Advanced Pharmacological Uses of Marine Algae as an Anti-Diabetic Therapy

Marine seaweeds are a promising source of bioactive secondary metabolites that can be utilized in drug development and nutraceuticals. Diabetes mellitus is a leading non-communicable disease, and it is the third leading cause of death worldwide. Among the types of diabetes, type 2 became the major health problem as it is associated with severe health complications. Since available oral hypoglycemic drugs cause several adverse effects, it is worth searching for a natural cure with fewer or no side effects that may benefit patients with type 2 diabetes. Among the marine seaweeds, brown and red seaweeds are extensively studied for the anti-diabetic activity compared to the green seaweeds. Bioactive compounds present in marine seaweeds possess anti-diabetic potential through diverse mechanisms, mainly by reducing postprandial hyperglycemia and associated complication. Most of the studies emphasized that the marine seaweeds control the hyperglycemic condition by inhibiting carbohydrate hydrolyzing α -amylase, α glucosidase enzymes, and the inhibitory effect of dipeptide peptidase-4 that are involved in the degradation of incretins. Similarly, bioactive compounds in marine seaweeds can reduce diabetes complications by inhibiting angiotensin-converting enzymes, aldose reductase, protein tyrosine phosphatase 1B enzyme. This chapter focuses on the anti-diabetic potential of marine brown, green, and red seaweeds through different mechanisms.


Introduction
The prevalence of diabetes has increased rapidly over the past few years, mainly in low to middle-income countries, and became one of the major causes of premature death worldwide. According to the WHO statistics, 422 million people were estimated as diabetes in 2014, and 1.6 million deaths were reported [1]. The International Diabetes Federation estimated that the world's diabetic population has increased to 592 million by 2035. The largest number of diabetes cases was reported in the Western Pacific region (132 million), while 71.4 million diabetes cases were reported in the South Asian area [2].
Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia due to defects in insulin secretion, insulin action, or both. It is mainly classified as insulin-dependent diabetes mellitus (Type 1 DM) and non-insulin-dependent diabetes mellitus (type 2 DM). Type 1 DM is associated with deficiency of insulin, which occurs due to the destruction of pancreatic ß-cells via an autoimmune process. In contrast, type 2 DM is linked with insulin resistance, which reduces insulin utilization by peripheral tissues and results in hyperglycemia and obesity [3]. Type 2 DM became a major health problem worldwide associated with microvascular and macrovascular health complications. Microvascular and microvascular complications include diabetic retinopathy, neuropathy, nephropathy, and cerebrovascular diseases, peripheral arterial diseases, respectively [4]. Therefore, natural therapeutic approaches [5] should be developed to maintain the blood glucose level and long-term complications in patients with type 2 DM.
As currently available treatment regimens for type 2 DM have adverse side effects, it is necessary to search for an effective drug that helps maintain the blood glucose level and complications in patients with type 2 DM. Even though most of the researchers focused on herbal medicine, none have a full beneficial effect on curing patients with type 2 DM [6]. Hence, it is worth emphasizing marine seaweeds as they have been identified as a rich source of promising bioactive compounds synthesized from their biochemical and physiological mechanisms. Besides, most marine seaweeds are survived in extremely harsh environments, which provide enormous potential to produce complex bioactive compounds to withstand extreme conditions. As a result, the composition of the bioactive compounds in marine seaweeds can vary depending on the geographic area and seasonal changes [7]. As most marine seaweeds are a potential source of bioactive compounds with various therapeutic effects, this chapter mainly emphasizes the pharmacological uses of marine algae as an anti-diabetic therapy.

Therapeutic targets for type 2 diabetes mellitus
As type 2 DM is a progressive disorder, the search for effective treatments is essential to maintain hyperglycemia and its associated diabetic complication. Insulin resistance and impaired beta-cell function lead to hyperglycemia due to alteration in glucose homeostasis, which in turn cause loss of postprandial glucose control. Therefore, postprandial blood glucose maintenance is essential to manage the hyperglycemic condition and associated complications in type 2 diabetes patients [8]. Postprandial hyperglycemia in type 2 DM patients can be controlled by inhibiting metabolic enzymes such as α-amylase, α-glucosidase, dipeptide peptidase-IV, gut-derived peptide hormones (incretins), and glucagon-like peptide-1 hormone. The glucose-dependent insulinotropic peptide, aldose reductase, angiotensin-converting enzyme, and protein tyrosine phosphatase 1B are involved with diabetic complications [9].
Alpha-amylase and alpha-glucosidase are exo-acting glycoside hydrolase enzymes involved in carbohydrate digestion. Alpha-amylase is involved in the digestion of long-chain carbohydrates, while alpha-glucosidase catalyzes the end step hydrolysis of starch or disaccharides into simple glucose units. Therefore, inhibitors of these enzymes delay glucose absorption, reducing the postprandial blood glucose level [10].
Dipeptide peptidase-IV is a protease enzyme involved in the degradation of incretins, a group of metabolic hormones that stimulate ß cells of Langerhans' islet to release insulin. Incretins are released after nutrient intake, and they delayed gastric emptying and decrease glucagon secretion in addition to stimulation of insulin secretion [11]. Contrarily, the incretin effect on insulin secretion gradually decreases once the patient becomes euglycaemic [12]. Hence, inhibitors of dipeptide peptidase IV are efficient therapeutic means to reduce the degradation of incretins, which help maintain hyperglycemic conditions in type 2 DM.
Similarly, aldose reductase is a rate-limiting enzyme involved in the polyol pathway, which catalyzes glucose reduction into sorbitol in an NADPHdependent pathway. As the aldose reductase has broad substrate specificity, it binds with glucose and converts it into sorbitol once the hexokinase is saturated and the blood glucose level is high. As a result, produced sorbitol is accumulated within the cells and creates an osmotic effect, leading to cataracts and diabetic neuropathy [13,14]. Thus, aldose reductase inhibitors prevent secondary diabetes complications.
Similarly, the angiotensin-converting enzyme plays a vital role in the renvital angiotensin-aldosterone system, a hormone system responsible for maintaining the blood pressure and fluid balance in the body. Angiotensin-converting enzymes convert angiotensin I into angiotensin II, a potent vasoconstrictor that mainly acts on arterioles that stimulate the release of aldosterone from the renal cortex and improve sodium reabsorption from the kidney. Therefore, activation of the renin-angiotensin-aldosterone system leads to increased blood pressure, resulting in microvascular and macro-vascular complications in patients with type 2 DM. Thus, inhibitors of the angiotensin-converting enzyme reduce the long-term microvascular and macrovascular complications by lowering the arterial and venous blood pressure [15]. A study reported by Ustundag et al. [16] confirms that angiotensin-converting enzyme activity is increased in diabetic patients compared to normal individuals.
Correspondingly, protein tyrosine phosphatase IB (PTP IB) is a negative regulator of the insulin signaling pathway that dephosphorylate tyrosine residues in insulin receptor and insulin receptor substrate-1. Which in turn reduces insulin sensitivity [17]. Hence, inhibition of the PTP IB enzyme leads to lower blood glucose levels by enhancing insulin sensitivity. The stable hyperglycemic condition in type 2DM patients leads to the accumulation of advanced glycated end products in various tissues resulting in diabetic complications such as neuropathy, nephropathy, retinopathy, and other chronic diseases [18]. Therefore, the natural compounds, which inhibit the formation of advanced glycation end products, would be a promising therapeutic target to suppress the diabetic complications associated with glycated products.

Bioactive compounds present in marine seaweeds
Marine seaweeds are categorized into three algal classes; red (Rhodophyceae), green (Chlorophyceae), and brown algae (Phaeophyceae) based on the presence of natural pigments. Phaeophyceae contains brown color fucoxanthin pigment, whereas Rhodophyceae possess red color pigments phycoerythrin and phycocyanin, and Chlorophyceae is rich in green color pigment chlorophyll. Among the three varieties, most marine algae are referred to as "edible" that can be used for human consumption. Asians and South Africans mainly consume these edible seaweeds as a promising complementary and alternative medicine [19].
Recently, marine seaweeds have been identified as a rich source of bioactive secondary metabolites with human health benefits. In particular, polyphenols, sterols, alkaloids, flavonoids, tannins, proteins, peptides, essential fatty acids, enzymes, vitamins, and pigments are extensively synthesized by marine seaweeds. These compounds exhibit significant chemical and biological properties such as anti-diabetic, antioxidant, cytotoxic, anti-fungal, anti-bacterial, anti-coagulant, anti-inflammatory, and antiproliferative activities, etc., [19,20]. The marine seaweeds are a rich source of sulfated polysaccharides (Figure 1), which have been reported to possess beneficial human effects. Fucoidan, alginates, and laminarans Pharmacognosy -Medicinal Plants are sulfated polysaccharide found in brown seaweeds and reported to exhibit anti-diabetic, antioxidant, and anti-inflammatory activities [21]. Carrageenans and agarans are sulfated polysaccharides found in red seaweeds. Similarly, ulvan is the sulfated polysaccharide found in green seaweeds [22]. The sulfated polysaccharides are known to possess anti-viral, anti-tumor, and anti-coagulant activities [23].
Marine seaweeds are rich in polyphenolic compounds, including flavonoids, bromophenols, phlorotannins, mycosprine-like amino acids, and phenolic terpenoids (Figure 2). The mycosprine-like amino acid is a small molecule with hydroxylated aromatic rings. Phlorotannins are polyphenolic metabolites found in brown seaweeds. They can be classified into six subgroups; fuhalols, phlorethols, fucophlorethols, and fucols, eckols, and carmalols based on their linkage between phloroglucinol units and hydroxyl groups. Flavonoids, bromophenol, phenolic terpenoids, phenolic acids, and mycosporine-like compounds are reported to possess antioxidant, anti-diabetic, anti-inflammatory, anti-allergic, and anticancer properties [24][25][26].  Among the bioactive compounds present in marine seaweeds, marine algaederived accessory pigments are important as they possess beneficial biological activities [27]. Fucoxanthin is the most abundant accessory pigment found in brown seaweeds and reported to have potent biological activities such as anticancer, antioxidant, anti-diabetic activities due to the presence of unusual allenic bond and a 5, 6-monoepoxide in its structure [21]. Phycobiliproteins, a water-soluble accessory pigment found in red seaweeds, can be divided into three main categories; phycocyanins, allophycocyanins, and phycoerythrin. Phycoerythrins are abundantly found in red seaweeds and reported to possess immuno-modulating and anticancer activities. Similarly, chlorophylls are found in green seaweeds and are said to have antioxidant activity [27].
Similarly, marine algae-derived peptides have been identified to possess a wide range of biological activities such as antioxidant, anti-diabetic, anti-microbial, antihypertensive properties, etc. Hence, most algal-derived proteins have been widely used in food and pharmaceutical industries [28]. The protein content of the marine seaweeds differs depending on the seasonal period and type of species. The brown seaweeds usually contain low protein content compared to the red and green seaweeds. Despite this, some brown algal species such as Choonospora minima, Padina gymnospora, Dictyota menstrualis, and Sargassum vulgare possess high protein content up to 10-15%. According to the reported studies, green seaweed contains an average protein content level, ranging between 10-26%. In contrast, the highest protein content was reported in red seaweeds such as Phorphyra tenure and Palmaria palmata, which was around 47% [29].

Anti-diabetic potentials of marine algae
Marine seaweeds have been widely studied for their anti-diabetic potential through different mechanisms due to bioactive secondary metabolites. Several in-vitro and in-vivo studies have been conducted so far to confirm the hypoglycemic effect of marine algae in addition to its ability to suppress diabetic complications. This section emphasizes the anti-diabetic potential of marine brown, red, and green seaweeds through diverse mechanisms.

b. Green seaweeds
Green seaweeds belong to the genus "Ulva." They have been reported to possess hypoglycemic activity, and they have been used for various food dishes in Asians due to the presence of high soluble fiber content. The aqueous extract of green seaweeds Ulva lactuca (Inhibition-α-amylase: 83.4%; α-glucosidase: 61.81%) and Ulva reticulate (Inhibition-α-amylase: 89.1%; α-glucosidase: 76.02%) were effective against α-amylase and α-glucosidase enzymes at a concentration of 100 μg/ml after 8 hours of extraction period at 37 °C in a water bath as it gets more time to release the phytochemicals and colloids to the extract [36]. Similarly, the crude extract of Ulva ohnoi exhibited α-amylase inhibition by 41.7% and complete α-glucosidase inhibition at 10 mg/mL [37].
The methanol extract of a green seaweed Chlorodesmis inhibited α-amylase enzyme by 72% at 500 μg/ml with IC50 of 408.9 μg/ml without any effect on α-glucosidase enzymes. Similarly, chloroform extract of Chaetomorpha aerea exhibited a potent inhibitory effect on α-amylase enzyme with IC 50 of 147.6 μg/ml. Besides, methanol extract of green seaweeds Enteromorpha intestinalis (59%) and Cladophora rupestris (14%) exhibited a moderate and lower effect on the α-amylase inhibitory activity at a concentration of 500 μg/ml [38]. Moreover, crude extracts of green seaweeds Derbesia tenuissima and Oedogonium intermedium were reported to exhibit lower α-amylase (53.6% and 49.2%) and potent α-glucosidase (73.98% and 69.5%) inhibitory effect at a concentration of 10 mg/ml [39]. Further studies reported that the green seaweed Chlorella pyrenoidosa could suppress the hyperglycaemic condition by inhibiting α-amylase and α-glucosidase enzymes. Besides, a green seaweed Cladophora rupestris has been reported to exhibit a hypoglycemic effect through α-amylase and α-glucosidase inhibitory mechanisms [40].

Inhibitory activity of dipeptidyl peptidase-IV (DPP-IV)
Dipeptidyl peptidase-IV (DPP-IV) is an enzyme involved in the degradation of incretin hormones, maintaining postprandial blood glucose levels. Among three types of seaweeds, brown seaweeds have been extensively reported to possess a dipeptidyl peptidase-IV (DPP-IV) inhibitory effect compared to red and green seaweeds [42].

a. Brown seaweeds
The brown seaweeds Padina sulcata, Sargassum binderi, and Turbinaria conoides have been reported to exhibit a potent inhibitory effect on the DPP-4 enzyme in a dose-dependent manner. The maximum inhibitory effect of Padina sulcata, Sargassum binderi, and Turbinaria conoides were observed as 83.09%, 81.75%, and 76.20%, at a concentration of 10 mg/ml, respectively. Further, crude water extracts of the above three brown seaweeds could secrete glucagon-like peptide-1 (GLP-1) to a greater extent than prevent hyperglycaemic conditions [42]. Similarly, ethyl acetate: methanol fraction (IC50: 0.013 mg/ml) of Sargassum wightii has been reported to exhibit an inhibitory effect on DPP-4 enzymes compared to the standard drug diprotein-A (IC50: 0.007 mg/ml) [43]. The methanol extract of Turbinaria ornate exhibited a strong inhibitory effect on the DPP-4 enzyme by 55.4% at 80 μg/ml than the standard drug diprotin A (65%) might attribute to the presence of fucoids and sulfated polysaccharides in T. ornata [44].

b. Green seaweeds
The previous study conducted by Chin et al. [42] reported the inhibitory activity of green seaweed Halimeda macroloba on the DPP-4 enzyme. Halimeda macroloba inhibited the DPP-4 enzyme by 60.53% at a 10 mg/ml concentration compared to the positive control Berberine (75.92% at 1 mg/mL). Moreover, crude water extract of H. macroloba was able to stimulate glucagon-like peptide-1 (GLP-1) secretion.

c. Red seaweeds
The sulfated polygalactans isolated from red seaweeds Kappaphycus alvarezii and Gracilaria opuntia have been reported to possess the inhibitory effect on the DPP-4 enzyme. According to the results, sulfated galactans isolated from Gracilaria opuntia (IC50 0.09 mg/mL) significantly inhibited the DPP-4 enzyme than the sulfated galactans of Kappaphycus alvarezii (IC50 0.12 mg/mL) compared to the standard diprotin A (IC50 1.54 mg/mL). The observed activity might be due to the reaction between functional groups of sulfated polygalactan with DPP-4 by H-bonding and hydrophilic interactions [45]. Similarly, aqueous, alkaline, and a mixture of aqueous/alkaline fractions of a red seaweeds Palmaria palmate have exhibited a potent inhibitory effect on DPP-4 enzyme with IC 50 of 2.52 ± 0.05 mg/ml, 4.60 ± 0.09 mg/ ml, and 4.24 ± 0.02 mg/ml respectively [46]. Further studies reported that the red seaweed Palmaria palmate's protein hydrolysate had a potential inhibitory effect on the DPP-4 enzyme [40]]. These results confirmed the possible inhibitory effect on DPP-4 enzymes of red seaweed extracts.

Inhibitory activity of aldose reductase (AR) a. Brown seaweeds
The ethyl acetate fraction of brown seaweed, Ecklonia stolonifera has been reported to possess a strong inhibitory effect on aldose reductase enzymes due to the presence of phlorotannins such as 7-phloroeckol and 2-phloroeckol in ethyl acetate fraction [47]. Similarly, phlorofucofuroeckol-A isolated from Eisenia bicyclis exhibited a potent inhibitory effect of aldose reductase enzyme (IC50: 6.22 μM). They also confirmed the inhibitory effect of fucosterol in the rat lens. Carotenoids isolated from Ecklonia stolonifera exhibited potent inhibitory activity on aldose reductase enzyme (IC50: 18.94 μM) compared to the standard positive control quercetin (IC50: 1.34 μM). The presence of porphyrin derivatives (pheophytin-A and pheophorbide-A) in the dichloromethane fraction of Saccharina japonica caused excellent inhibitory effects on aldose reductase (AR) in rat lens [ [48]. Moreover, fucoxanthin isolated from Undaria pinnatifida and Eisenia bicyclis reported acting as a competitive inhibitor on the aldose reductase enzyme [49].

b. Green seaweeds
The chloroform and ethanol fractions of green seaweed, Capsosiphon fulvescens showed a potent inhibitory action on the AR enzyme [50]. The authors further carried out isolation of compounds, and the isolated compounds (capsofulvesin A, B, and chalinasterol) demonstrated high inhibitory action on AR enzyme with IC 50 values of 52.53, 101.92, and 345.27 μM, respectively.

b. Green seaweeds
Several studies have been reported to elucidate the anti-diabetic potential of green seaweeds by enhancing insulin sensitivity through the mechanism of PTP 1B inhibition. Among the marine green seaweeds, Crude chloroform and methanol extract of a green seaweed Derbesia marina has been reported to exhibit an inhibitory effect on PTP 1B enzyme by 61.7% and 80.65 respectively at a concentration of 15 μg/ml. Further, the crude chloroform and methanol extract of edible green sea lettuce "Ulva pertusa" has exhibited potent PTP 1B inhibition at 15 μg/ml by 25.8% and 48.1%, respectively. Similarly, the crude chloroform and methanol extract of Enteromorpha linza (42.1%:35.4%) and Codium adhaerens (51.5%:71.2%) increased insulin sensitivity by inhibiting PTP 1B enzyme at 15 μg/ml concentration [52]. Further, the compounds isolated from the green seaweeds belonged to the genus "Caulerpa" had a potent anti-diabetic effect by the mechanism of PTP 1B inhibition. Racemosin C, Caulerpin, Caulerpic acid isolated from Caulerpa racemosa, and Caulersin isolated from Caulerpa serrulata have reported significant PTP1B inhibitor [55].

c. Red seaweeds
The red seaweeds have been widely studied to elucidate the inhibitory effect on angiotthe ensin-converting enzyme, as it plays a crucial role in regulating blood pressure. According to the recorded studies, the aqueous extract at 20 °C of red seaweeds Lomentaria catenata, Lithophyllum okamurae, Ahnfeltiopsis flabelliformis, and Gracilaria textorii significantly inhibited the angiotensin-converting enzyme by 98.92%, 89.23%, 73.45%, and 65.40% at a lower concentration of 200 μg/ml. Similarly, the methanol extract at 700C of red seaweeds, Ahnfeltiopsis flabelliformis, and Laurencia okamurae has been reported to exhibit a strong inhibitory effect angiotensin-converting enzyme by 97.59% and 78.01% at a concentration of 200 μg/ml. Further, the methanol extract at 70 °C of red seaweeds Grateloupia filicina, Sinkoraena lancifolia, Grateloupia elliptica, Grateloupia lanceolata, and Laurencia okamurae exhibited an inhibitory effect on ACE by 83.14%, 80.86%,68.13%, 89.04%, and 69.80% at 200 μg/ml of concentration [59]. This study revealed the presence of ACE like inhibitors in red seaweeds. Protein-derived hydrolysate in Palmaria palmate (red seaweed) showed marked antihypertensive activity. The antihypertensive activity was exerted via inhibition of angiotensinconverting enzymes [46]. Further, an enzymatic hydrolysate of a red seaweed Pyropia columbina exhibited an inhibitory effect on the angiotensin-converting enzyme with an IC50 value of 1.2 mg/ml [58].

b. Green seaweeds
So far, minimal studies have been reported to demonstrate the inhibitory effect of green seaweeds on the formation of advanced glycation end products. The chloroform, ethanol, and butanol fractions of a green seaweed Capsosiphon fulvescens have been reported to exhibit an inhibitory effect on the formation of advanced glycation end-products [50].

c. Red seaweeds
Regarding the red seaweeds, the ethyl acetate fraction (IC 50 : 586.54 μg/ml) of Gracillaria edulis has been reported to exhibit the inhibitory effect on the formation of advanced glycation end products compared to the standard drug rutin (IC 50 : 11.55 μg/ml) [34]. Similarly, carrageenan extract from red algae could inhibit progressive glycation end product uptake by macrophage-like RAW 264.7 cells [63].

Conclusions
Recently, marine seaweeds have been extensively studied for their therapeutic effects due to promising bioactive compounds. Among the non-communicable diseases, diabetes mellitus is the third leading cause of death associated with vascular complications. As it is a progressive disorder, it is necessary to search for an adequate drug for natural resources with minimum side effects. Therefore, this chapter illustrates the different anti-diabetic mechanisms of marine seaweed extracts and their bioactive compounds.