Encyclopedia
Please note this is a comparison between Version 7 by Ron Wang and Version 6 by Ron Wang.
Macroalgae, or seaweeds, are a rich source of components which may exert beneficial effects on the mammalian gut microbiota through the enhancement of bacterial diversity and abundance. An imbalance of gut bacteria has been linked to the development of disorders such as inflammatory bowel disease, immunodeficiency, hypertension, type-2-diabetes, obesity, and cancer. This review outlines current knowledge from in vitro and in vivo studies concerning the potential therapeutic application of seaweed-derived polysaccharides, polyphenols and peptides to modulate the gut microbiota through diet. Polysaccharides such as fucoidan, laminarin, alginate, ulvan and porphyran are unique to seaweeds. Several studies have shown their potential to act as prebiotics and to positively modulate the gut microbiota. Prebiotics enhance bacterial populations and often their production of short chain fatty acids, which are the energy source for gastrointestinal epithelial cells, provide protection against pathogens, influence immunomodulation, and induce apoptosis of colon cancer cells. The oral bioaccessibility and bioavailability of seaweed components is also discussed, including the advantages and limitations of static and dynamic in vitro gastrointestinal models versus ex vivo and in vivo methods. Seaweed bioactives show potential for use in prevention and, in some instances, treatment of human disease.
- seaweed
- prebiotics
- gut microbiota
- polysaccharides
- polyphenols
- peptides
- colonic fermentation
- short chain fatty acids
- bioaccessibility
- simulated gastrointestinal and fermentation digestion models
1. Introduction
Seaweed-derived components with potential to impact positively on diseases of the body including hypertension [1], cancer [2], type-2-diabetes [3], obesity [4], oxidation [5], inflammation [6] and other disorders have been evaluated in a number of studies to date [7][8][9][10][11][12][13][14][15]. The pathogenesis of these disorders has been linked to the health of the gut microbiota [16]. The microorganisms that inhabit the human gastrointestinal tract—bacteria, archaea, fungi, protozoa, and viruses—are collectively termed the gut microbiota [17]. The gut microbiota is established during infancy [18]. There is a broad variance amongst individuals in microbiota composition because it is shaped by infant transitions such as the gestational period, delivery method, weaning age, breast-feeding duration, or use of formula milk [19]. The microbiota remains relatively stable throughout adulthood but is affected by factors such as enterotype, antibiotic use, diet, lifestyle, genetic traits, and body mass index [20]. Three enterotypes have been described in the human gut microbiome based on variations in levels of the bacterial genera Bacteroides, Prevotella, and Ruminococcus [21]. The gut microbiota is regarded as an endocrine organ that co-develops with the host throughout its life. It exerts an effect on immunity, metabolism, neuroendocrine responses, and synthesises vitamins, amino acids, and enzymes [22][23].
The gut microbiota also aids in the absorption of dietary minerals and produces important short-chain fatty acids (SCFA) such as butyrate, propionate, and acetate. These SCFA are the energy source for gastrointestinal epithelial cells, provide protection against pathogens, influence intestinal mucosal immunity and barrier integrity, and induce apoptosis of colon cancer cells [24][25]. SCFA also regulate liver mitochondrial function, insulin secretion, and induce the production of gut hormones γ-aminobutyric acid and serotonin by interacting with their receptors on enteroendocrine cells [26][27]. An increase in the gut bacterial population enhances the beneficial effects of the microbiota and increases SCFA production [20]. An imbalance or decreased diversity of beneficial versus harmful bacterial species in the gut microbiota is termed dysbiosis and is linked to several diseases [28][29][30][31][32]. Therefore, maintaining the health of the microbiota through diet or supplementary means is thought beneficial to overall health [30]. Seaweed components may exert a beneficial effect on gut health by acting as prebiotics [33][34]. The potential bioactivity of seaweed components has been demonstrated previously in in vitro studies [35][36], however the impact of gastrointestinal enzymatic digestion and colonic bacterial fermentation in vivo must also be considered, since it may have an effect on the bioavailability of prebiotic and other actives [37][38][39]. As a pharmacological concept, bioavailability is a measure of drug absorption defined as the percentage of the drug that reaches blood circulation, measured by a dose-response curve [40]. However, the evaluation of bioavailability in food-derived extracts differs, since characteristic dose-response curves are not exhibited [41]. In addition, the bioaccessibility of food-derived active compounds must be taken into account, i.e., the accessible portion of the active compound released from the food or extract matrix during digestion [42][43]. Although pharmacokinetic studies are required for the development of prebiotics destined for human and animal use, such studies are not within the scope of this review. The pharmacokinetics of seaweed-derived prebiotics in terms of absorption, distribution, metabolism, and elimination has previously been documented in animal studies after oral administration [44][45][46][47][48] and topical application [49], and recently reviewed by Corino et al. [50] and Shikov et al. [51].
2. Current Insights
Seaweed components that have the potential to exert beneficial effects on the gut by modulating the abundance and diversity of bacterial populations in the gut microbiota include polysaccharides, polyphenols, and peptides. Their structure, function, and studies regarding their potential impact on the gut are considered in this review. Despite the positive results reported from cited studies concerning in vitro and animal work, more research is required in human dietary intervention studies, with health-related end points, to determine prebiotic potential.
2.1. Polysaccharides
Polysaccharides, or carbohydrates, are repeating units of monosaccharides linked by glycosidic bonds found in all plants, fungi, and algae. They are considered primary metabolites with structural and energy storage functions [52]. The majority of seaweed polysaccharides are composed of water-soluble and -insoluble fibre [53][54]. The total fibre content of seaweed varies between species and has been reported to range from 35–62% in brown, to 10–57% in red and 29–67% in green (DW) [55][56][57][58][59]. The principal fibres in brown seaweeds are fucoidan, laminarin, and alginate; porphyran, carrageenan, hypnean and floridean starch in red; and ulvan, sulphated-rhamnans, -arabinogalactans and -mannans in green [60][61]. Humans do not produce the endogenous enzymes in the upper gastrointestinal tract required to degrade dietary fibre to monosaccharides. However, fibre is an excellent food substrate, or prebiotic, for human gut bacteria [62][63]. Prebiotics are food components that are indigestible in the small intestine but can be metabolised by microorganisms in the large intestine, modulating their composition and/or activity, thus conferring a beneficial physiological effect on the host [64]. Many species of gut bacteria produce endogenous carbohydrate-degrading enzymes, such as β-glucanase and β-glucosidase, capable of hydrolysing the glycosidic linkages of polysaccharides [65][66][67][68]. Several polysaccharides within seaweed that are indigestible in the upper gastrointestinal tract are thought to exert bioactive effects including glycaemic control [69] and the promotion of gut microbial- and immune-modulation by acting as prebiotics in in vitro and in vivo studies [70][71]. The bioactivity of polysaccharide fractions is influenced by a number of factors such as chemical structure, molecular weight (MW), solubility, extraction method, seaweed genus and seasonal variation [72][73]. The principal polysaccharides of brown, red, and green seaweeds are detailed below.
2.1.1. Fucoidans
Three polysaccharides—fucoidans, laminarin and alginate—occur within brown seaweeds, each of which have differing structures and functions [74]. Fucoidans comprise 5–20% (DW) of the entire seaweed thallus [75][76]. They are water-soluble sulphated-polysaccharides composed of repeating fucose and sulphate groups, and may also contain galactose, mannose, xylose, rhamnose, arabinose, glucose, acetyl groups, or glucuronic acid [77]. The molecular weight of fucoidans varies from 7 to 2300 kDa [11]. Fucoidans provide structure for the outer cell wall and a hydrophilic coating to prevent desiccation of the seaweed during low tide. They also play a role in adapting to osmotic stress caused by changes in salinity as their sulphate groups can bind to cations such as sodium, potassium, magnesium, and calcium [78][79]. Fucoidans have previously been shown in in vitro studies to have potential for use as anticancer [80], antiviral [81], antioxidant [77], and anti-inflammatory [82] agents; and in vivo as anticoagulants (human trial) [83], anticancer (human trial) [84], antitumour (mouse model) [85], antihyperglycaemic, and antihyperlipidaemic agents (mouse model) [86]. However, the oral bioavailability of fucoidan can be low due its highly polar nature and limited ability to pass through intestinal epithelial cells [68]. In recent years, the prebiotic status of fucoidan has been recognised in vitro [82][87] and in human [88] and animal [89][90][91][92] gastrointestinal studies.
2.1.2. Laminarin
The energy storage polysaccharide of brown seaweeds is laminarin, composed of β(1–3)-linked glucose units with β(1–6)-branches [93]. It occurs within the chloroplasts in micro-compartments called pyrenoids [94]. Laminarin is water-soluble, though increased branching of the molecule requires colder temperatures for solubility. It comprises 3–35% of brown seaweed dry mass and is most prevalent in Laminaria species [95]. It is a small polysaccharide with a molecular weight of approximately 5 kDa [96]. Laminarin has shown efficacy in in vitro studies carried out previously and has potential for use as an anticancer [97], antimetastatic [98], antioxidant [99] and immunostimulatory [100] agent [97][99][100]; and in vivo as an immunomodulatory agent [101] and prebiotic to modulate dysbiosis (animal models) [102][103][104][105].
2.1.3. Alginate
Alginate comprises up to 45% of brown seaweed dry mass [106], occurring in the cell walls as salts of alginic acid bound to sodium, calcium or magnesium ions [107]. It is a water-soluble linear polysaccharide composed of (1–4)-linked β-D-mannuronate and α-L-guluronate residues [108]. Molecular weight ranges from 20 to 350 kDa [109][110]. It is the most abundant polysaccharide in brown seaweed and imparts flexibility to the thallus to withstand the force of the ocean. Alginate is a phycocolloid that can bind up to 20 times its own mass with water, making it very useful for food and industrial applications [111]. The prebiotic effect of alginate on gut microbiota was demonstrated previously in vitro by Bai et al. [112] and Li et al. [113]; and in a human study by Mizuno et al. [114]. Bai et al. fermented seaweed-derived alginates in vitro and observed that the alginates were degraded by human-derived gut bacteria, producing a significant (p < 0.05) increase in SCFA compared to a starch control, and suggested that further investigations of the prebiotic effects of alginate are warranted. Li et al. also fermented seaweed-derived alginates with human faecal bacteria in vitro and found a significant (p < 0.05) increase in total SCFA in the alginate sample (78.6 ± 5.9 mM) compared to the control (62.5 ± 5.1 mM). The bacterial Richness index in the alginate ferment (15.83 ± 2.3) was also significantly greater (p < 0.05) than that of the control (12.67 ± 2.88). The authors propounded that alginate may be capable of sustaining the growth of human gut bacteria, and recommended further study to evaluate the potential impact that alginate food additives may exert on host health. The in vivo study by Mizuno et al. was an interventional study of 11 elderly patients who required enteral feeding. After 4 weeks of receiving the alginate formula (equivalent to 14.52 g fibre/day) there was a significant increase (p = 0.039) in Clostridium cluster XI bacteria compared with the baseline. However, there was no increase in Bifidobacterium, Lactobacillales, or Bacteroides. The patients’ stool form improved (p = 0.044) (Bristol Stool Scale), as did mean blood concentrations of total SCFA (p = 0.042), acetic acid (p = 0.042), propionic acid (p = 0.027), serum albumin (p = 0.039), total cholesterol (p = 0.002), and cholinesterase (p = 0.034). The alginate did not induce any significant changes in stool frequency, body weight, or arm circumference. The authors suggested that the alginate-containing liquid formula may potentially exert a beneficial prebiotic effect on intestinal function through increased production of SCFA. However, the limitations of the study were noted due to the small sample size and single-center study design. In order to validate the findings, the authors recommend a larger, multicenter study.
2.1.4. Carrageenans
Within red seaweeds, carrageenans and porphyran are the prevalent polysaccharides. The family of linear, sulphated polysaccharides, carrageenans, occur as a structural component of the extracellular matrix [118]. Of the 15 different carrageenan forms, iota (ι), kappa (κ) and lambda (λ) are the most widely used as phycocolloids in the food industry [119] and as a vegan alternative to beef gelatin in pharmaceutical capsules [120]. κ and ι-carrageenan are composed of alternating d-galactose and 3,6-anhydro-galactose units with varying numbers of sulphate groups, while λ-carrageenan lacks 3,6-anhydro-galactose and has alternating α-1,3 and β-1,4 inter-galactose bonds [121]. Average molecular weight ranges from 453 to 652 kDa [122]. All forms of carrageenan are soluble in water above their gel-melting temperatures (40–70° C). In cold water, only λ-carrageenan and the sodium salts of κ and ι-carrageenan are soluble [93]. ι-carrageenan was shown to reverse the symptoms of metabolic syndrome in a rat model by significantly decreasing systolic blood pressure, body mass (BM), abdominal and liver fat, and total cholesterol, while also beneficially modulating the gut microbiota [123]. As potential antitumour agents, κ/ι hybrid carrageenans have shown activity in vitro against colorectal cancer stem cell-enriched tumourspheres [2]. However, simulated gastrointestinal studies have found that κ-carrageenan can be both beneficial and harmful by increasing or decreasing markers of inflammation and the growth of beneficial gut bacteria and SCFA. This is dependent on the degree of polymerisation of the carrageenan [124].
2.1.5. Porphyran
Porphyran is a sulphated polysaccharide that occurs in red seaweed, within the genus Porphyra, and comprises approximately 11–21% of the seaweed dry mass [125]. It is composed of repeating units of galactose and 3,6-anhydrogalactose, with alternating units of galactose-6-sulphate and 6-O-methyl-galactose [126]. Average molecular weight ranges from 14 to 201 kDa [127][128]. Porphyran is soluble in hot water and has similar structural functions to carrageenan, though its higher viscosity limits its pharmaceutical applications [128][129]. Porphyran has shown potential antioxidant and anti-inflammatory effects in cell studies using RAW264.7 cell line [125] and was found to promote cell migration and proliferation in intestinal epithelial cells [127]. It also has antitumor activity against HeLa cells [130], HT-29 colon cancer cells and AGS gastric cancer cells [131]. As a prebiotic, porphyran was previously found to increase beneficial gut bacteria and SCFA production in vitro in simulated digestion studies [126][132][133] and in animal studies as whole red seaweed [134][135][136].
2.1.6. Ulvans
Green seaweeds are dominated by the ulvans, which account for 38–54% of the thallus dry mass [137]. Ulvans are water-soluble, gelling polysaccharides composed of repeating units of sulphated l-rhamnose, d-xylose, d-glucuronic acid and its epimer L-iduronic acid [138]. Molecular weights range widely from 1 to 2000 kDa depending upon the degree of sulphation [139]. Ulvans have demonstrated potential anticoagulant [140], antibacterial [141], antiviral [142], and immunoregulatory (porcine intestinal epithelial cells) [143] activities in vitro. They have also shown potential for the use as prebiotics in animal studies [144] and in vitro [132][145][146].
2.2. Gastrointestinal Digestion Studies with Seaweed Polysaccharides
A number of recent studies have used simulated in vitro gastrointestinal digestion or in vivo clinical trials to investigate the effect of polysaccharides on beneficial bacterial populations and their metabolites. Table 1 summarises the polysaccharide fraction used in each study and its impact on gut bacteria. Further characterisation and in vivo animal and human dietary intervention studies are required to confirm any potential therapeutic benefits.
Table 1. The impact of polysaccharides on gut bacteria.
| |||||
| SCFA production after 24 h (all | p < 0.05):
|
[10] | ||
| * (i) Low MW polysaccharide (LMW) (primarily laminarin) (ii) High MW polysaccharide acidic water extract (HMW) (primarily fucoidan and alginate) (iii) High MW polysaccharide water and ethanol precipitate (HMWW) (primarily fucoidan and alginate) |
E. radiata | (i) Enzymatic (cellulase) (ii) Acidic water (pH 4.5) (iii) Water and ethanol precipitation |
Simulated in vitro colonic digestion | 24 h post fermentation (all differences p < 0.05): (i) LMW increased Bifidobacteria from 5.51 ± 0.15 log10 cells/mL (in cellulose fermented control) to 6.55 ± 0.08 log10 cells/mL; Lactobacillus from 4.73 ± 0.13 (cellulose) to 5.28 ± 0.19 log10 cells/mL and Bacteroidetes from 5.09 ± 0.06 (cellulose) to 6.02 ± 0.09 log10 cells/mL. Negative results: no significant increase by LMW on populations of F. prausnitzii, Clostridium leptum, Ruminococcus bromii, E. coli or Enterococcus. (ii) HMW increased C. coccoides from 5.74 ± 0.75 (cellulose) to 7.07 ± 0.04 log10 cells/mL, E. coli from 6.09 ± 0.41 (cellulose) to 7.52 ± 0.07 log10 cells/mL and Enterococcus from 5.02 ± 0.31 (cellulose) to 6.63 ± 0.11 log10 cells/mL. Negative results: no significant increase by HMW in any other bacterial populations. (iii) HMWW increased E. coli from 6.09 ± 0.41 (cellulose) to 7.01 ± 0.17 log10 cells/mL and Enterococcus from 5.02 ± 0.31 (cellulose) to 5.80 ± 0.33 log10 cells/mL. HMWW also had a negative effect on several bacterial populations—Bifidobacteria reduced from 5.51 ± 0.15 (cellulose) to 3.21 ± 0.61 log10 cells/mL, Bacteroidetes from 5.09 ± 0.06 (cellulose) to 4.08 ± 0.12 log10 cells/mL, Lactobacillus 4.73 ± 0.13 log10 cells/mL (cellulose) to not detected (ND), C. coccoides from 5.74 ± 0.75 log10 cells/mL (cellulose) to ND, C. leptum from 6.23 ± 0.28 log10 cells/mL (cellulose) to ND and R. bromii from 6.20 ± 0.06 (cellulose) to 4.87 ± 0.29 log10 cells/mL. SCFA increases in seaweed ferments vs. cellulose control after 24 h (all p < 0.05):
|
[148] |
| ** (i) Polysaccharide fraction (PF) (primarily fucoidan and alginate) (ii) Whole seaweed (WS) |
E. radiata | (i) Enzymatic (Viscozyme) (ii) Whole dried E. radiata |
In vivo trial with healthy Sprague-Dawley rats (7 d, 5% PF or 5% WS added to feed) | After 7 days supplementation (all differences p < 0.05): Reduction in potentially pathogenic Enterococci in WS group (6.04 ± 0.09 log10 cells/mL) vs. control (5.59 ± 0.08 log10 cells/mL) Increase in butyrate-producing F. prausnitzii in PF group (5.32 ± 0.11 log10 cells/mL) vs. control (4.87 ± 0.11 log10 cells/mL) 2-fold increase in caecal digesta mass 1.36 ± 0.17 (PF) vs. 0.60 ± 0.06 g/100 g BM (control) Putrefactive microbial products reduced (all values µg/g caecal digesta):
|
[149] |
| * (i) conventional chemical extraction (CCE) (11.9% fucoidan) (ii) microwave-assisted extraction (MAE) (5.71% fucoidan) (iii) ultrasound-assisted extraction (UAE) (4.56% fucoidan) (iv) enzyme-assisted extraction (EAE) (3.89% fucoidan) |
A. nodosum | (i, ii, and iii) Ethanol followed by acidic water (0.01 M HCl) (iv) Cellulase, acetate buffer (pH 4.5) |
L. casei and L. delbrueckii ssp. bulgaricus broth cultures, 3.75% (v/v). A. nodosum extracts added at 0.1%, 0.3% and 0.5% (w/v) |
All differences p < 0.05 compared to non-supplemented control medium: Increase in L. delbrueckii ssp. bulgaricus by CCE, MAE, UAE and EAE at 0.1%, 0.3% and 0.5%. Increase (24.5%) in L. casei only by MAE at 0.5% inclusion. Negative results:
|
[87] |
| * Crude sulphated polysaccharide (716 kDa) (90% galactose, 9.07% sulphate) | C. pilulifera | Acidic extraction (0.0.1 M HCl) and ethanol precipitation | Simulated in vitro saliva, gastric, small intestinal and colonic digestion | After 24 h, all differences p < 0.05 compared to inulin control: Increase in Bacteroides, Parabacteroides, Megamonas and Veillonella. Increase in total SCFA (22.17 ± 0.82 mmol/L) vs. control (16.17 mmol/L ± 0.39). Negative results:
|
[150] |
| * (i) Polysaccharides (SJP) (138 kDa) (Fucose:galactose:glucuronic acid:mannose, molar ratio of 4.1:3.6:1.2: 1.0). (ii) Oligosaccharides (SJO) |
S. japonica | (i) Methanol, dichloromethane, water and ethanol (ii) Methanol, dichloromethane, water and ethanol, followed by 0.6 M HCl |
Simulated in vitro colonic digestion | After 24 h, all differences p < 0.05 compared to FOS control
|
[91] |
| ** Crude sulphated polysaccharide (SP) (28.807 kDa) (Galactose (59.7%), galacturonic acid (19.8%), xylose (7.1%) and sulphate (8.8%)) | G. pacificum | Ultrasound-assisted water extraction followed by ethanol, acetone and petroleum precipitation | In vivo trial with lincomycin hydrochloride induced diarrhoeal mice (9 days, 75 mg SP/kg BM) | After 9 d, seaweed polysaccharide group vs. non-supplemented normal recovery group (all differences p < 0.05): Increase in beneficial Bacteroides, Oscillospira and Bifidobacterium. Decrease in Parabacteroides, Sutterella and AF12. Reduction in inflammatory cytokines, TNF-α, IL-1β and IL-2. Improved (lower) diarrhoea status scores, water intake, and less weight loss. Increase in total SCFA, acetate and propionate. |
[151] |
| ** Fucoidan (300 kDa) (60% fucose, 14.3% sulphate) | C. okamuranus | Method not specified | In vivo trial with Traf3 ip2-mutant psoriasis mice (fucoidan diet group n = 14, normal diet group n = 9, 63 days, 1% fucoidan added to feed) | Fucoidan group vs. cellulose control group (all differences p < 0.05). After 56 days:
|
[89] |
| ** Laminarin and fucoidan (10% laminarin,8% fucoidan and 82% ash) | Laminaria hyperborea | Method not specified | In vivo trial (10 pregnant sows/treatment) (10 g/days seaweed extract from day 107 of gestation until weaning (day 26)) and ex vivo lipopolysaccharide (LPS) immunological challenge |
Compared with non-supplemented group, seaweed extract supplemented (SWE) sows had:
Piglets suckling SWE sows had:
|
[101] |
* = in vitro studies; ** = in vivo animal studies.
2.3. Polyphenols
Polyphenols are secondary metabolites that occur ubiquitously in terrestrial plants and algae. They are composed of repeating units of phenol—an aromatic phenyl group (a benzene ring, minus one hydrogen atom) bound to one or more hydroxyl groups [152]. Polyphenols are involved in numerous functions. They protect the seaweed thallus against biotic and abiotic stresses such as predation from herbivores, microbial infection, oxidation, and UV damage [153]. The total polyphenolic content of brown seaweed (dry mass) can be as high as 20%, while green and red seaweeds contain 1–5% [154][155][156]. The molecular weight of seaweed polyphenols ranges from approximately 26 Da to 650 kDa [157][158]. Seaweed polyphenols have been found to increase high-density lipoprotein cholesterol [159], post-prandial cognitive function [160], and exert anti-hypertensive [161] anti-hyperglycaemic [162] and peak blood glucose reducing effects (females only) [163] in human studies. However, only 5–10% of polyphenols are absorbed in the upper gastrointestinal tract due to their structural complexity. Large polyphenol compounds that reach the large intestine can be converted by microbial activity into beneficial bioactive metabolites [164][165], while also inhibiting pathogenic species [166]. Gut microbial enzymes catabolise polyphenols via hydrolysis, dehydroxylation, decarboxylation, reduction, demethylation, and isomerisation [167]. Studies with germfree animals have shown that bioactive phenolic metabolites—normally found after oral administration of polyphenols—are absent in their gut [168]. This shows the importance of the gut microbiota in polyphenol metabolism.
In terrestrial plants, the predominant polyphenols are flavonoids, stilbenes, lignans, and phenolic acids [169]. Seaweeds also produce flavonoids, coumarins, phenolic terpenoids, phenolic acids, luteolin, regiolone, and neoeriocitrin as well other polyphenols that are unique to algae [170][171][172]. These include bromophenols and phlorotannins [173].
2.3.1. Bromophenols
Bromophenols are molecules composed of one to five phenol groups, bound to one or more bromine [174]. Bromophenols are produced by seaweed as part of their chemical defence system to protect them from herbivores [175], oxidation, bacteria, and fungi [176][177]. Tri-bromophenols are the most common isomers found in seaweed, followed by di- and mono- bromophenols [178]. Bromophenols occur most abundantly in red and green seaweeds, and to a lesser extent in brown genera. A study of 49 red, green, and brown seaweeds by Whitfield et al. [179] reported bromophenol contents ranging from 8 to 2590 ng/g in red, 0.9 to 2393 ng/g in green, and 2 to 454 ng/g in brown. Seaweed-derived bromophenols have antioxidant [180], anti-inflammatory [181], antibacterial [182], anti-cancer [183], antithrombotic [184], and antidiabetic [185] activity.
2.3.2. Phlorotannins
Phlorotannins, found only in brown seaweeds, are composed of repeating units of phloroglucinol—a phenyl ring bound to three hydroxyl groups. Due to their ability to precipitate proteins, they are considered tannins [186]. Phlorotannins have structural functions within the seaweed cell wall [187], and protect against oxidation [188] and predation by herbivores [189]. Phlorotannins are sub-classified into four main groups depending upon the type of chemical bonds that link their phloroglucinol units [190]. Fuhalols and phlorethols have ether bonds; fucols have phenyl bonds; fucophlorethols have phenyl and ether bonds; while eckols have dibenzodioxin bonds [191]. The molecular weight of phlorotannins ranges broadly depending upon the number of phloroglucinol units they contain [192]. Molecular weights have been reported from 1.2 to 6 kDa [193], 30 to 100 kDa [194], and as high as 300 kDa [195]. Phlorotannin content differs broadly amongst species, and is influenced by seasonal variations and geographic location [196][197]. Content is generally expressed as gallic acid or phloroglucinol equivalents, or as a percentage of seaweed dry mass. A study of eight brown seaweeds over 14 months from the same location in France by Connan et al. [196] reported significant inter- and intra-species seasonal differences in phlorotannin content, with the highest values occurring in summer. Values ranged from 0.13% phlorotannin content (DW of total seaweed) in L. digitata, to 5.80% in A. nodosum and F. vesiculosus. Phlorotannins have been studied for their potential health effects. Reported bioactivities include antioxidant [198], antidiabetic [199], anticancer [200], antihypertensive [201], anti-inflammatory [202], antiviral [203], neuroprotective [204], antimicrobial [205], and prebiotic activities [10][206][207].
2.4. In Vitro and In Vivo Gastrointestinal Digestion Studies with Seaweed Polyphenols
The effect of polyphenols, particularly phlorotannins, on the gut, metabolic syndrome, and DNA damage has been reported in some in vitro and in vivo studies which are discussed below.
2.4.1. Prebiotic Function and Attenuation of Metabolic Syndrome by Phlorotannins
Charoensiddhi et al. [10] evaluated the prebiotic potential of phlorotannin enriched (PE) ethanolic extracts in vitro from E. radiata harvested in Australia. After 24 h fermentation, the phlorotannin extracts induced significant increases (all p < 0.05) in some populations of beneficial bacteria, which were selected for the study due to their relevance to gut health [10]. These were: Bacteroidetes (6.52 ± 0.04 log10 cells/mL) compared to the cellulose control (6.40 ± 0.05 log10 cells/mL); F. prausnitzii (6.57 ± 0.05 log10 cells/mL) compared to inulin and cellulose controls (6.17 ± 0.04 and 6.07 ± 0.06 log10 cells/mL, respectively); C. coccoides (7.97 ± 0.05 log10 cells/mL) compared to inulin and cellulose controls (7.57 ± 0.06 and 7.40 ± 0.05 log10 cells/mL, respectively); and E. coli (8.09 ± 0.02 log10 cells/mL) compared to inulin and cellulose controls (6.81 ± 0.03 and 6.94 ± 0.03 log10 cells/mL, respectively). However, the production of SCFA was not enhanced by fermentation with the phlorotannin extract.
Lin et al. [170] reported the effect of a polyphenolic extract from the green seaweed, Enteromorpha prolifera, harvested in China, on the gut microbiome and glucose metabolism of diabetic mice. Polyphenols were extracted from E. prolifera using ultrasound-assisted ethanol and ultrafiltration to a MW of 3 kDa. The extract was characterised by UPLC-MS and found to contain four polyphenols—luteolin-6-c-glucoside, regiolone, neoeriocitrin, and estr-5(10)-ene-3,17-diol. Diabetes was induced in ICR mice (20/group) using STZ. Ten of the diabetic mice received a high-sucrose/high-fat diet with no polyphenol supplement (model group); while 10 received a high-sucrose/high-fat diet with E. prolifera polyphenol extract (300 mg/kg BM/d) (diabetic group). A control group of non-diabetic mice received standard chow (normal group).
After 28 days, there was an increase (p < 0.05) in the abundance of beneficial Alistipes intestinal bacteria in the polyphenol-fed diabetic group compared to the model group. After 14 days, there was a significant reduction (p < 0.05) in the mean BM of the E. prolifera-fed diabetic group compared to the model group. After 28 days, fasting blood glucose levels of the diabetic group were lower (p < 0.05), and glucose tolerance was increased (p < 0.05) compared to the model group.
Histopathological analysis of the liver revealed that the polyphenol-fed diabetic group had less cell damage and inflammation of the hepatic cord than the model group. The mRNA expression of two proteins associated with glucose metabolism was also measured in liver tissue—phosphatidylinositol 3-kinase (PI3K) and c-Jun N-terminal kinase (JNK). The PI3K pathway regulates insulin signal transduction and glucose homeostasis [208], while over-activity of the JNK pathway is linked to insulin resistance and type-2-diabetes [209]. After 28 days, mRNA expression of PI3K was increased in the diabetic group (p < 0.01) compared to the model group, and was even significantly higher than the normal group (p < 0.05). JNK1 expression in the diabetic group was successfully downregulated by polyphenol supplementation and was lower (p < 0.05) than the model group.
Yuan et al. [210] investigated the ability of polyphenol extracts from the brown seaweed, Lessonia trabeculata, harvested in China, to alter the gut microbiota of rats in response to type-2-diabetes. Microwave-assisted methanol extraction was followed by solvent fractionation and macroporous resin adsorption separation. The polyphenol-rich fractions produced were composed primarily of phlorotannins, followed by phenolic acids and gallocatechin derivatives. Diabetes was induced in C57BL/6J rats using streptozotocin (STZ). STZ damages the insulin-producing β cells of the pancreas, resulting in hypoinsulinaemia and hyperglycaemia. Diabetic rats (8/group) (PE) were fed 200 mg/day polyphenol extract/kg BM along with their regular food for 4 weeks. A diabetes control (DC) group and a normal control (NC) group (of non-diabetic rats) received no polyphenol supplement with their food.
Hyperglycaemia, insulin resistance, and hyperlipidaemia were significantly (p < 0.01) reduced in the diabetic rats after 4 weeks administration of the seaweed polyphenol extract. Mean fasting blood glucose was lower (p < 0.05) in the PE group (10.55 ± 0.94 mmol/L) compared to the DC control group (13.99 ± 0.87 mmol/L) as was serum insulin (14.69 ± 0.11 vs. 17.70 ± 0.22 mU/L (p < 0.01)). The homeostatic model assessment of insulin resistance (HOMA-IR) value was lower in the PE group (p < 0.01) (6.89 ± 0.42 vs. 11.01 ± 0.98) compared to the DC group. The reductions in lipid profiles in the PE group compared to the DC group were: total cholesterol (4.92 ± 0.14 vs. 5.64 ± 0.16 mmol/L (p < 0.01)), triglycerides (0.99 ± 0.04 vs. 1.43 ± 0.10 mmol/L (p < 0.01)), low-density lipoprotein cholesterol (0.68 ± 0.03 vs. 1.06 ± 0.06 (p < 0.0)), glycated serum protein (2.15 ± 0.16 vs. 2.74 ± 0.15 (p < 0.01)) and non-esterified fatty acids (1.86 ± 0.05 vs. 2.02 ± 0.11 mmol/L (p < 0.05)). The dyslipidaemia observed in the DC group who did not receive polyphenol supplementation was most likely due to the deficiency of circulating insulin, which increases lipase activity and fatty acid mobilisation from adipose tissue [211]. 16S rRNA gene sequencing of faecal samples from the diabetic rats revealed that there was a significant (p < 0.01) increase in gut bacterial diversity within the polyphenol-fed PE group compared to the DC and NC groups. The PE group had a significantly greater abundance of Bacteroidetes, less Proteobacteria, and an improved (lower) ratio of Firmicutes to Bacteroidetes compared to DC (p < 0.01). An overabundance of Proteobacteria has been reported as a pro-inflammatory phylum and linked with the imbalance of glucose homeostasis in type-2-diabetes [170]. At the genus level, the PE group had approximately 10 times more Odoribacter (p < 0.008) and Muribaculum (p < 0.005), and twice the population of Alistipes (p < 0.006), Lachnospiraceae (p < 0.015) and Parabacteroides (p < 0.022) compared to the DC group. Lachnospiraceae and Alistipes are butyric acid producing bacteria that contribute to the maintenance of colonic epithelial tissue [212]. The Odoribacter genus, part of the Bacteroidetes phylum, is an acetic, propionic and butyric acid producer. Its abundance ameliorates inflammation by increasing SCFA availability [213]. An increase in Muribaculum and Parabacteroides numbers has been reported to combat dyslipidaemia, weight gain, inflammation, and insulin resistance resistance [214][215].
Quantification of gut SCFA showed a 61.1% increase in total SCFA production (from 491.31 ± 10.39 to 1276.34 ± 16.86 μg/g (p < 0.01)) by the rats after 4 weeks of polyphenol supplementation. The PE group also produced 68.6% more acetic acid (1202.49 ± 11.55 compared to 377.77 ± 3.46 μg/g (p < 0.01)) and 74.4% more butyric acid (39.77 ± 1.85 compared to 10.18 ± 0.58 μg/g (p < 0.01)) than the DC group. The authors of the study concluded that seaweed polyphenols may have regulated dysbiosis of the gut microbiota in diabetic rats.
2.4.2. Impact of Digestion on Phlorotannin Bioactivity, Attenuation of DNA Damage, and Cancer Cell Proliferation In Vitro
Corona et al. [216] studied the effect of in vitro gastrointestinal digestion and colonic fermentation on the polyphenolic content and bioactivity of high molecular weight (HMW > 10 KDa) and low molecular weight (LMW 1–10 KDa) ethanol-extracted phlorotannins from A. nodosum harvested in Scotland. To assess changes in phlorotannin bioactivity post-gastric digestion and -fermentation, the ability of the extracts to prevent H2O2 induced DNA damage in HT-29 colon cancer cells and inhibit cell proliferation was also measured. The HMW extract had the greatest total polyphenol and total phlorotannin contents before and after digestion. The HMW extract also had the highest Trolox equivalent antioxidant capacity. The molecular weight of total phlorotannins before and after gastric digestion and colonic fermentation was evaluated by normal phase HPLC. Gastric digestion reduced the level of very high molecular weight components present in the HMW fraction by only 5.4%, while colonic fermentation caused an 89.9% reduction. In the LMW extracts, gastric digestion reduced the level of very high molecular weight components by 52.8% and colonic fermentation by 62.0%. In both cases, colonic fermentation had a far greater impact on the breakdown of phlorotannins compared to enzymatic gastric digestion, suggesting that phlorotannins have the potential to be metabolised by human gut bacteria.
A sulforhodamine B assay was used to measure changes in HT-29 colon cancer cell biomass. The addition of post-gastric digested HMW and LMW at a concentration of 500 μg/mL significantly inhibited (p < 0.01) HT-29 cell proliferation (number of cells by division), with HMW being the most effective. Post-gastric digested LMW did not inhibit cell growth (mass accumulation) at any concentration, but HMW did (p < 0.05) at concentrations of 250 and 500 μg/mL. High molecular weight phlorotannins may therefore have a potential protective effect on colonocytes against cancer. H2O2 induced DNA damage in HT-29 cells was evaluated by single cell gel electrophoresis (Comet) assay. Three of the four phlorotannin extracts (at 100 μg/mL) were successful in reducing DNA damage. Post-gastric digested HMW significantly (p < 0.01) reduced DNA damage compared to the control, while post-gastric digested LMW had no effect. However, both the HMW and LMW post-colonic fermented extracts significantly (p < 0.001) reduced DNA damage, suggesting that colonic bacteria may potentially metabolise phlorotannins into molecules with different bioactivity than their parent structures.
Although in vitro studies and animal trials do not replicate the human gut environment identically, these results show that the abundance of bacteria which normally colonise the mammalian gut may potentially be enhanced by the inclusion of dietary polyphenols. The findings are an indication of prebiotic potential, which may be used to inform the design of future human clinical studies. Table 2 summarises the polyphenol used in each study and its potential impact on the gut microbiota in vitro and in vivo, the modulation of hyperglycaemia in animal models, and attenuation of DNA damage in vitro.
Table 2. The potential impact of polyphenols on the gut microbiota in vitro and in vivo, modulation of hyperglycaemia in animal models and DNA damage in vitro.
| Polyphenol | Seaweed | Extraction Method | Study Type | Statistically Significant Effects | Ref. |
|---|---|---|---|---|---|
| * Phlorotannin enriched fraction |
E. radiata | Ethanol (90%) | Simulated in vitro colonic digestion | Increases (all p < 0.05) in Bacteroidetes (6.52 ± 0.04 log10 cells/mL) compared to the cellulose control (6.40 ± 0.05 log10 cells/mL); F. prausnitzii (6.57 ± 0.05 log10 cells/mL) compared to cellulose and inulin controls (6.17 ± 0.04 and 6.07 ± 0.06 log10 cells/mL, respectively); C. coccoides (7.97 ± 0.05 log10 cells/mL) compared to inulin and cellulose controls (7.57 ± 0.06 and 7.40 ± 0.05 log10 cells/mL, respectively); and E. coli (8.09 ± 0.02 log10 cells/mL) compared to inulin and cellulose controls (6.81 ± 0.03 and 6.94 ± 0.03 log10 cells/mL, respectively). | [10] |
| ** Polyphenols (3 kDa) (luteolin-6-c-glucoside, regiolone, neoeriocitrin and estr-5(10)-ene-3,17-diol) | E. prolifera | Ultrasound assisted ethanol extraction (55%) and ultrafiltration (3 kDa) | In vivo trial with diabetic mice (4 weeks, 300 mg polyphenol extract/kg BM/day) | Reduction after 14 days (p < 0.05) in mean BM of E. prolifera-fed diabetic group compared to model diabetic group. Reduction after 28 days (p < 0.05) in mean fasting blood glucose levels of E. prolifera-fed diabetic group and glucose tolerance increased (p < 0.05) compared to the model diabetic group. Increase in Alistipes (p < 0.05) in E. prolifera-fed diabetic group compared to model diabetic group. Hypoglycaemic effect via increase (p < 0.01) in phosphatidylinositol 3-kinase and suppression (p < 0.05) of c-Jun N-terminal kinase in E. prolifera-fed diabetic group livers compared to model diabetic group. |
[170] |
| ** Polyphenol-rich fraction (primarily phlorotannins, phenolic acids and gallocatechin derivatives) | L. trabeculata | Microwave assisted methanol extraction, solvent fractionation and macroporous resin adsorption separation | In vivo trial with diabetic rats (4 weeks, 200 mg/day phlorotannin extract/kg BM) | Increase in genera of the phylum Bacteroidetes in the PE group compared to the DC group: Odoribacter (p < 0.008), Muribaculum (p < 0.005), Alistipes (p < 0.006), Lachnospiraceae (p < 0.015) and Parabacteroides (p < 0.022). Decrease in Proteobacteria, and ratio of Firmicutes to Bacteroidetes (p < 0.05 PE vs. DC group). Increase in total SCFA (491.31 ± 10.39 (DC), 1276.34 ± 16.86 μg/g (PE) (p < 0.01)), acetic acid (377.77 ± 3.46 (DC), 1202.49 ± 11.55 μg/g (PE) (p < 0.01)) and butyric acid (10.18 ± 0.58 (DC), 39.77 ± 1.85 μg/g (PE) (p < 0.01)). Reduction in the PE group versus the DC group in: fasting blood glucose (10.55 ± 0.94 vs. 13.99 ± 0.87 mmol/L (p < 0.05)), serum insulin (14.69 ± 0.11 vs. 17.70 ± 0.22 mU/L (p < 0.01)), HOMA-IR insulin resistance value (6.89 ± 0.42 vs. 11.01 ± 0.98 (p < 0.01)), total cholesterol (4.92 ± 0.14 vs. 5.64 ± 0.16 mmol/L (p < 0.01)), triglycerides (0.99 ± 0.04 vs. 1.43 ± 0.10 mmol/L (p < 0.01)), LDL cholesterol (0.68 ± 0.03 vs. 1.06 ± 0.06 (p < 0.01)), glycated serum protein (2.15 ± 0.16 vs. 2.74 ± 0.15 (p < 0.01)) and non-esterified fatty acids (1.86 ± 0.05 vs. 2.02 ± 0.11 mmol/L (p < 0.05)). |
[210] |
| (i) * Phlorotannin (HMW > 10 kDa) (ii) Phlorotannin (LMW 1–10 kDa) |
A. nodosum | Ethanol | (a) In vitro gastrointestinal digestion and colonic fermentation (b) H2O2 induced DNA damage in HT-29 colon cancer cells |
(a) Reduction in MW of phlorotannins (89.9% HMW, 62.0% LMW) by colonic fermentation, compared to enzymatic gastric digestion (5.4% HMW, 52.8% LMW), suggesting phlorotannins may potentially be metabolised by human gut bacteria. (b) Compared to the control, HMW and LMW phlorotannin extracts at a concentration of 500 μg/mL inhibited (p < 0.01) HT-29 colon cancer cell proliferation (number of cells by division), HMW inhibited (p < 0.05) HT-29 cell growth (mass accumulation) at concentrations of 250 and 500 μg/mL. H2O2 induced DNA damage in HT-29 cells reduced by post-gastric digested HMW extract (p < 0.01) and HMW and LMW post-colonic fermented extracts (both p < 0.001). |
[216] |
* = in vitro studies; ** = in vivo animal studies
2.5. Seaweed-Derived Peptides
Seaweed-derived peptides have reported bioactivity as inhibitors of renin [217], angiotensin converting enzyme-I (ACE-I) [9] dipeptidyl peptidase (DPP-IV) [218], platelet activating factor acetylhydrolase (PAF-AH) [219] and α-amylase [220]. They also have reported immunostimulatory [221], antitumor [222], anti-coagulant [223], antioxidative [5], and anti-hyperglycaemic [224] activity. There is recent evidence that some peptides found in vitro correlate with animal studies [225]. A study conducted by Fitzgerald et al. [217] previously identified the potential heart health beneficial effects of peptides included in a bread product with peptides derived from the red seaweed Palmaria palmata using both in vitro and animal models [226]. Peptides were isolated from the seaweed and characterised to completion. However, another study carried out by Allsopp et al. [227] found that the same seaweed had a pro-inflammatory effect when consumed as a whole seaweed in a bread product. This highlights the importance of extraction and characterisation of seaweed bioactives for potential use as therapeutic agents. Allsopp et al. suggested that the iodine content of the seaweed may have been responsible for the observed pro-inflammatory effect in a human dietary intervention study. Table 3 details the amino acid sequences of recently elucidated seaweed peptides and their bioactivities in vitro, in silico, or in vivo.Table 3. Amino acid sequences of recently elucidated seaweed-derived peptides and their bioactivities in vitro, in silico or in vivo.
| Seaweed | Extraction Method | Amino Acid Sequence | Bioactivity | Ref. |
|---|---|---|---|---|
| * † U. lactuca | Enzymatic (Papain), MWCO filtration, preparative RP-HPLC and in silico enzyme cleavage simulation | (i) Ala-Thr-Lys-Pro-Ala-Asn (ii) Ser-Gly-Ala-Ala-Ser-Ala-Ser-Gly-Ala-Ala (iii) Ala-Gly-Gly-Pro-Asn-Gln-Pro-Pro-Asn (iv) Ala-Ala-Asn-Ile-Thr-Val-Pro-Ala-Ala-Asn (v) Glu-Ala-Glu-Pro-Ala-Glu-Ala-Ala (vi) Gly-Ala-Ala-Pro-Thr-Pro-Pro-Ser-Pro-Pro-Pro-Ala-Thr-Lys-Pro-Ser-Thr-Pro-Pro-Lys-Pro-Pro-Thr (vii) Pro-Pro-Asn-Pro-Pro-Asn-Pro-Pro-Asn Amino acid sequences not defined: (a) crude seaweed protein (b) full peptide hydrolysate (c) 1 kDa-UFH (ultra-filtered hydrolysate) (d) 3 kDa-UFH (e) 10 kDa-UFH |
Peptides (i) to (vii) ACE-I, DPP-IV, and enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibition (in silico predictive activity) In vitro ACE-I inhibitory activity (%) (all assayed at conc. of 1mg/mL): (a) crude seaweed protein 79.87 ± 0.18% (b) full peptide hydrolysate 82.37 ± 0.05% (c) 1 kDa-UFH (ultra-filtered hysrolysate) 93.03 ± 0.87% (d) 3 kDa-UFH 86.64 ± 2.17% (e) 10 kDa-UFH 88.12 ± 0.02% |
[9] |
| * P. palmata | Enzymatic (Papain) | Ile-Arg-Leu-Ile-Ile-Val-Leu-Met-Pro-Ile-Leu-Met-Ala | Renin inhibition (58.97 ± 1.26% inhibition in vitro at 1 mg/mL) |
[217] |
| * P. palmata | Enzymatic (Protease) | (i) Ile-Leu-Ala-Pro (ii) Leu-Leu-Ala-Pro (iii) Met-Ala-Gly-Val-Asp-His-Ile |
DPP-IV inhibition IC50 values in vitro: (i) 43.40 ± 1.40 μM (ii) 53.67 ± 0.82 μM (iii) 159.37 ± 13.67 μM |
[218] |
| * P. palmata | Enzymatic (Papain) | Asn-Ile-Gly-Lys | PAF-AH inhibition IC50 value in vitro 2.32 ± 2.12 mM |
[219] |
| * Porphyra (Laver—species not specified) | Enzymatic (Viscozyme, Alcalase, Neutrase, Pepsin and Trypsin) | (i) Gly-Gly-Ser-Lys (ii) Glu-Leu-Ser |
α-amylase inhibition IC50 values in vitro: (i) 2.58 ± 0.08 mM (ii) 2.62 ± 0.05 mM |
[220] |
| * P. palmata | Thermolysin hydrolysis | (i) Leu-Arg-Tyr (ii) Val-Tyr-Arg-Thr |
ACE-I inhibition IC50 values in vitro: (i) 0.044 μM (ii) 0.14 μM |
[228] |
| *,** U. pinnatifida | Enzymatic (Protease) | (i) Val-Tyr (ii) Ile-Tyr (iii) Phe-Tyr (iv) Ile-Trp (v) Ala-Trpvi) Val-Trp (vii) Leu-Trp |
ACE-I inhibition IC50 values in vitro: (i) 35.2 μM (ii) 6.1 μM (iii) 42.3 μM (iv) 1.5 μM (v) 18.8 μM(vi) 3.3 μM (vii) 23.6 μM In vivo antihypertensive effect in spontaneously hypertensive rats (single oral dose, 1 mg/kg of BW). Blood pressure decreases (pre-administration vs. 9 h post): (i) Val-Tyr (228.2 ± 3.4 vs. 206.7 ± 9.5 mmHg) (p < 0.05) (ii) Ile-Tyr (205.6 ± 5.2 vs. 184.3 ± 4.5 mmHg) (p < 0.05) (iii) Phe-Tyr (208.7 ± 4.4 vs. 193.0 ± 5.1 (p < 0.01) (iv) Ile-Trp (213.3 ± 3.4 vs. 199.5 ± 5.9) (p < 0.05) |
[229] |
| * U. pinnatifida | Enzymatic (Pepsin) | (i) Ala-Ile-Tyr-Lys (ii) Tyr-Lys-Tyr-Tyr (iii) Lys-Phe-Tyr-Gly (iv) Tyr-Asn-Lys-Leu |
ACE-I inhibition IC50 values in vitro:((i) 213 μM (ii) 64.2 μM (iii) 90.5 μM (iv) 21.0 μM |
[230] |
| * P. palmata | Enzymatic (Protease) | Ser-Asp-Ile-Thr-Arg-Pro-Gly-Gly-Asn-Met | Antioxidant activity after simulated gastrointestinal digestion: Oxygen radical absorbance capacity 152.43 ± 2.73 nM Trolox equivalents (TE)/µmol peptide and ferric reducing antioxidant power activity 21.23 ± 0.90 nM TE/µmol peptide, |
[231] |
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