Extraction and Nano-Sized Delivery Systems for Phlorotannins to Improve Its Bioavailability and Bioactivity

This review aims to provide an informative summary of studies on extraction and nanoencapsulation of phlorotannins to improve their bioavailability and bioactivity. The origin, structure, and different types of phlorotannins were briefly discussed, and the extraction/purification/characterization methods for phlorotannins were reviewed, with a focus on techniques to improve the bioactivities and bioavailability of phlorotannins via nano-sized delivery systems. Phlorotannins are promising natural polyphenol compounds that have displayed high bioactivities in several areas: anticancer, anti-inflammation, anti-HIV, antidiabetic, and antioxidant. This review aims to provide a useful reference for researchers working on developing better utilization strategies for phlorotannins as pharmaceuticals, therapeuticals, and functional food supplements.

Last by not least, phlorotannins are strong natural antioxidants [55][56][57][58][59][60]. Dieckol can suppress UV-B radiation induced photo-oxidative stress in human fibroblast cell line and reduce cell damage [55]; diphlorethohydroxycarmalol (DPHC) isolated from I. okamurae can scavenge UV-B radiation induced ROS in human fibroblast cells [57]; and dieckol can reduce cell damage induced by UV-B radiation both in vitro (HaCaT cells) [61] and in vivo (zebra fish) [59]. Phlorotannins can also potentially be used as functional ingredients in skin care products to offer protection against photo-induced skin damages.
Although phlorotannins have many attractive biofunctionalities, as members of the polyphenol family, phlorotannins also suffer from low bioavailability, which limits their utilization. Polyphenols' low bioavailability is mainly due to their low absorption in the human gastrointestinal (GI) tract following consumption and their extensive biotransformation within the gut, which may lead to their rapid clearance from the body (Figure 1 [62]). To improve the bioavailability of phlorotannins, nano-sized delivery systems have been investigated [26,61,[63][64][65][66][67][68][69][70]. Figure 2 showed a schematic representation of examples of these systems [71]. These nano-enabled approaches could protect phlorotannins against transformation in the gut; they can also improve delivery/absorption of phlorotannins [61]. Nano-phlorotannin systems were also shown to enhance the functionality of the phlorotannins. For example, gold nanoparticle-phlorotannin and silver nanoparticle-phlorotannin complexes have been investigated by serval researchers [26,61,[63][64][65][66][67][68][69][70]; they were shown by DPPH assays to have elevated antioxidant activity. Nano-enabled improvement on bioavailability and bioactivity may offer a more effective way for the utilization of phlorotannins in pharmaceutical, food, and biochemical industries.

Extraction and Purification of Phlorotannins
The operation of phlorotannin extraction aims to achieve two major goals: one is to extract as high a quantity of phlorotannins, primarily from brown algae, as possible; another is to maintain high bio-integrity and bioactivities of the phlorotannins [7][8][9][10][11][12]. These two goals could be in conflict: phlorotannins are unstable polyhydroxylated molecules, and they can easily be oxidized [17]. Considering the different types of phlorotannins and their susceptibility to various physical/chemical processing, development of different isolation/extraction methods suitable for different types with specific biofunctionality [17] remains a challenge [18,19]. A quick summary of the pros and cons of common isolation/extraction methods reported by different research groups follows.
HPLE methods: HPLE is generally considered environmentally friendly to an extent, if water is used instead of organic solvents [2,27,29,31,32]. Low cost is another advantage of HPLE [28,30]. HPLE can also be scaled up to a certain extent to go beyond lab scale, but its yield as of today is still not high [29]. Another restriction of HPLE is that it requires the solvent to be kept in liquid state during the entire extraction process, which can be an operational challenge [29]. In addition, macroalgae might contain heavy metal [72], which could be extracted together with phlorotannins by HPLE [72] and become enriched.
HPLE-RP methods: to reduce the risk of heavy metal being enriched along with phlorotannins, resin purification (RP) methods were introduced [33,34]. With RP, heavy metal content in the phlorotannins extracted from macroalgae can be dramatically reduced [33] Kim et al. [33] compared four types of resins: HP-20, SP-580, XAD-7HP, and XAD-2. All four types of resins displayed effective purification of phlorotannins [33]. Among them, HP-20 resins displayed the highest absorption and desorption capacities, and this led to the best purification performance.
NEDES extraction: Another new development in phlorotannin extraction was the use of deep eutectic solvents (DESs). DES is a new class of ionic liquid (IL), typically formed by mixing choline chloride with hydrogen bond donors [81]. Environmentally friendly natural DESs (NADESs) were first explored for extracting active compounds from plant raw material [82,83]. Oblichinskaya et al. reported the first effort of using NADES to extract phrolotannins from brown algae F. vesiculosus L. and A. nodosum (L.) Le Jolis [84]. Ten NADES with different compositions were studied for their efficiency of extracting phlorotannins. They found that aqueous solutions of NADES based on choline chloride and lactic acid were the most efficient, comparable to those of traditional extractants such as Me 2 CO 3 and EtOH and almost 10 times more efficient than pure NADES. As NADESs are clean green solvents, they could become an alternative to organic solvents to reduce the environmental footprint of phlorotannin extraction.
Following extraction, purification and characterization are the next steps. Molecular size-based separation is often used to purify different types of phlorotannins: these methods usually involve dialysis or ultrafiltration (UF) or a combination of the two [85]. Dialysis is an approach to isolate phlorotannins by different molecular weight [79,86,87]. The proper selection of membrane pore size is key to separating high molecular weight (MW) phlorotannins from low MW ones [79,86,87]. As for the UF system, with a fixed flow rate, UF can have a better performance [85]. The capacity of the UF system reported in the literature is still limited to lab-scale operations, ranging from 100 mL to 10 L [85]. One advantage of the UF system is its wide range of MW cutoff (MWCO), but industrial-ready UF for phlorotannins remains to be developed for large-scale applications [85].
After purification, NMR spectroscopy, mass spectrometry, and advanced chromatography are three common tools to evaluate the phlorotannin product to characterize the level of polymerization and the molecule weight distribution [88]. Phlorotannins could be indirectly quantified via the Folin-Ciocalteu (F-C) method, which measured the antioxidant activities in a sample. Although the F-C method is not selective for phlorotannins, it could yield quick readings with a simple operation, short turnaround time, and high throughput [20,79,[89][90][91]; hence, it has been used for rapid determination of phlorotannin content in brown algae extracts [92]. In addition, computational chemistry was another useful tool [93][94][95]. Computational chemistry method can provide a cost-efficient supplementary tool to NMR spectroscopy [93][94][95][96]. Density functional theory (DFT) quantum chemical calculation was introduced to determine electronic structure of phlorotannins [96]. Natural bond orbital (NBO) was introduced to determine the donor acceptor stabilization energy caused by intramolecular and intermolecular hydrogen bonds in phlorotannins [96].

Bioactivities of Phlorotannins
As a group of tannins, phlorotannins from brown algae were shown to be effective in regulating several biochemical processes linked to the disruption of homeostasis in major chronic diseases [18,39]. They have been revealed to have a range of diverse biological functions and activities, such as antibacterial [7], anti-inflammatory [8], antioxidant [9], antidiabetic [10], anti-HIV [11], and anticancer [12,97,98] activities. They can form complexes with pro-oxidant proteins, chelate metal ions, or directly trap reactive oxygen species (ROS) to modulate cellular responses to stresses and/or injuries [99]. The most studied bioactivity of phlorotannins is their ability to scavenge radicals due to donation of their hydrogen atoms or electrons [100][101][102][103][104][105], which can be evaluated via 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical-scavenging assay [88,106,107], ferric reducing/antioxidant power (FRAP) assay [108], dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay [109], trolox equivalent antioxidant capacity (TEAC) assay [110], and electron spin resonance spectroscopy [107]. Table 2 listed some of reported anti-inflammation effects of phlorotannins from literature. This list is by no means comprehensive, but it showcases the great potential of phlorotannins as anti-inflammation agents. In addition, phlorotannins isolated from brown algae have also been widely reported to have anticancer bioactivities. For example, eckol from brown algae was shown to suppress tumor growth in vivo in S180 xenograft-bearing mice [46].

Nano-Sized Delivery Systems for Phlorotannins to Enhance Bioavailability and Bioactivities
Although phlorotannins have all the highly desirable bioactivities and functionalities summarized in Section 3, their utilization as pharmaceuticals, therapeuticals, and food supplements is often limited due to their low bioavailability. Phlorotannins are unstable in the acidic condition of the gut and the alkaline condition of the small intestine; if not protected, they could easily be transformed and/or metabolized [138]. Phlorotannins are water soluble, but their absorption in the human gastrointestinal (GI) tract following consumption could be low, as evidence suggested that phlorotannins were mainly absorbed after metabolized in the large intestine [139], not in the stomach and the small intestine. A number of strategies have been used to increase the chemical stability of phlorotannins and to improve their transportation/absorption in the GI tract via nano-sized delivery systems that involved the encapsulation of phlorotannins in nanovectors, which offer protection against chemical/enzymatic transformation. These nano-systems could improve the distribution and overall bioavailability and bioactivity of the phlorotannins [65]. Such systems differ for the internal structures (e.g., core-shell-like vs. embedding matrixes) and the physical/chemical states of the encapsulated active ingredients (i.e., the phlorotannins), and their encapsulation efficiencies (EE, defined as the experimental loading/theoretical loading × 100%) were different. Effective drug delivery systems usually require an EE to be higher than 60%, which could be used as a reference to evaluate various phlorotannin nano-delivery systems.
Nano-sized delivery systems utilize nanoparticles or nanostructures with diameters ranging from 1-1000 nm to encapsulate/carry phlorotannins as the "payload". These nanoparticles, due to their size, exhibit properties and phenomena attributable to their dimension [140]. Nano-sized encapsulation systems can be made via several different approaches based on chemical, physical, and physiochemical principles. Chemical encapsulation (e.g., interfacial and in situ polymerization methods) usually requires the polymerization of monomers that cross-link with the payload [141]; physical encapsulation, on the other hand, usually uses already formed polymers (and natural polymers) to form a matrix in which the payload can be entrapped via various methods including air suspension, pan coating, spray drying, spray congealing, and micro-orifice systems [65]. Physiochemical methods (e.g., coacervation, phase separation, complex emulsion, meltable dispersion, and nanoprecipitation) aim to form stable nano-sized payload-carrying systems through particle size reduction processes [142]. Nanoencapsulation techniques, especially physical and physiochemical methods, have been [140] widely investigated for improving bioavailability and bioactivities of polyphenols by protecting the polyphenols against digestive transformation/degradation, reducing their toxicity and improving their water solubility [65]. Various nanocarrier systems, including cyclodextrins, nanospheres, nanocapsules, solid lipid nanoparticles, liposomes, and micelles, have been explored for polyphenols in general. Here, we will review a few specific cases in which phlorotannins were the payloads.
Shibata et al. [71] studied phlorotannins extracted from brown algae E. bicyclis, E. cava, and E. kurome; they showed that, with a liposome carrying system, the nanoencapsulated phlorotannins showed potent inhibition of phospholipid peroxidation at 1 µM, had significant radical scavenging activities against the superoxide anion (50% effective concentration values: 6.5-8.4 µM) and DPPH (50% effective concentration values: 12-26 µM), and were more effective compared to ascorbic acid and α-tocopherol. Furthermore, a nanocarrier system of phlorotannins and soybean protein prepared by co-extrusion was shown to have a pronounced DPPH radical scavenging activity. The authors concluded that the nanoencapsulated phlorotannins could be a good functional food or supplement with anti-inflammatory activity.
Physical encapsulation was also applied to phrolotannins. In a recent effort, Cuong [69] reported nano phlorotannin powder made by spray-drying of phlorotannins isolated from S. serratum grown in Vietnam. The optimum spray drying condition was established as follows: a carrier-to-solution ratio of 10%, compressed air pressure of 0.8 bar, liquid feed speed of 10 mL min −1 , and inlet temperature of 110 • C. Under these conditions, the antioxidant activity of phlorotannin powder possessed total antioxidant activity at 4.347 ± 0.018 g ascorbic acid equivalent 100 g −1 DP, reducing power activity at 9.390 ± 0.024 g FeSO 4 equivalent 100 g −1 DP and DPPH free radical scavenging activity at 70.02 ± 0.26%. The phlorotannin content and antioxidant activities of the nano-sized powder particles were affected by the spray drying condition (p < 0.05). These particles were nano-sized with morphology of irregular shapes. These nano phlorotannin powders displayed antioxidant activities for potential application in pharmaceuticals and nutritional supplements. In another effort [81], electrospun polycaprolactone (PCL)/phlorotannin micro/nanofibres containing different phlorotannin (from E. cava) concentrations (1, 3, and 5 wt%) were fabricated. Due to their hydrophilicity and water absorption ability, phlorotannin-containing fibrous mats exhibited outstanding wettability compared with pure PCL fibrous mats. The biocompatibility of the mats was examined in vitro using osteoblast-like cells (MG63). The phlorotannins were shown to promote alkaline phosphatase (ALP) activity and calcium deposition, which could induce bone regeneration. Cell viability (at 5 wt% phlorotannin), total protein content, ALP activity, and calcium deposition were all higher with PCL/phlorotannin mats than with pure PCL mats. These results suggest that electrospun nano PCL/phlorotannin fiber is a promising bioactive material for enhancing bone tissue growth. Lin et al. also demonstrated phlorotannin-encapsulating nanofibers with blended sodium alginate (SA) and poly(ethyleneoxide) (PEO) as the matrix via electrospinning, The optimum blending ratio to produce quality nanofibers was found to be 50:50:10 (SA/PEO/Ph); the nanofibers were shown to have an average diameter of 331 nm. The fibers were then used as an antibacterial reagent against S. enteritidis on chicken at 4 and 25 • C with great performance; the cell count drastically decreased from 6.20 to 3.28 Log CFU/g at 4 • C and decreased from 8.80 to 2.53 Log CFU/g at 25 • C. Their application significantly increased the shelf-life of chicken, which suggested that these nanofibers could be good food packaging materials due to the activities of the phlorotannins. Cui et al. [67] used a natural polymer (Momordica charantia polysaccharide (MCP)) as a nanofiber matrix to encapsulate phlorotannins via cold plasma treatment. The successfulness of PT loading into MCP nanofibers was confirmed using SEM, AFM, TGA, and DSC, as shown in Figure 3. After cold plasma treatment, the release efficiency of PT from the nanofibers was enhanced by 23.5% (4 • C) and 25% (25 • C). In addition, both antioxidant and antibacterial activities of the MCP-phlorotannin nanofibers were markedly improved. These nanofibers could be a novel functional food packaging material. In this paper [67], the authors reported that both MCP and phlorotannin exhibit antidiabetic and antioxidant properties. They demonstrated that an MCP nanofiber-phlorotannin complex can enhance antioxidant effects. Thus, this MCP-phlorotannin complex was also used to make novel and enhanced food packaging materials.
Solid dispersion technique is another method that is widely used for generating nanocapsulation systems for wrapping a core material (the payload) with functionality (e.g., phlorotannin) inside a coating material. This technology can not only mask or retain flavor and increase solubility of the payload, but also protects it from degradation by environmental factors, and controls its release at the target site [82]. In a recent report, Qi et al. [66] investigated the potential of using polyvinylpyrrolidone (PVP) as the coating to produce nanocapsulated PVP-phlorotannin complexes (Figure 4) with improved bioactivities and bioavailability. Different loading ratios of PVP vs. phlorotannin were investigated, and an optimum ratio of 8:1 (w/w) was established. As shown in Figure 5, the results indicated that the PVP-phlorotannin nanoparticles showed a slow and sustained kinetic release of phlorotannin in simulated gastrointestinal fluids; they were non-toxic to HaCaT keratinocytes (i.e., skin cells), and they could reduce the generation of endogenous reactive oxygen species (ROS). A PVP-based solid dispersion system would be a good choice for making better nano-sized delivery systems for phlorotannins in medicine, food, and cosmetics [66,83].   An interesting development with phlorotannin nano carriers was the synthesis of metallic nanoparticles (typically Au and Ag), with phlorotannins serving as the reducing agent. In these processes, the metallic nanoparticles formed have phlorotannin coating, hence can serve as delivery vehicles for phlorotannins (PhTs). Kim et al. [68] demonstrated that Au nanoparticles were formed within 1 min while mixing phlorotannins extract from E. cava with chloroauric acid at 80 • C, and the Au-PhT nanoparticles were 30 ± 0.25 nm and displayed promising antimicrobial activities. Machado et al [70] compared phlorotannins extracts from two macroalgae, C. baccata (CB) and C. tamariscifolia (CT), for their abilities to form gold nanoparticles; the results showed that CT possess three times more reducing power, almost four times more phenolic content, and four times more DPPH scavenging activity than CB, and the gold nanoparticles produced presented a non-cytotoxic profile in lower concentrations in mouse cell line L929 and human cell line BJ5-ta, which were efficient in cell regeneration, although with some differences between both species. The CT-gold nanoparticle complex had significantly better antioxidant bioactivity than the CB-gold nanoparticle complexes. Shim et al. [69] reported a green synthesis process for silver nanoparticles. The phlorotannin-silver nanoparticle complex has an average size of around 40 nm with spherical structure. These phlorotannin-silver nanoparticle complexes displayed antibacterial and antioxidant bioactivities. In particular, they exhibited a strong apoptotic anticancer activity against human cervical cancer cells that was not observed for phlorotannin, which suggested that the synergistic effects of phlorotannins and silver nanoparticles could further enhance their anticancer activities ( Figure 6). Abedel-Raouf et al. [74] reported that phlorotannin isolated from G. elongate can be utilized to synthesize phlorotannin-gold nanoparticle complexes. Several shapes of phlorotannin-gold nanoparticles were formed, as shown by TEM. Triangular, truncated triangular, hexagonal, and rod shapes were detected. The sizes of these phlorotannin-gold nanoparticle complexes were around 3-77 nm. Antibacterial bioactivity was tested against E. coli, K. pneumoniae, and MRSA. Phlorotannin-gold nanoparticle complexes of 13 nm demonstrated the best antibacterial performance. It could be concluded that phlorotannininduced Au and/or Ag nanoparticles could potentially become effective antibacterial, antiviral, and antioxidant reagents [75].

Conclusions
In this review, we summarized extraction techniques and bioactivities of phlorotannins and the strategies that have been explored via utilization of nano-sized delivery systems to improve their bioavailability and bioactivities. With nano-carriers protecting phlorotannins against GI tract biotransformation and degradation, phlorotannins can be released in a controlled manner to enhance their therapeutic functionalities, which included anticancer, anti-inflammatory, anti-HIV, antidiabetic, antioxidant, antibacterial activities, etc. This review serves as a roadmap for the development of more effective phlorotannin utilization strategies to fully take advantage of the tremendous potential of these algae-derived natural compounds as drugs and nutritional supplements for the pharmaceutical, food, and cosmetics industries.

Conflicts of Interest:
The authors declare no conflict of interest.