Profiling the activity of edible European macroalgae towards pharmacological targets for type 2 diabetes mellitus

ABSTRACT In traditional medicine marine extracts are extensively used as therapies for diabetes. With the increasing rate of incidence of type 2 diabetes mellitus and rising cost of treatments, we investigated the anti-diabetic properties of extracts of common edible seaweeds in Europe including their ability to inhibit alpha-glucosidase and dipeptidyl peptidase 4 (DPP-4) enzymatic activity, block sodium glucose transporter-2 (SGLT-2) activity and stimulate glucagon-like peptide-1 (GLP-1) secretion and synthesis. The most promising seaweed extracts were tested for anti-hyperglycaemic activity in vivo. Some brown seaweed (Phaeophyceae) extracts had inhibitory effects on alpha-glucosidase ranging from 13.9 ± 0.2% to 89.5 ± 0.4% (p < 0.001). However, none of the seaweed extracts was able to block SGLT-2 activity. Ethanol extracts of the kelp Alaria esculenta and water extracts of Laminaria digitata strongly inhibited DPP-4 activity by 91.3 ± 0.1% and 90.0 ± 0.2%, respectively (p < 0.001), while ethanol extracts of Ulva rigida (Chlorophyta) had the greatest potential to stimulate GLP-1 secretion and GLP-1 synthesis (p < 0.001). A water extract of Porphyra linearis (Rhodophyta) significantly reduced the overall glycaemic excursion during an oral glucose tolerance test in normal mice (p < 0.05). These results demonstrate future potential for common edible seaweeds to be used as medicinal foods or bio-therapeutics to tackle type 2 diabetes mellitus by targeting GLP-1 secretion, DPP-4 activity or alpha-glucosidase activity.


Introduction
Diabetes mellitus is a group of human metabolic disorders causing elevated blood glucose levels due to a lack of insulin secretion and/or the development of insulin resistance in peripheral tissues. It is one of the most common chronic diseases in the world, with an estimated 422 million adults globally living with diabetes in 2014, compared with 108 million in 1980 (World Health Organization, 2016). The estimated global direct health expenditure on diabetes in 2019 was USD 760 billion (Williams et al., 2020). Targets of the UN's Sustainable Development Goal 3 include reducing the mortality rate attributed to cardiovascular disease, cancer, diabetes and chronic respiratory disease by one-third by 2020 (https:// sustainabledevelopment.un.org/sdg3).
Currently, type 2 diabetes mellitus (T2DM) accounts for more than 90% of all cases of diabetes, so there is a focus on treatment or management of this disease (World Health Organization, 2016). T2DM has many features but a key characteristic is hyperglycaemia, in which an excessive amount of postprandial glucose is absorbed into the bloodstream as a result of impaired insulin production/signalling (Muskiet & Tonneijck, 2017). A major source of this glucose is the intestinal hydrolysis of dietary carbohydrates carried out by starch-converting enzymes, in particular alpha-glucosidase. Thus, inhibition of alpha-glucosidase delays carbohydrate hydrolysis and in turn reduces postprandial hyperglycaemia (Muskiet & Tonneijck, 2017). Glucose absorption is further affected by the action of sodium glucose transporter-2 (SGLT-2). These transport proteins are found in the renal tubular epithelium and are quantitatively the most important for glucose reabsorption in the kidney (Wright, Hirayama, & Loo, 2007). Inhibition of SGLT-2 has proven to lower blood glucose in a dosedependent manner (Rosenstock et al., 2012). Furthermore, stimulating the secretion of glucagon-like peptide-1 (GLP-1), a hormone released from L-cells which elicits postprandial insulin secretion and thus has a significant role in glucose homoeostasis, could have a considerable impact on T2DM patients. The administration of GLP-1 to type 2 diabetic patients effectively lowered blood glucose levels (Gutniak, Orskov, Holst, Ahren, & Efendic, 1992). Finally, inhibiting dipeptidyl peptidase-4 (DPP-4), a ubiquitous type II transmembrane glycoprotein responsible for GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) degradation, has also proven fundamental as a management tool for T2DM (Green et al., 2003;Green, Irwin, Gault, FPM, & Flatt, 2005). Improved glucose tolerance was observed in DPP-4 knockout mice, in correlation with increased GLP-1 levels and enhanced insulin secretion, after oral administration of glucose (Marguet et al., 2000). A combination of all of these strategies could allow effective management of T2DM in affected patients.
With the increasing global incidence and cost of managing T2DM, further action must be taken to source alternatives to the costly, synthetic drugs already available and which can be easily made accessible to patients on a global scale. As such, naturally derived marine products with anti-diabetic activity may be exploited as an efficient and cost-effective approach to managing this disease (Nguyen et al., 2019). Previous studies have demonstrated that extracts from brown, red and green seaweed species, collected primarily in tropical areas, have the ability to inhibit alpha-glucosidase (e.g. Li, Niu, Fan, & Han, 2005;Lee, Karadeniz, Kim, Kim, & Kim, 2009;Apostolidis & Lee, 2010;Nwosu et al., 2011;Lee et al., 2012aLee et al., , 2012bKawamura-Konishi et al., 2012;Kang et al., 2013;Schultz Moreira et al., 2014;Lauritano & Ianora, 2016;Shannon & Abu-Ghannam, 2019). The hypoglycaemic effects of seaweed extracts and their secondary metabolites have been demonstrated in normal and diabetic animals (e.g. Iwai, 2008;Lopes, Andrade, & Valentao, 2017;Roy et al., 2011;Xu et al., 2012). To date, comparatively few naturally occurring compounds have been discovered which inhibit DPP-4. Berberine, an isoquinoline alkaloid from the Chinese herb Coptis chinensis, inhibited human recombinant DPP-4 (Al-Masri, Mohammad, & Tahaa, 2009). Procyanidins from grape seed inhibited intestinal DPP-4 in vivo (Gonzalez-Abuin et al., 2012 and protein hydrolysates from the red seaweed Palmaria palmata inhibited DPP-4 in vitro with IC 50 values of 1.65-4.60 mg ml -1 (Harnedy, FitzGerald, & FitzGerald, 2015). Growing interest over the last 15 years in the effects of natural products (with phlorizin obtained from higher plants as a model) on SGLT-2 inhibition and GLP-1 secretion (Ehrenkranz, Lewis, Kahn, & Roth, 2005) has led to the development and testing of new drugs as next-generation antihyperglycaemic agents (reviewed by Blaschek, 2017;Choi, 2016).
Marine organisms are currently the subject of intense research effort into bioactives including those with antidiabetic properties (reviewed by Cotas, Leandro, Pacheco, Gonçalves, & Pereira, 2020;Lauritano & Ianora, 2016;Shannon & Abu-Ghannam, 2019). Brown algae are of particular interest due to their production of phlorotannins, a class of polyphenols exclusively produced by brown seaweeds (Gunathilaka, Samarakoon, Ranasinghe, & Peiris, 2020;Lopes et al., 2017). The majority of studies to date have focussed on tropical species (Gunathilaka et al., 2020), although recently the effects of dried seaweeds from Norway were investigated in a diabetic mouse model (Sørensen, Jeppesen, Christiansen, Hermansen, & Gregersen, 2019). Here we screened extracts from eleven common edible species of seaweeds (three red, two green and six brown algae) collected in Ireland for an array of anti-diabetic effects. The aim was to ascertain whether edible seaweeds could act through known and established therapeutic mechanisms, which included inhibition of αglucosidase, inhibition of DPP-4, inhibition of SGLT-2 transporters and promotion of GLP-1 synthesis and secretion. We examined the effects of seaweed extracts on an important type of intestinal cell (the enteroendocrine L-cell). We used the widely established STC-1 murine cell model which produces significant amounts of the insulinotropic hormone GLP-1 (Gillespie, Pan, Marco-Ramell, Meharg, & Green, 2017;McCarthy et al., 2015). In a final phase, oral glucose tolerance tests were performed in vivo on promising seaweed extracts. We limited ourselves to using only food grade solvents to permit the future prospect of extracts being used as food additives/ ingredients, with the aim of exploiting these natural products to be used as dietary supplements, medicinal foods or bio-therapeutics for cost-effective management of T2DM and hence potentially contribute to reducing premature mortality. In order to ensure reproducibility, one of our goals was to use samples that had been carefully identified, with publicly available voucher specimens, to determine the potential therapeutic value of these seaweeds. Jolis and Alaria esculenta (Linnaeus) Greville (Supplementary figs S1-2).

Collection and identification of seaweeds
Several of these species have been subject to extensive taxonomic and genetic investigation at this site: Codium fragile subsp. fragile (Provan, Wattier, & Maggs, 2005a); Palmaria palmata (Provan, Murphy, & Maggs, 2005b); Chondrus crispus (Provan, Glendinning, Kelly, & Maggs, 2013) and Ulva spp. (Brodie, Maggs, & John, 2007;Hughey et al., 2019). Voucher specimens of the readily identifiable species F. vesiculosus, L. digitata, H. elongata and A. esculenta have been deposited in the algal herbarium (BM) at the Natural History Museum (Supplementary table S1). Ulva species are extremely difficult (arguably impossible) to identify morphologically (Brodie et al., 2007;Hughey et al., 2019); therefore, samples of the Doaghbeg population were sequenced for the rbcL marker. Samples for molecular identification were collected on 22 September 2002 and processed as described in Krupnik et al. (2018), using PCR to obtain rbcL sequences from the samples. Identifications were made by BLAST searches using only quality-assured sequences in GenBank. The majority of the Fanad material was U. rigida, whereas U. fenestrata Postels & Ruprecht (formerly known as U. lactuca) was relatively uncommon.

Preparation and extraction of seaweed extracts
Seaweed was rinsed under cold fresh water to remove excess sand and grit. Samples were dried with paper towels, frozen to −80°C then lyophilized for two days in a Moduloyd freeze dryer (Milford, USA). Freezedried samples were blended to a fine powder in a blender (IKA®-Werke GmbH & Co. KG, Germany), milled and stored in airtight containers at room temperature for less than 6 months prior to use.
Only water and ethanol extractions were performed as these solvents are classified as food grade solvents. 1.25 g of dried seaweed powder was added to 50 ml of boiling absolute ethanol (> 99.5%), placed on a rotary mixer for 30 min then centrifuged at 4000 g for 10 min after which the supernatant was removed. Ethanol extracts were dried in a MiVac sample concentrator (Genevac, Ipswich, UK) and water extracts freezedried. The extracted seaweed powder was stored at −20°C until further use. On experimental days, dried seaweed extracts were reconstituted in appropriate buffer for experimentation.

Alpha-glucosidase assay
Alpha-glucosidase enzyme was initially prepared from rat intestinal acetone powder (Sigma-Aldrich Company Ltd, Dorset, UK) in a 9-fold volume of citrate buffer (pH 5.6) and supernatant obtained as the crude enzyme solution for use in assays. The control assay contained 300 µl of maltose at 10 mg ml -1 and 150 µl of phosphatebuffered saline (PBS) buffer (pH 7.4). Seaweed extracts were assayed at 12.5 mg ml -1 in the 150 µl volume and reactions were started by the addition of 10 µl of rat intestinal alpha-glucosidase enzyme. The solutions were incubated at 37°C for 75 min and analysed for glucose content every 15 min on a PGM7 Micro-Stat Analyser (Analox Instruments Ltd, London, UK). Acarbose (Sigma-Aldrich Company Ltd) was dissolved at 1 mg ml -1 and used as a positive control.

SGLT-2 assay
Determination of SGLT-2 inhibition was carried out according to the protocol of Castaneda and Kinne (2005) involving a [ 14 C]AMG assay (NEN, Bad Homburg, Germany) which specifically measures SGLT-2 mediated glucose uptake (Castaneda & Kinne, 2005). Briefly, Chinese hamster ovary cells transfected with human kidney SGLT-2 were seeded into 96-well plates. After overnight attachment, culture medium was removed and plates washed three times with Krebs-Ringer-Henseleit (KRH) solution containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl 2 , 2.2 mM CaCl 2 , 10 mM HEPES (pH 7.4 with Tris). Transport buffer containing KRH-Na + with [ 14 C]AMG (0.1 µCi µl -1 ) was added to each well and incubated for 1 h at 20°C. At the end of the uptake period the transport buffer was removed and uptake of [ 14 C]AMG stopped by addition of ice-cold stop buffer (KRH-Na + with 0.5 mM phlorizin (positive control) or seaweed sample at 12.5 mg ml -1 ). Wells were washed three times with stop buffer and then solubilized by adding ATPlite substrate solution (Perkin-Elmer, Boston, USA). Luminescence of ATP was measured using a MicroBeta Trilux (Perkin-Elmer, Boston, USA). After 24 h a scintillation counter was used to determine radioactive [ 14 C]AMG. The mean counts per minute were calculated and converted to picomoles and percentage inhibition calculated by comparison of negative control.

DPP-4 assay
Seaweed samples were analysed for DPP-4 inhibition fluorometrically using a method described by Fujiwara and Tsuru (1978) for measurement of free AMC (7-amino-4-methyl-coumarin) liberated from the DPP-4 substrate, Gly-Pro-AMC. Using half-volume 96-well plates with opaque walls and transparent bottoms, 20 µl of seaweed extracts dissolved in 100 mM HEPES (4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer and 30 µl of Gly-Pro-AMC (1 mM; Bachem AG, Bubendorf, Switzerland) were added to each well. The reaction was initiated by the addition of 20 µl of bovine serum and the plate was incubated at 37°C with agitation (microplate shaker) for 1 h. 100 µl of 3 mM acetic acid was added to halt the reaction and plates were immediately measured using a desktop fluorometer (Tecan UK Ltd, Reading, UK) at excitation and emission wavelengths of 351 and 430 nm, respectively. Berberine (Fluorochem, Derbyshire, UK), an established natural DPP-4 inhibitor (Al-Masri et al., 2009), was dissolved at 1 mg ml -1 and used as a positive control.

Cell secretion and accumulation studies
STC-1 cells were seeded in 12 well plates (2 x 10 6 cells per well) with 1.5 ml DMEM and incubated overnight at 37° C in a 5% CO 2 humidified atmosphere to allow attachment (Hand, Bruen, O'Halloran, Giblin, & Green, 2010). Media were removed and cells were washed twice with HEPES buffer (20 mM HEPES, 10 mM glucose, 140 nM NaCl, 4.5 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgCl 2 ) and pre-incubated in the same HEPES buffer for 1 h. After removal of buffer, seaweed extracts reconstituted in buffer were added to cells in triplicate for 3 h. After the incubation period the supernatant was removed, centrifuged at 1000 g for 10 min to remove cellular debris and stored at −20°C prior to analysis.

Cellular content studies
To determine cellular GLP-1 content, cells were washed twice with HEPES buffer after sample incubations and incubated overnight at 4°C in an acid/ethanol solution (1.5% hydrochloric acid (v/v): 75% ethanol (v/v): 23.5% water (v/v)) to lyse the cells. The incubation solution was removed and centrifuged (900 g for 5 min) to remove cellular debris. The supernatant was collected and solvent evaporated using a Speedvac sample concentrator (Genevac, Ipswich, UK). Samples were reconstituted in PBST 0.1% BSA and stored at −80°C (for < 4 weeks) prior to analysis.

Measurement of GLP-1 by radioimmunoassay
GLP-1 was determined by means of an in-house fully optimized radioimmunoassay using a polyclonal rabbit antibody for GLP-1 (7-36)amide with no cross-reactivity for glucagon or GIP (sensitivity = 59 ± 26.8 pM, r 2 = 0.99). In brief, PBST (phosphate-buffered saline Tween) buffer with 0.1% BSA, seaweed extracts and GLP-1 standards (GLP-1 (7-36)amide , American Peptide Company, California, USA) was incubated with primary antibody (rabbit anti-GLP-1, made in-house) in plastic tubes overnight at 4°C. Freshly labelled GLP-1 125 I (PerkinElmer, LAS, UK Ltd) diluted to approximately 10 000-15 000 cpm was added to each tube, and the mixture vortexed and incubated at 4°C for a further 48 h. Secondary antibody was added (antirabbit IgG Sac-Cel, IDS, Boldon, UK) and left for 30 min at room temperature prior to addition of 1 ml of deionized water. Tubes were centrifuged at 3000 g for 20 min at 4°C and the supernatant aspirated off. Counts for radioimmunoassay tubes were measured using a Packard Cobra ll Model 5002 gamma counter (PerkinElmer LAS, Beaconsfield, UK).

Glucose tolerance tests in mice
C57/BL6 mice (5-10 weeks old) were obtained from Harlan® (Harlan Laboratories, Wyton, UK) and housed at 23 ± 1°C with 50% humidity and 12 h light/12 h dark cycle. Mice had free access to food (Teklad standard rodent diet; Harlan, Wyton, UK) and water. Prior to experimentation mice were randomly divided into groups. Control animals were orally gavaged with saline containing 2 g kg -1 of glucose alone. Treatment groups received glucose combined with 500 mg kg -1 of seaweed. Blood glucose levels were measured at time points 0, 15, 30, 60 and 105 min using a glucometer (FreeStyle Freedom Lite Blood Glucose Monitoring System, Abbott, USA). Mice used were housed throughout these studies under constant climatic conditions. All experimental procedures were performed in accordance with the Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986 (UK) and approved by the Queen's University Belfast Animal Welfare and Ethical Review Body.

Statistical analysis
All data are expressed as mean ± standard error mean (SEM). For α-glucosidase and in vivo tolerance tests data were subject to Area Under Curve (AUC) analysis. For DPP-4 and SGLT-2 studies inhibition was calculated as a percentage of the negative control. GLP-1 concentrations were determined by interpolation from a standard curve. All statistical analyses were carried out using a one-way ANOVA with Tukey's Multiple Comparison Test to compare differences between groups with the exception of in vivo tolerance tests where a two-way ANOVA was carried out to compare different time points (*p < 0.05, **p < 0.01, ***p < 0.001). All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad, California, USA).

SGLT-2 inhibition
None of the seaweeds tested at 12.5 mg ml -1 were effective blockers of SGLT-2 ( Figure 2). The inhibitor phlorizin was successfully used as the positive control ( Figure 2).

GLP-1 secretion, accumulation and cellular content in STC-1 cells
Red seaweed species were very potent at increasing acute GLP-1 secretion from STC-1 cells at concentrations of 25 mg ml -1 (Figure 4(a)). The water extract of P. linearis and ethanol extract of U. rigida increased GLP-1 secretion by 2.3-fold and 1.9-fold, respectively, during a 3 h acute exposure. Over a 3-day period ethanol extracts of A. esculenta and U. rigida were the best GLP-1 secretagogues, increasing secretion by around 41.2-fold to 52.3-fold, while the water extract of L. digitata also significantly increased GLP-1 levels over 3 days by 25.4-fold (Figure 4(b)). Water extracts of C. fragile, L. digitata and A. nodosum and ethanol extracts of U. rigida and A. esculenta were able to increase the cellular content of GLP-1 significantly (Figure 4(c)).

Oral glucose tolerance tests in vivo
In normal mice during oral glucose tolerance tests the water extract of C. crispus and ethanol extract of P. palmata at 500 mg kg -1 concentration significantly reduced the rise in blood glucose levels at 15 min and 30 min, respectively (p < 0.01 and p < 0.05, respectively), however blood glucose level returned to that of the control animals after this time ( Figure 5). Only the water extract of P. linearis at 500 mg ml -1 significantly reduced the overall blood glucose levels when orally gavaged with glucose (p < 0.05), and in fact its response was significantly lower than the control by 30 min. This indicates the potential of these extracts for acutely reducing postprandial blood glucose.

Discussion
These screening studies demonstrate that extracts from common edible Irish seaweeds exhibit a range of potential anti-diabetic effects and could potentially form the basis for future development of novel treatments or prophylactics for T2DM. Our results confirm previous work demonstrating that water extracts of Ascophyllum nodosum potently inhibit alpha-glucosidase activity at concentrations as low as 12.5 mg ml -1 (Apostolidis & Lee, 2010). This study is in accordance with the finding that A. nodosum and Alaria esculenta have potent inhibitory activity on alpha-glucosidase (Nwosu et al., 2011). Our findings are also supported by a study which combined Ascophyllum nodosum and Fucus vesiculosus extracts and achieved alpha-glucosidase inhibition of nearly 100% (Roy et al., 2011). Extraction and assay methodologies differ substantially from study to study making it difficult to closely compare the inhibitory activities of each seaweed species without identification of the bioactive secondary metabolite. However, it can be concluded that brown seaweed species have future potential for managing T2DM by means of inhibiting alpha-glucosidase. One drug currently used for managing T2DM is acarbose, which is effective in inhibiting alpha-glucosidase but has several undesirable side-effects such as excessive gas, abdominal distention and flatulence (Fujisawa, Ikegami, Inoue, Kawabata, & Ogihara, 2005). We suggest that natural products consumed with or as part of a meal potentially could offer Figure 2. Screening of seaweed extracts for Sodium glucose transporter-2 (SGLT-2) inhibitory activity. Figure shows the % SGLT-2 inhibitory activity of aqueous and ethanol seaweed extracts at 12.5 mg ml -1 . Bars represent mean ± SEM. Inhibition is calculated as a percentage of negative control. Phlorizin (0.5 mM) obtained from plants (Ehrenkranz et al., 2005) was used as the positive control (*p < 0.05, **p < 0.01, ***p < 0.001; n = 8).
lesser side-effects and similar efficacy. To date, few results of human clinical trials have been reported (https://clinicaltrials.gov/ct2/show/NCT03075943). Sakai et al. (2019) investigated the results of inclusion of fucoidan in the diet.
The glucose transporter SGLT-2 is known to be inhibited by plant flavonoids isolated from Cynodon dactylon (Annapurna et al., 2013), and also by two cyclic diarylheptanoids isolated from the bark of Acer nikoense (Morita et al., 2010). The potential development of drugs from natural products in this therapeutic area has been reviewed recently (Blaschek, 2017;Choi, 2016). To our knowledge ours is the first study to investigate the effect of seaweed on SGLT-2 inhibition, although none of the seaweeds tested were effective.
More promisingly, our study demonstrated that seaweeds have a significant ability to inhibit DPP-4 activity. Palmaria palmata has previously been shown to cause potent DPP-4 inhibition, up to 81% after solid-phase fractionation (Harnedy et al., 2015). Our results supported previous findings that brown seaweeds tend to have higher inhibitory activity than red or green seaweed species (Chin et al., 2014;Harnedy et al., 2015), indicating they have specific compounds able to bind and inhibit DPP-4. These compounds may be structurally similar to those in berberine, where they readily interact with the binding pocket of DPP-4 (Al-Masri et al., 2009). However, more studies will need to be carried out to isolate the compounds responsible and to determine their mechanism of DPP-4 inhibition.
A number of plant sources and food constituents are known to stimulate GLP-1 secretion (Rafferty et al., 2011). The finding that extracts from red, brown and green seaweeds caused increased GLP-1 synthesis and secretion by STC-1 cells potentially conflicts with another study which demonstrated that a 10-day treatment with sodium alginate from L. digitata did not alter  GLP-1 levels in overweight and obese subjects (Odunsi et al., 2010). Similarly, Sakai et al. (2019) found that consumption of fucoidan resulted in a decrease of the baseline GLP-1 level, possibly as a result of altered gastrointestinal function in patients. For this reason, it is likely that molecules other than sodium alginate, such as phenolic compounds in L. digitata, are responsible (Heffernan, Smyth, Soler-Villa, Fitzgerald, & Brunton, 2015). For example, natural phenolic compounds such as resveratrol, produced by various plants, increase both portal vein GLP-1 and intestinal content of GLP-1 (Dao et al., 2011). Our study found that P. palmata, whether extracted in water or ethanol, resulted in significant GLP-1 secretion. Water and ethanol extracts of other red algae, nori (dried Porphyra/Pyropia species), were previously reported to possess GLP-1 secretory activity (Schultz Moreira et al., 2013). It is a promising finding that seaweed extracts not only caused GLP-1 secretion from STC-1 cells, but they were also able to increase GLP-1 synthesis within the cell (as indicated by increased cellular GLP-1 content). Our results open up the future possibility of using a seaweed species with dual L-cell activities, e.g. U. rigida which increases both GLP-1 synthesis and GLP-1 secretion, although this requires further investigation. The mechanisms whereby P. linearis extracts generate lower glucose excursions in vivo indicated by our in vitro data may result from an increase in GLP-1 secretion and/ or DPP-4 inhibition, although it should be noted that there are other physiological mechanisms by which glucose lowering can occur. Porphyran, a soluble dietary fibre present in Porphyra, has been shown to improve glucose metabolism in KK-Ay mice via upregulation of adiponectin levels (Kitano et al., 2012), while several studies have shown the ability of seaweed to reduce postprandial blood glucose levels and improve glucose intolerance, particularly in . In vivo effects of seaweed extracts on blood glucose following oral glucose challenge. a) and c) Each seaweed extract (500 mg kg -1 ) was orally gavaged with glucose (2 g kg -1 ) to normal mice and glucose responses were monitored at 0, 15, 30, 60 and 105 min. b) and d) Incremental areas under plasma glucose curves (ΔAUC0-105) were calculated with baseline subtraction. Bars and points represent mean ± SEM. Line graph data were compared with saline treated mice and analysed by two-way ANOVA with a Bonferroni post-test. Bar graphs are AUC data compared by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001; n = 4-5) (H 2 Owater extract, EtOH -ethanol extract).
animal models of T2DM (Iwai, 2008;Kang et al., 2013;Motshakeri, Ebrahimi, Goh, Matanjun, & Mohamed, 2013;Park, Kim, Kim, Kim, & Kim, 2009;Tas, Celikler, Ziyanok-Ayvalik, Sarandol, & Dirican, 2011: Xu et al., 2012. Any of these mechanisms could be responsible for the observed glucose-lowering effect caused by P. linearis. In conclusion, it is clear that common edible European seaweeds have anti-diabetic properties. There is a need to identify the compound(s) responsible for these effects and to maximize their yield by studying the composition of the seaweed, the extraction time/ temperature/solvents used, and also to assess the effect of harvesting location and season of collection. These studies are supportive of the notion that edible seaweeds (such as those studied here) are beneficial to human health if consumed as part of the normal diet, and may promote normoglycaemia (Lee, Kim, Chang, & Nam, 2010). Much work is still needed, including the isolation and identification of the bioactive secondary metabolites and confirmation of their in vitro and in vivo activity but there is huge potential for seaweeds as natural therapeutics for treatment or management of T2DM.