethanol. A pressurized liquid method has been used to extract phloroglucinol, which usually yields a high content of phenols. 27 Recent studies have shown that an improved yield and purity of phlorotannins can be achieved by applying several energy-assisted systems. A pressurized liquid method has been used to extract phloroglucinol with an improved yield and a higher content of phenols when compared to ordinary solvent extraction methods. 28 Other systems, such as microwave-assisted, ultrasonic-assisted, and enzyme-assisted extraction have been hyphenated to the extraction system. Presented in Table 1 are recent studies which have adopted different energy-assisted systems for phlorotannins extraction with their improved concomitant yields.
Analytical Methods of Quantification and Characterization of Phlorotannins
Phlorotannins after extraction can be qualitatively identified, and their amounts quantitatively estimated using several techniques. Two general qualitative analytical approaches for detecting phlorotannins are the Folin-Ciocalteu method and the 2,4-dimethoxybenzaldehyde (DMBA) method. 26 Several spectrometry hyphenated techniques have also been used to successfully estimate specific phlorotannins in an extract. 29 The Folin-Ciocalteu method is summarized as follows. The extracts are diluted in a ratio of 0.05 mL of extract to 4.95 mL of water. One mL of the diluted extract is mixed with 1 mL of Folin-Ciocalteu reagent and allowed to stand for 3 min, after which 2 mL of 20% of sodium carbonate is added. The mixture is then incubated in a dark environment at an ambient temperature for 45 min before centrifugation for 8 min (1600 g). The resultant blue coloration of the supernatant is an indication of the presence of a phenolic group (Figure 2).
Biological application of phlorotannins as nutraceuticals, cosmeceuticals and angiotensin-I converting enzyme inhibitory activities.
Moreover, the total phlorotannin content can be estimated by UV–visible spectrophotometry reading at an absorbance of 730 nm, and extrapolating against a standard curve. The Folin-Ciocalteu method has been the most frequently adopted method for estimating the amount and presence of phlorotannins. However, the DMBA method has been adopted and described explicitly in other studies. 30
Several chromatographic techniques, which are frequently hyphenated with spectrometric analytical procedures, have been applied to elucidate specific phlorotannin members. Low-molecular-weight phlorotannins have been quantified using HPLC, while some other polar forms are quantified with normal phase-HPLC. 23 Ultra-high-performance liquid chromatography (UHPLC) has also significantly contributed to elucidating the isomeric diversities in phloroglucinols. 31 Centrifugal partition chromatography (CPC) and two-dimensional liquid chromatography have also been used to isolate phlorotannins.32–34 Advances in analytical technology is evident in the use of high-resolution mass spectrometry (HRMS), ultra-efficient liquid chromatography-mass spectrometry (UPLC-MS), and by combining liquid chromatography of hydrophilic interaction (HILIC) 35 and NP-HPLC, a variant of HILIC, which has also been used to quantify phlorotannins with remarkable results.23,34,36
Factors Affecting the Content and Concentration of Phlorotannins
Pre-Processing
Before being employed in any nutritional test, industrial process, or storage, algae are harvested from the sea and subjected to several preprocessing procedures. Drying is the most generally used method for prolonging the shelf life of algae by lowering the moisture content, thereby reducing the risk of deterioration. 37 The two drying methods usually used are air drying and freeze-drying (Figure 3). Because of the significant output rates, industrial producers choose air-drying as a cost-effective and quick method. The air-drying process, however, results in the loss of phlorotannins as a result of exposure to high temperatures. However, freeze-drying keeps the phlorotannins concentration at a consistent level, but is expensive and time-consuming. 38 A study compared the effect of different drying pre-treatments (sun-drying, shadow-drying, oven-drying, and others) on phlorotannin content. The results indicated that the drying procedure significantly affected the phlorotannin content of the crude extract. Sun-dried tissues had the least concentration of phlorotannins, whereas freeze-dried tissues had the most. The researchers also proposed boiling the seaweed tissue before drying to denature enzymes responsible for bioactive compound decomposition. 39 However, when compared to freeze-dried tissue, this process dramatically reduces the phlorotannin concentration, as significant levels of phlorotannins are released into the water during boiling.
Extraction and fractionation of phlorotannins from dry seaweed.
Extraction Procedure
Phlorotannin content and concentration are greatly affected by the extraction procedure. Soxhlet extraction and maceration are the two conventional procedures widely employed in the extraction of bioactive compounds. However, these procedures are time- and energy-intensive, and they necessitate the use of significant volumes of solvents and the application of heat. Because some bioactive compounds are thermosensitive, there is an increasing interest in creating and using new, efficient, and environmentally friendly extraction procedures that improve extraction yield without compromising compound bioactivity (Table 1). Nonconventional technologies, such as ultrasound, pressured liquid, microwave, supercritical fluid, and subcritical fluid, are being considered to mitigate these problems. 40 A recent study compared the extraction yield achieved from Saccharina japonica phlorotannins using microwave and traditional extraction methods, and a greater yield was reported using microwave-assisted as compared to conventional solvent extraction methods. 40 Microwave-assisted extraction improves extraction yields by allowing improved solvent diffusion into seaweed tissue in a quick and efficient manner. 44
Green Approaches Employed in Phlorotannin Extraction.
The type of extraction solvents also affects the amount of phlorotannins extracted. Polar solvents have been reported to extract phlorotannins better than nonpolar solvents. 40 Ethanol, methanol, acetone, and ethyl acetate are the most regularly used organic solvents.25,48 For the extraction of phenolic compounds, a dual solvent system consisting of a mixture of water and organic solvent exhibits a greater extraction power due to the synergistic effect of its components than a monocomponent solvent system containing only water or organic solvent. 31 Water would swell plant cells, while ethanol could aid in disrupting the bonds between solutes and plant tissues. 49 Thus, a mixture of water and organic solvents is usually preferred. 50
Extraction time, temperature/pressure, and solvent-to-solid ratio are critical parameters determining the phlorotannin content and concentration. A study of the yield of phlorotannins using a conventional extraction process revealed that the concentration of phlorotannins grew as the extraction period increased to a maximum value (30 min), and subsequently declined. 31 Phlorotannins are likely exposed to oxidative processes compromising their stability for longer durations. As a result, good extraction time selection is required to ensure effective bioactive chemical extraction and lower energy expenditures. 26 An increase in temperature generally increases the solubility of compounds. High temperatures may enhance phlorotannin concentration by altering the structure of algal cell walls, weakening their tissue and, thereby, enabling the release of more phlorotannins. 51 When phlorotannins are exposed to specific high temperatures, however, they can degrade. Catarino et al 26 found that increasing the extraction temperature from 17 °C to 25 °C boosted the recovery of phlorotannins from F vesiculosus. However, a decline in extraction yield was observed above 25 °C. Solvent to solid ratio has been noted as one of the factors influencing phlorotannin concentration. Several studies have demonstrated that an increase in the solvent-to-solid ratio increased the extraction yield of polyphenols from seaweed until reaching a maximum value, after which no further increase in recovery occurred. 26
Storage
Phlorotannins are affected by storage-related factors, such as dissolved oxygen, light, temperature, and time. 52 A study conducted by Cuong 53 measured the phlorotannin content of six dried Sargassum species (19% final moisture content) stored in polyethylene bags at 30 °C for 2 years and observed that with extended storage duration, the content of phlorotannins decreased significantly. Phlorotannin stabilization is necessary since storage might degrade these sensitive molecules. As a result, practical tactics for developing a stable product that retains the active molecular form of phlorotannins from storage to consumption are critical.52,54 Encapsulation has emerged as the most prevalent means of preventing the degradation of phlorotannins during storage. Encapsulation in this context refers to the encapsulation of an active ingredient (in this case, polyphenols) in micro- or nanocapsules made of another solid- or liquid-immiscible substance, such as a coating, carrier, or shell. The coating works as a physical permeability barrier, restricting molecular oxygen diffusion and thereby extending the shelf life of the encapsulated substance. 52 Many attempts have been made to create new encapsulation technologies to stabilize and deliver phenolic chemicals from terrestrial plants. However, there is a scarcity of studies on encapsulating polyphenols from seaweed.
Biological Roles of Phlorotannins
Phlorotannins are polyphenolics commonly found in brown algae of marine and estuarine habitats. 26 Phlorotannins are polymers of phloroglucinols and have diverse structures. They have multiple phenolic rings; however, they exhibit diverse linkages, such as ether, phenyl, and dibenzodioxin. 55 Moreover, phlorotannins have more hydroxyl groups than other tannins, hence an essential structural feature that translates to their bioactive potency and biological significance. 56
In brown algae, phlorotannins constitute 5 −12% of their dry weight and function as a repellent to herbivory activity. Studies have implicated phlorotannins as an inducible metabolite, overproduced as an outcome of stress in several species of brown algae.57,58 There is potential for some phlorotannins to act as insecticides based on their structural–function inference, although there are no studies at the time of this review to validate claims. Other bioactive roles from recent literature have shown phlorotannins to possess antioxidant, antimicrobial, anti-inflammatory, antiproliferative, antitumor, antidiabetic, radio-protective anti-adipogenic, and anti-allergic activities. 59 The subsequent subsection of this review extensively examines studies on various activities of phlorotannins.
Phlorotannins as Potent Antioxidants
Phlorotannins, especially from brown algae, have been reported to possess anti-oxidative properties. Many scholars have implicated the polyhydroxy groups in the structure and their inherent metal-chelating ability to be responsible for their potency in scavenging free radicals.55,60 Shibata et al 61 discovered that phlorotannins from Laminarian brown algae, as highlighted in Table 2, inhibited phospholipid peroxidation at 1 μM in the liposome system and scavenged more potently free radicals from superoxide anions and DPPH at 6.5 to 8.4 μM and 12 to 26 μM, respectively. The antioxidant results for phlorotannins were significantly higher than ascorbic acid and α-tocopherol. 61 Other studies have found anti-oxidative phlorotannins in species such as S kjellmanianum, 63 F vesiculosus,48,66 S ringgoldianum, 65 I okamurae 87 and Symphyocladia latiuscula, 68 with the capability of scavenging superoxide anions and other free radicals (Table 2). Most studies also investigated the capacity of the different chemical classes of phlorotannins to suppress reactive oxygen species (ROS) by adopting the 2′,7′-dichlorofluorescein diacetate assay. 76 In general, results from individual studies have shown that members of the eckol class of phlorotannins are more potent antioxidants than the other classes (Table 2).
Biological Activities of Phlorotannins.
DPPH - 2,2-diphenyl-1-picrylhydrazyl; BHT - Butylated hydroxytoluene; IC50 - half maximal inhibitory concentration; EC50 - Half maximal effective concentration; DCFH-DA - 2’-7’dichlorofluorescin diacetate; PGE2 - Prostaglandin E2; LDH - Lactate dehydrogenase (LDH)
Primarily, the anti-oxidative properties of phlorotannins are beneficial to humans and valuable to producers. A few studies have shown that phlorotannins are inducible secondary metabolites induced by herbivory activities. 57 Stern et al 72 reported that phlorotannins act as an antifeedant as they form a covalent bond with the gut proteins of herbivores. Similarly, another study conducted by Koivikko et al 58 discovered increased phlorotannin content with increased herbivory activities. Conversely, Pavia et al 74 showed that when brown algae are exposed to UV radiation, they significantly produce phlorotannin from their thallus compared to those algae stimulated by just herbivory activities. Summarily, oxidative stress, herbivory activities, and UV-radiation are the central reported stimulants for phlorotannin secondary metabolite production. 57
Anti-Inflammatory Activities
Inflammation forms part of the body's defense mechanism against foreign intruders and could be worrisome depending on its extent and how long it lasts. A few studies have reported the valuable roles phlorotannins play as an anti-inflammatory agent. Wijesinghe et al 71 reported the anti-inflammatory activities of a fermented phlorotannins-rich extract on liposaccharide-stimulated RAW 264.7 cell lines. Phlorofucofuroeckol A and trifuhalol A are the specific phlorotannins that have been reported to have remarkable anti-inflammatory activities.64,69,70 Both phlorotannins have shown significant inhibition of nitric oxide and prostaglandin-2. Studies on trifuhalol report its remarkable abilities to down-regulate nitric oxide synthase, cyclooxygenase-2, and tumor necrosis factor- α, hence confirming its roles as an anti-inflammatory agent.56,70
Antimicrobial Activities of Phlorotannins
Several studies have investigated the potential of phlorotannins to exert antimicrobial activities. The polyhydroxy group of phlorotannins contributes tremendously to its ability to annihilate or inactivate microbes. 13 Significant studies have shown that the most probable mechanism of action for its antimicrobial activities is through lipid peroxidation of the cell membrane of the microbes. 23 Nagayama et al 75 showed that dieckol and 8,8′-bieckol significantly killed Campylobacter sp, methicillin-resistant Staphylococcus aureus (MRSA), and Vibro parahaemolyticus compared to catechin as the control. Other studies have shown that phlorotannin-rich fractions showed intense antibacterial activity against MRSA with a 500 to 600 μg/mL minimum inhibitory concentration (MIC). Similarly, a unique phlorotannin, diekol, was discovered to exhibit antifungal activities against Trichophyton rubrum at an MIC of 200 μM. 80 Therefore, there is a need for more studies to investigate the antimicrobial ability of other phlorotannins on possibly recalcitrant organisms like Mycobacterium tuberculosis and other causes of infection.
Antitumor/Antiproliferative Activities
The search for potent and efficacious therapeutic approaches to treat and manage metastasis has been a long research problem. Phlorotannins from some brown algae have been discovered to possess some anti-proliferation or anticancer activities. Yuan and Walsh 95 reported up to 78% inhibition of HeLa cell proliferation by a phlorotannin-rich fraction from the methanolic extracts of some brown algae such as Laminaria setchellii, Macrocystis integrifolia, and Nereocystis leutkeana. Similarly, dioxinodehydroeckol, and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-24,9 trihydroxydibenzo-1,4-dioxin isolated from E cava inhibit the proliferation of breast cancer cells in a dose-dependent fashion by induction of apoptosis. 96 Despite the health potential reported for phlorotannins, there have been only a few studies on its anticancer and anti-proliferative activities. Hence, more studies are needed to adopt the genome-wide associated approach (GWAS) and the next-generation of high throughput techniques to investigate further the potency of phlorotannins as anticancer agents.
Antidiabetic Activities
Diabetes mellitus is a disease that causes high glucose levels due to insufficient insulin in the pancreas and has been considered the lead cause of mortality and morbidity in the world. Attention has been shifted to natural products with anti-diabetic potential from marine organisms. Studies have shown that phlorotannins extracted from the brown seaweed Cystoseira compressa possess anti-diabetic activities in streptozotocin-induced diabetic rats by decreasing serum glucose, α-amylase, and glucosidase activities by increasing the serum insulin and antioxidant levels. 102 Furthermore, a wide diversity of brown seaweed extracts and isolated phlorotannins have been explored for their ability to suppress carbohydrate digestion and glucose absorption. Of the species studied in the past decade, Ecklonia and Eisenia species are, by far, the most widely explored in this respect.76,81,103 A study of E cava revealed that its phlorotannin derivatives inhibited intestinal α-glucosidase and pancreatic α-amylase. The inhibition mechanism of one of the isolates, dieckol, was non-competitive. 77 Moon et al 104 reported six active phlorotannin derivatives from the genera Ecklonia and Eisenia, which exhibited intense activity against α-glucosidase and compared favorably with the reference drug, acarbose. Eckol, dieckol, and 7-phloroeckol, isolated from E bicyclis, exhibited α-amylase inhibitory capacity. 105 Diphlorethohydroxycarmalol (DPHC), isolated from I okamurae, 87 and 2-(4-[3,5-dihydroxyphenoxy]-3,5-dihydroxyphenoxy) benzene-13,5-triol (DDBT), isolated from S patens, 106 exhibited α-glucosidase and α-amylase inhibitory capacity. Lordan et al 107 reported how the solvent used for the extraction procedure influenced the bioavailability of different phlorotannin compounds in algae. Methanol extracts of Ascophyllum nodosum showed better potency against α-amylase, while the extract of F spiralis demonstrated more activity against α-glucosidase. Research has shown that phlorotannin rich extracts of S ringgoldianum lowered postprandial blood glucose levels in STZ-induced diabetic mice, 108 while other reports with diabetic mice revealed that phlorotannins restored normalcy in postprandial high glucose levels.81,109 Approaches towards the curative capacity of phlorotannins in diabetes-related complications have been recorded.103,110,111 Furthermore, some research reports have explored the potentials of phlorotannin-rich extracts to prevent glucose-induced toxicity.60,112 From the above statement, phlorotannins are a potent algal source for inhibiting digestive enzymes, considering their efficacy and reduced cytotoxicity.
Radioprotective Activities
Antioxidants prevent radiation that causes harm to cells. Recent studies have shown that phlorotannin derivatives comprised of phloroglucinol and eckol isolated from Ecklonia species protected intestinal stems against gamma-irradiation. 113 Similar radioprotective effects have been reported for extracts of Polyopes lancifolia. 114 Derivatives of phlorotannins, dioxinodehydroeckol (DHE), and diphloroethohydroxycarmalol (DPHC) have protective roles in ultraviolet B (UVB)-induced damage in HaCaT cells, 115 and DNA in HaCaT cells, 116 respectively. Topical application of metabolites from brown seaweeds, including phlorotannins, alleviated radiation dermatitis symptoms in mice and promoted the healing process. 117 Phloroglucinol from E cava played a protective role against oxidative damage in the whole-body irradiation of experimental mice by blocking the mitochondria-mediated caspases reaction. 118 Furthermore, this compound inhibited apoptosis and strengthened hematopoiesis in mice exposed to ionizing radiation. 119 Eckol from E cava prevented γ radiation damage in mice by inhibition of P53 and Bax gene, thereby blocking oxidative damage of the DNA. 120
Adipogenic Activities
Obesity and its related diseases are the underlining cause of fat metabolism. The inhibition of the adipogenic cells responsible for obesity has been suggested to be the primary key in its management. The research focus has shifted toward isolating bioactive compounds with antioxidant potential. Karadeniz et al 121 isolated triphlorethol-A, eckol, and dieckol, which inhibited the manifestation of adipogenic differentiation markers in 3T3-L1 fibroblasts and reduced lipid accumulation.
Similarly, Ko et al 122 and Choi et al 123 confirmed that dieckol isolated from E cava inhibited adipogenesis by activating the adenosine monophosphate-activated protein kinase (AMPK) of 3T3-L1 preadipocyte cells. Different fractions of phlorotannin from the same organism were also tested for their inhibitory effects on 3T3-L1 cells, and they exhibited anti-obesity properties via regulation of adipogenic transcription factors.124,125 Aside from E cava, I kamurae has been reported to contain a phlorotannin derivative, diphlorethohydroxycarmalol, which exhibited an inhibitory action on preadipocyte cells.
Anti-allergic activities
Seaweeds have been extensively utilized to manage allergic responses, which trigger chemical mediator inflammatory reactions. The development has created a new research focus toward providing a solution to allergic cascade reactions. Two derivatives of phloroglucinol (fucodiphloroethol and phlorofucofuroeckol A) isolated from E cava were extensively studied by Li et al 100 These derivatives exhibited anti-allergic activity via the inhibition of histamine release in human (KU812) and rat basophil leukemia-2H3 (RBL-2H3) cell lines. Sugiura et al 98 also reported an anti-allergic inhibitory effect of phlorofucofuroeckol-B isolated from E arborea against histamine release from rat basophile leukemia (RBL)-2H3 cells. Furthermore, Sugiura et al 98 reported that phlorotannin derivatives from E arborea exhibited an anti-allergic effect by inhibiting the activity of β-hexosaminidase released from the rat basophilic leukemia-2H3 cells. This inhibitory effect exhibited by these derivatives was more significant than that of the known inhibitor, epigallocatechin gallate.
Anti-HIV Activities
Since its discovery, the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), has become a global health issue. Drugs used in anti-HIV therapy for HIV/AIDs patients are no longer potent due to the emergence of HIV resistance to these drugs and side effects with prolonged usage. The need to pave the way to novel management of this infection using natural sources and marine algae has proved their potency due to the presence of phlorotannins. A phlorotannin derivative (diphlorethohydroxycarmalol) isolated from I okamurae exhibited anti-HIV 1 potency by inhibiting HIV-1 reverse transcriptase and integrase; however, no inhibition was recorded against HIV-1 protease. 67 A phloroglucinol derivative (6,6′-bieckol) found in E cava likewise blocked HIV-1 reverse transcriptase activity with no cytotoxic effect on the cells. 94 The same trend of inhibitory activity against HIV-1 p24 antigens with no cytotoxicity was recorded for 8,4‴-dieckol isolated from the same organism. 94 Langarizadeh et al 126 carried out an in silico study of phlorotannin derivatives from marine algae in viral protein U (Vpu). Their study revealed that these derivatives suppressed Vpu, are responsible for viral particle dissemination, and invariably reduced viral pathogenesis.
Applications of Phlorotannins
Nutraceutical Potential of Phlorotannins
Although recognized for their high antioxidant (free radical scavenging) activity, various studies have shown phlorotannins to be involved in a wide range of biological activities, making them promising candidates for diverse applications such as in food, nutraceuticals, and pharmaceuticals, as well as for cosmeceutical applications. 117 Some of the pharmacological activities attributed to these compounds include antidiabetic,82,102 neurological effects, such as prevention of chemical-induced memory impairments, 127 and mitigation of sleeplessness, 127 as well as their impressive in vitro effects against different targets involved in neurodegeneration.124,128 Phlorotannins are also effective against different types of cancer either directly 129 or as adjuvants helping to increase the sensitivities of cancer cells to chemotherapeutic agents.130,131 Oxidant species and inflammation play significant roles in the pathophysiology and progression of many diseases, including cancer, diabetes, and cardiovascular diseases. Thus, agents with potent antioxidant and anti-inflammatory effects are considered helpful in medicine. The anti-inflammatory and antioxidant capacity of phlorotannin extracts was evaluated by Barbosa et al 128 using RAW 264.7 macrophages stimulated with bacterial lipopolysaccharide and lipoxygenase inhibition as models of inflammation and NO radical scavenging activity. Phlorotannin extracts showed intense antioxidant activity and modulated the inflammatory processes in both models of inflammation, indicating the potentials of this class of compound as anti-inflammatory agents.
ACE-I Inhibitory Activity of Phlorotannins
The zinc metal protease, angiotensin-I converting enzyme (ACE-I), is a critical player in blood pressure control via its action on angiotensin I, which it converts to angiotensin II (a potent vasoconstrictor), as well as its role in degrading bradykinin (a vasodilator). Persistent high blood pressure is a significant contributor to chronic diseases such as stroke, cardiovascular diseases, diabetes, and kidney problems, 132 making it an effective target in blood pressure management. In their work, Paiva et al 132 reported a strong ACE-I inhibitory effect of F spiralis extract and fraction in vitro, attributing this effect to the rich phlorotannin content of the extract and fraction. Ko et al 133 also reported ACE-I inhibition by the phlorotannin, octaphloretol A from I sinicola in vitro and in silico. This compound also inhibited nitric oxide production in human vascular endothelial cells after being incubated for 24 h with the cells. In another study carried out by the same researchers, 6,6′-bieckol, another phlorotannin isolated from E cava, also enhanced nitric oxide production in human endothelial cells by phosphorylating endothelial nitric oxide synthase. This compound also inhibited ACE-I activity in vitro and in silico, indicating significant potential in blood pressure regulation and cardiovascular disease management. 134
Cosmeceutical Potential of Phlorotannins
Cosmeceutical formulations are generally prepared for UV protection, treating or concealing skin blemishes such as acne, pimples, and eczema, skin moisturizing, toning and tightening, antimicrobial activity, perfume, hair health and growth, and anti-aging with the goal of beauty enhancement and a generally healthy outlook.135,136 There is, recently, a growing interest in the use of natural products for cosmeceutical purposes due to the many drawbacks associated with synthetic agents such as skin bleaching, burning sensations, dryness, and photo-sensitivity.137–139 Several plant constituents are employed in cosmeceutical formulations due to their antioxidant, antimicrobial, and photo-protective properties. Polyphenols are particularly popular for this purpose due to their wide range of biological activities, including anti-inflammatory, antioxidant, enzyme inhibitory, antimicrobial, and even anticancer activities. Recently, Ohno et al 140 reported that two phlorotannins, phlorofucofuroeckol-A and fucofuroeckol-A from E bicyclis suppressed melanin production without inhibiting tyrosinase activity in mouse B16 melanoma cells. Other cosmeceutical potentials of phlorotannins are as follows:
Anti-acne Potential of Phlorotannins
Acne vulgaris is a common chronic inflammatory skin disease that affects the pilosebaceous follicle. There are four main factors influencing acne formation: follicular epithelial cells’ hyper-proliferation, hormonal stimulation of the sebaceous gland to produce excess sebum, Propionibacterium acnes, and inflammation.138,141 Medications for acne treatment aim to decrease bacterial load and inflammatory processes leading to a decrease in retention hyperkeratosis and sebum production while also reducing scar formation. While topical agents decrease keratinocyte proliferation by decreasing microcomedones formation, antibiotics target the disease-causing bacteria. 142 The inflammatory response in the skin involves producing pro-inflammatory cytokines and chemokines with keratinocytes, Langerhans, and mast cells. 143 Anti-inflammatory agents are vital in acne treatment because the acne-causing bacterium (P acnes) activates various immune pathways, inducing inflammatory mediators in keratinocytes. P acnes activates monocytes via the Toll-like receptor (TLR2 or TLR4), inducing the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), prostaglandins, leukotrienes, and chemokines (IL-8). P acne can also activate the NLRP3 (nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing)-inflammasome pathway in antigen-presenting cells and myeloid cells via IL-1 β.144,145 Cho et al 146 examined the effect of eckol (a phlorotannin) on the TNF-α/interferon-γ (IFN-γ)-induced inflammatory response in HaCaT cells. In their study, TNF-α/IFN-γ-induced pro-inflammatory cytokines (IL-1β, IL-4, IL-5, IL-6, TNF-α, and IFN-γ) and chemokines of different classes at both gene and protein levels, all of which were suppressed in eckol-treated cells. Antioxidant strategies are also employed in acne treatment due to oxidative stress conditions in acne development. This is because sebum produced by damaged follicular walls of sebaceous glands contains reactive radicals such as nitrous oxide, hydroxyl, and superoxide radicals responsible for the occurrence of irritations in acne infection. 147 Phlorotannins are recognized for their high antioxidant activities suggesting another mechanism for their cosmeceutical relevance.14,48,148
Photo-protective and Antiaging Properties of Phlorotannins
Another aspect of phlorotannins application in cosmetics is protection from the damaging effects of UV radiation from the sun (photo-protection). Although necessary for the synthesis of vitamin D in humans, exposure to UV radiation can produce unwanted effects such as redness of the skin (erythema), sunburn and pigmentation, skin aging, carcinomas, and loss of skin elasticity (dermal elastosis). UV radiation promotes oxidative damage characterized by increased lipid peroxidation and degenerative processes characterized by skin wrinkling and laxity.149,150 The unwanted effects necessitated the development of sunscreens to reduce the damaging effects of UV radiation on the skin. Schneider et al 151 evaluated the photo-protection capabilities of extracts from two marine photosynthetic organisms by measuring their various effective solar absorption radiation (ESAR in %), that is, the extracts’ potentials to absorb different UV wavelengths with correspondingly different biological effects, and the extracts’ photo-protection index (EPI). Extracts were applied on the rough side of polymethylmethacrylate (PMMA) plates and exposed to radiation from a solar simulator equipped with a mercury xenon lamp after incubation in a dark room for 15 min. Both extracts showed high %ESAR and EPI, indicating potent inhibition of photoaging and immunosuppression, respectively, and this result was attributed to their high phlorotannin content, as indicated from the spectral analysis of the extracts. Wang et al 152 also reported the protective effect of diphlorethohydroxycarmalol (DPHC) and other phlorotannins against UVB-induced photodamage of human dermal fibroblast cells in vitro and zebrafish in vivo. From their results, DPHC displayed strong photo-protection in vitro and in vivo, decreasing reactive oxygen species (ROS) levels and lipid peroxidation in UV-B irradiated cells. DPHC also inhibited UVB-induced collagenase and elastase activity in human dermal fibroblast cells indicating great potential in maintaining skin elasticity. Gager et al 153 evaluated the cosmeceutical potentials of phlorotannins from seaweeds harvested in Brittany (France). Photoprotective and anti-aging (elastase inhibitory) effects were observed to be high, with the phlorotannins extracts showing up to 80% elastase inhibition. Phlorotannins were also shown to promote wound healing by suppressing oxidative stress and inflammatory signaling in experimentally induced radiation dermatitis in rats. 117 Also, phlorotannins from Halidrys siliquosa showed photo-protective and antibacterial activities in vitro, 154 highlighting their potential benefits in cosmeceutical formulations.
Bioavailability of Phlorotannins
The word “bioavailability” refers to the rate and extent to which bioactive compounds navigate several metabolic barriers to reach their targeted site for action (receptors) and then to elicit concomitant biological responses. 155 Specific phlorotannin compounds are bioavailable if they reach their target receptor through the blood circulation in their bioactive form, without any form of degradation or metabolism. Hence, estimation of phlorotannin bioavailability can be made by measuring the amount and activities of the compound in the serum, the population of the undegraded phlorotannin compound in the urine, and possibly its specific metabolite after interaction with its specific receptor. 156 In this context, the bioavailability of phlorotannins is determinant on their biological activity, and numerous studies have been conducted to understand better the changes which different phlorotannin compounds undergo during their passage through the gastrointestinal tract (GIT) into the blood for circulation to their specific organs and final excretion in urine.156,157
In a preliminary bioavailability study conducted by Bangoura et al,158,159 a noticeable increase in the concentration of phlorotannin was reported in the flesh of abalones after been fed with either E stolonifera or E cava.158,159 A more recent study on the bioavailability and gastrointestinal modification of phlorotannins of Ascophyllum nodosum reported that high molecular weight phlorotannin derived from A nodosum was poorly absorbed in the small intestine leading to the generation of sulfate and glucuronide conjugates for phase II detoxification. 160 The unabsorbed conjugated metabolites traveled down the colon, where they subsequently underwent fermentation by the gut's microbiota leading to the formation of metabolites of lower molecular weight and some other phlorotannins with very little polymerization (eg, the dimers of phloroglucinol, 7-hydroxyalkyl, and hydroxytrifuhalol A) were detected in plasma and subsequently excreted through urine. 160 Inferably, high-molecular-weight phlorotannins are not bioavailable and are further metabolized in the large intestines by the gut microbiota into smaller molecular weight phlorotannins and other metabolites. Evidence has shown that these lower-molecular-weight phlorotannins are reabsorbed back into the blood, where they can be transported to organ receptors and excreted through the urine. 160
The prebiotic effects of extracts of E radiata, particularly those enriched with low-molecular-weight polysaccharides and phlorotannins, were evaluated using in vitro anaerobic 24 h-fermentation with fecal inoculum from humans. 161 It was observed that the fermentation of phlorotannin-rich fractions in the colon led to the production of a considerably low amount of SCFA metabolites in contrast to polysaccharides fermentation. Additionally, Fecalibacterium prausnitzii, Escherichia coli, Clostridium coccoides, and Bacteroidetes populations utilized phlorotannins as a substrate for their growth, although it was found to inhibit the growth of Lactobacillus bacteria 24 h post-fermentation. This could explain the antimicrobial activity of phlorotannins. 161
The first human clinical trials study on the bioavailability and bioactivity of seaweed phlorotannins on inflammation and oxidative stress biomarkers in overweight and obese individuals reported a 98% retention rate of capsulated phlorotannins. However, there was no significant decrease in the analyzed biomarkers when compared to the placebo. Moreover, the study reported that the bioavailable low-molecular-weight phlorotannins considerably prevented DNA damage, especially in the obese population. Finally, the study reported that conjugated phlorotannin metabolites such as phloroglucinol sulfate, hydroxytrifurahol A-sulfate, dioxinodehydroeckol, fucophloroethol-glucuronide, diphlorethol sulfates, and dioxinodehydroeckol glucuronide were found in the urine of participants in the experimental group of the trial. 162
The application of biotechnological treatments using enzymes (eg, fungal and bacterial glycosidases) and bacteria (eg, probiotics, which be able to modulate gut microbiota) could be an exciting strategy to induce changes in the phlorotannins’ structure, which may considerably enhance their uptake into the blood, hence their bioavailability. 163
Safety and Biotoxicity of Phlorotannins
The overall efficacy and relevance of phlorotannins’ vast bioactivity rely on the safety-to-toxicity profile ratio. Any substance is relatively safe and nontoxic when the adverse effects on physiological and biological processes are reduced to the barest minimum. There are sparsely available studies on the relative safety profile of phlorotannins in humans.162,164–166 However, a few studies have examined the safety and biotoxicity of some phlorotannins in some lower animals, while other in vitro toxicity studies were conducted in human cell lines. 167 Moreover, only about four studies have been conducted as clinical trials in humans.162,164–166
Most of the studies reported the majority of the bioactive phlorotannins to be nontoxic in animal models, human cell lines such as HeLa,168,169 HaCaT,170,171 fibroblast, 87 adenocarcinoma, 172 and melanoma cells.173,174 Dieckol has been studied extensively and has been reported to be completely nontoxic in the majority of studies. Conversely, in a study conducted by Ha et al, 175 dieckol extracted from E cava showed some level of toxicity when administered at a high dose of 100 µg/mL. At that concentration, dieckol, rather than ameliorating PM10-induced cytotoxicity, a reduction in cell viability alongside an increased level of prostaglandin (PGE2) was observed. The results from this recent finding showed an immense contrast to a previous study conducted by Ko et al 176 According to this, a concentration of 100 µg/mL of dieckol from E cava did not cause any form of toxicity, and instead, the viability of the cells was increased. However, another study reported that dieckol, still from E cava, exerted some low toxicity at 500 µM. 101 Similar studies on dieckol in lower animals such as in zebrafish,123,177 mice,75,123 and beagle dogs 178 resulted in some level of very mild toxicity in the animals. In zebrafish, at a concentration of 4 µM, a noticeable reduction in the level of body lipids was reported. 177 In mice, a significant reduction in body weight was reported in animals orally administered a 60 mg/kg dose of dieckol. 75 Likewise, beagle dogs administered 750 mg/kg bodyweight suffered from diarrhea and defecated soft stools. 178
Reports on other phlorotannins, such as 6,6′-bieckol, 179 7-phloroeckol, 89 and diphloroethohydroxycarmalol (DPHC) 171 did not pose any form of toxicity on the cell line and in lower animals. Similarly, the crude extract rich in phlorotannins did not cause any loss of viability in lower invertebrates such as Artemia salina, Hydroides elegans, 180 Mytilus edulis 181 and Portunus tribuberculatus 182
The few clinical studies reported so far showed no side effects in human volunteers administered with capsules of a crude extract rich in phlorotannin at a concentration of up to 250 mg/capsule/day.162,164–166 However, a clinical trial with dieckol resulted in very mild adverse effects in 4 out of 20 human subjects. Some of the mild acute toxicity symptoms reported were mild fatigue, dizziness, nausea, and abdominal distension among human subjects, but did not lead to them discontinuing the clinical trials. 166
In conclusion, from the few toxicity and safety studies, phlorotannins, especially dieckol, are safe, and any possible adverse effects are extremely mild. There is a need for specific studies on other classes of phlorotannins, such as phlorofucofuroeckol A, 7-phloroeckol, fucodiphloroethol, bifuhalol, and others.
Future Prospects for Phlorotannins
The availability of functional ingredients such as phlorotannins from natural sources such as algae is entirely sustainable and cost-effective. These compounds have found practical application in the pharmaceutical, nutraceutical, and cosmetic industries. However, their metabolism across the GIT has remained a considerable challenge. We have been able to review the chemical compositions and properties of phlorotannins, analytical techniques for their extraction, characterization, and quantification, their biological roles, bioavailability, and other prospects of phlorotannins with their huge potential in pharmaceuticals, nutraceuticals, and cosmeceuticals (skin protective effects), as well as metal sequestration potential. Producing phlorotannin-containing products on an industrial scale and at a reasonable cost, and the lack of comprehensive knowledge of their complete chemical characterization requires the potential biological activities to be reported in documented in vitro assays. This lack is currently limiting their ability for development and commercialization. More advanced, sophisticated, and reliable analytical techniques (for instance, HRMS) are needed to identify complex mixtures of phlorotannins and resolve the profile of their metabolites. Additionally, extraction, storage and digestion, and preprocessing operations are determinants of phlorotannins’ contents, which also have a corresponding effect on their biological activities. The employment of these processes should ensure the stability of phlorotannins.
Many studies have investigated the bioactivities of phlorotannins, but only a few are available on their toxicity and bioavailability in humans. With more than 20 members of the phlorotannins class already discovered, only a few such as eckol, dieckol, and diphloroethohydroxycarmalol have been studied to a reasonable extent. There is a need for future studies on other members of the phlorotannins class, especially on their safety and toxicity profile.
The advent of computer-aided drug discovery approaches (CADD) and in silico molecular docking and dynamic studies has been a valuable tool in phytochemistry. Since most phlorotannins’ 2D and 3D structures have been elucidated through various spectra analytical techniques, these structures can be targeted to disease receptors and potent druggable targets to investigate their efficacies for managing and treating disease conditions prior to in silico and in vivo studies. Hence, with the CADD approach, members of the phlorotannin classes could be investigated for anti-coronavirus disease-2019 activities by targeting various severe acute respiratory syndrome coronavirus 2 proteins (the causative agent for 2020 pandemics) with phlorotannins.
Most of the studies showing phlorotannin bioactivities were performed on cell lines and lower invertebrates. Hence, the activities from those subjects cannot be translated into what will be found in humans. Therefore, there is a need for more studies on the bioavailability of phlorotannins in humans. Some of the questions that need to be addressed are first, are phlorotannins easily absorbed across the gastrointestinal barrier in humans; second, are they susceptible or substrate to P-glycoprotein; and finally, considering phlorotannins as therapy against neurological disorders such as Alzheimer's disease, studies should ascertain their ability to cross the blood–brain barrier. These studies can be quickly predicted by in silico approaches to open-source online tools such as SWISS ADME (http://www.swissadme.ch/) and others available.
To increase and improve the shelf-life of phlorotannins, it is advised that algal materials are freeze-dried and stored at freezing temperature in a vacuum-packed package immediately after harvest. This will help in reducing the moisture content, thereby reducing spoilage and deterioration before extraction. The efficiency of phlorotannin extraction is affected by several factors such as extraction time, solvent type, pressure/temperature, the ratio of solvent to solid, and the dry seaweed particle size. Therefore, optimizing these factors will ensure that these factors are stable, have better content, and have more bioactive phlorotannin yield. Additionally, a more stable product could be obtained through encapsulation, which may offer better protection necessary for maintaining the bioactive molecules within the storage and utilization period. More study is required to create sustainable seaweed processing methods that allow for cleaner exploitation, greater resource efficiency, simpler industrial scale-up, and the creation of co-products instead of wastes. Furthermore, more studies are required to explore the molecular mechanism of their exhibited biological activities at the cellular and gene level.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship and/or publication of this article.
