Phytopharmacological Strategies in the Management of Type 2 Diabetes Mellitus

Type 2 Diabetes Mellitus (T2DM) is a chronic disease which corresponds to 90% of the worldwide cases of diabetes, mainly due to epigenetic factors such as unhealthy lifestyles. First line therapeutic approaches are based on lifestyle changes, most of the time complemented with medication mostly associated with several side effects and high costs. As a result, the scientific community is constantly working for the discovery and development of natural therapeutic strategies that provide lower financial impact and minimize side effects. This review focus on these nature-based therapeutic strategies for prevention and control of T2DM, with a special emphasis on natural compounds that present pharmacological activity as dipeptidyl peptidase-4 (DPP4), alpha-amylase, alpha-glucosidase, lipase, and protein tyrosine phosphatase 1B (PTP1B) inhibitors.


Introduction
Diabetes mellitus (DM) is a chronic disease characterized by excessive concentration of sugar glucose in the bloodstream, a pathophysiological sign termed: hyperglycemia [1]. There are two main types of DM: type 1 (T1DM) caused by the absence of insulin production due to auto-immune mediated loss of pancreatic β-cells and type 2 (T2DM), which results from the deficient action of insulin, triggering the aberrant synthesis of hepatic glucose, secretion deviations, and insulin resistance in target tissues (liver, muscle, and adipose tissue), with consequent progressive deterioration of pancreatic β-cells functions [1][2][3]. Patients with T2DM are not insulin dependent, unlike those with T1DM, as long as lifestyle interventions and oral hypoglycemic agents are sufficient for effective glycemic control [1,3,4]. Accounting for about 90% of the worldwide cases of DM, and the sixth leading cause of disability, T2DM is clinically detected mainly by the 3 Ps: polyuria, polydipsia, and polyphagia, as well as body weight loss, distorted vision, and fatigue [1,[3][4][5][6][7]. The disease can be attributed, on the one hand, to behavioral/environmental factors, and, on the other hand, to not fully understood genetic factors with an influence on β-cells [2,[7][8][9]. Nevertheless, the main risk factors for the development of T2DM are oxidative stress, lack of exercise, obesity, and unhealthy diet [2,9]. Inadequate glycemic control can lead to an array of microvascular (e.g., retinopathy, nephropathy, neuropathy) and macrovascular (e.g., cardiovascular diseases such as stroke and heart attack) complications [10]. Thus, it is fundamental to develop effective strategies to restore and maintain blood glucose homeostasis. The aim of this review is to summarize some of the natural therapeutic strategies for prevention and control of T2DM, with a special emphasis on natural compounds that present pharmacological inhibitory activity against dipeptidyl peptidase-4 (DPP4), alpha-amylase, alpha-glucosidase, lipase, and protein alpha-amylase, alpha-glucosidase, lipase, and protein tyrosine phosphatase 1B (PTP1B). These natural inhibitors include several classes of compounds such as bromophenols, phlorotannins, sterols, terpenes, stilbenoids, flavonoids, furans, catechols, and fungal metabolites, among others. The structures of some of the natural compounds mentioned across this review are represented in Figure 1.

Conservative Treatment
The treatment of T2DM safeguards patient-centered therapeutic individualization and is initiated by the alteration of the individual lifestyle, counterworking sedentarism, and obesity through the increase of physical activity and adoption of a balanced diet [11]. However, with progressive decline of pancreatic β-cells function, medication is required generally for extended periods of time [1,[11][12][13]. The pharmacologic therapies are mainly based on increasing insulin availability either by direct administration of insulin or via agents promoting insulin secretion, improving insulin sensitivity, delaying gastrointestinal absorption of carbohydrates, and/or increasing glucose excretion [14]. The administration of insulin allows glycemic control, but is related to weight gain due to an increase in body fat mass, especially abdominal obesity, with consequent increase in insulin resistance, as well as episodes of hypoglycemia when the treatment is not performed properly [14].

Lifestyle Interventions: Diet and Physical Activity
Diet influences body weight, glucose, and insulin homeostasis being recognized as a risk factor for the development of T2DM [15,16]. In fact, there are several studies that verify the capacity of prevention and control of metabolic diseases by the food or by specific substances in the diet [16]. There is unanimity on the importance of body weight control, reduction of energy intake coupled with exercise, and healthy diet with low intake of processed foods (rich on refined sugars and flour) and high consumption of whole grains, fiber, polyunsaturated fatty acids, fruits, vegetables, and low-fat dairy products for the control and prevention of T2DM [2,9,16].
Processed red meat belongs to the group of foods to be avoided by the patient with T2DM, although the effect of unprocessed red meat on the pathology is not fully known [16]. The group of forbidden foods for those with T2DM also includes refined grains and sugars (high glycemic index). Preference should be given to the consumption of whole grains (low glycemic index), and, above all, fiber, with a higher consumption being recommended for T2DM patients (50 g per day) than for healthy individuals (30 g per day) [1,16]. Dietary fiber derives from plants and is not hydrolyzable by human digestive enzymes, but is digested by intestinal microflora. Dietary fibers are divided into soluble (e.g., β-glucans, pectins and some hemicelluloses) and insoluble (e.g., cellulose, some hemicelluloses and lignin) [17]. With the exception of lignin, the set of soluble and insoluble fiber, it is called non-starch polysaccharides (NSPs) [17]. When NSPs are ingested and mixed with water, they form a dispersion with an increase in the viscosity of the bolus, reducing the diffusion of digestive enzymes and promoting the sense of satiety, resulting in the fight against obesity, as well as prevention and control of T2DM [12].
On the other hand, there are foods and eating habits whose effect on T2DM remains a case study. Low-calorie and low-carbohydrate diets have beneficial effects on T2DM control, but there is no consensus for the optimal calorie intake of macronutrients in both diets [16]. According to several studies, fish consumption and the risk of developing T2DM is considered positive, inverse, or absent depending on geographical location and other factors that influence the type of fish consumed, preparation/confection methods, and contaminants (e.g., methylmercury) [16]. Nevertheless, the consumption of fish oil is recommended for a positive effect on lipoproteins and prevention of cardiac coronary diseases, being a subject of debate the discouragement the supplementation of ω-3 in diabetics [16]. Dairy consumption is recommended in T2DM prevention, especially fermented as yogurt, although the promotion of low-fat dairy products in this population is debatable [16]. Tropical and plant oils are also subjects of study in T2DM prevention and control, with evidence supporting the benefit of olive oil as part of the Mediterranean diet [16].
Regardless of the effects on body weight, physical inactivity is identified as an independent risk factor for T2DM that can be reduced by 20-60% with regular physical activity in a dose-response manner [8]. Glucose is transported by proteins called GLUT (Glucose Transporter), with GLUT4 being the predominant isoform in muscle, modulated by insulin and muscle contractions [18]. Insulin activates the intracellular transport of GLUT4 to the cell membrane through a complex signaling sequence, which is usually compromised in individuals with T2DM, but is stimulated during aerobic or anaerobic exercise [18]. Exercise allows for the increase of GLUT4 by muscle contractions and increased glucose uptake, which is corroborated with the normal values of glucose absorption in patients with T2DM submitted to exercise protocols [18]. Glycogen is the first source of energy for exercise through glycogenolysis, resorting to plasma glucose absorption and a release of free fatty acids when the first source of energy runs out [18]. In long-term activities, glycemic control is achieved by the use of intramuscular lipid reserves [18]. In healthy subjects during moderate/intense exercise, increased peripheral glucose uptake is accompanied by increased hepatic glucose output, allowing for the maintenance of plasma glucose values, except during prolonged exercise [18]. In subjects with T2DM, during moderate exercise, glucose uptake by muscles increases more strongly than their production, leading to a decline in plasma glucose levels [18]. However, the risk of hypoglycemia reduces in non-medicated individuals with insulin or their secretagogues, due to the decrease in plasma insulin levels, even in prolonged exercise [18]. The action of aerobic exercise on insulin effect varies with duration, intensity and subsequent diet, where a single session increases the action and glucose tolerance between 24 h and less than 72 h [18]. However, brief and intense aerobic exercise increases catecholamine plasma levels, causing an increase in glucose production, which can result in hyperglycemia for 1 h to 2 h since levels do not return to normal immediately after the activity ends [18].

Adiponectin
Adiponectin (Acrp30, AdipoQ, GBP-28, or apM1) is an endocrine factor, mainly secreted by the adipose tissue, but also by skeletal and cardiac myocytes and endothelial cells, with direct actions in the liver, skeletal muscle, and vasculature [19]. It exists in the circulation as varying molecular weight forms produced by multimerization, where the high-molecular weight complexes have apparent predominant action on metabolic tissues [19]. Adiponectin administration in human and rodents has insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects with possible decreases of body weight [19]. In fact, low plasma adiponectin concentrations are associated with obesity and T2DM regardless of ethnic groups, where hypoadiponectinemia is more closely related to the degree of insulin resistance and hyperinsulinemia than the degree of adiposity and glucose intolerance [20]. As a result of the importance of adiponectin levels in the pathology, studies have been conducted aiming the discovery of new natural sources which promote adiponectin release. Some of these studies are reviewed by Ríos and colleagues, reporting different natural products for the treatment of T2DM from medicinal plants such as Ipomoea batatas, Aronia melanocarpa, and Salacia reticulate, as well as from mushroom Agaricus blazei [21].

Lipase Inhibition
Fat digestion involves gastrointestinal enzymes like pre-duodenal lipases (lingual and gastric lipases), pancreatic lipase, cholesterol-ester lipase, and bile-salt stimulated lipase [22]. Triglycerides are the most dietary fat ingested (90-95%). Their hydrolysis starts in the mouth, followed by acid stable gastric lipase in the stomach and synergistic action of gastric and colipase-dependent pancreatic lipase in duodenum [22]. As a result, monoglycerides and free fatty acids are formed, the latter being absorbed by enterocytes to synthetize new triglyceride molecules, which are transported by lipoproteins (chylomicrons) to different organs after a meal [22]. Pancreatic lipase is responsible for the hydrolysis of 50-70% of total dietary fats, highlighting it as the main lipid digesting enzyme [22,23].

Pharmacological Approach
The inhibition of lipase leads to restored insulin production from β-cells protecting pancreas through decrease of lipid absorption. Orlistat is the most prescribed synthetic drug for the pathology, but with several side effects reported including steatorrhea, bloating, oily spotting, fecal urgency, fecal incontinence, and hepatic adverse effects [22,24,25]. Aditionally, the inhibition of fat absortion results in the need of vitamin supplementation because of the defficiency of fat-soluble vitamins in patients undergoing orlistat theraphy [22].

Naturally-Occurring Lipase Inhibitors
Considering the numerous side effects related to the use synthetic lipase inhibitors, interest in the search for new natural inhibitors against pancreatic lipase is growing. Some of them are summarized in Table 1 and described below.  5-methoxy-7-hydroxy-9,10-dihydro-1,4-phenanthrenequinone isolated from a methanol extract of the whole plant of Dendobium formosum at 50 µg/mL is related with non-competitive inhibition of alpha-glucosidase (96%) and pancreatic lipase (83%), reveling IC 50 (half maximal inhibitory concentration) values of 126.88 ± 0.66 µM, and 69.45 ± 10.14 µM, respectively [24].
Oxalic and furoic acid, which are compounds derived from the oxidation of vitamin C to dehydroascorbic acid, were investigated for their inhibitory activity against pancreatic lipase [37]. In this study, furoic acid and oxalic acid revealed IC 50 values of 2.12 ± 0.04 and 15.05 ± 0.78 mM, respectively, where the ultraviolet (UV) wavelength scanning and fluorescence quenching experiments proved that furoic acid inhibition was stronger than that of the other compound [37]. Both acids presented reversible inhibition with non-competitive and competitive action by furoic and oxalic acid, respectively [37]. Other compounds evaluated for their potential as lipase inhibitors include terpenes, like carnosic acid isolated from methanol extract of Salvia officialis at 36 µM (IC 50 = 12 µg/mL) and bioactive-guided fractionation of this plant with isolation of carnosol, roylenoic acid,7-methoxyrosmanol, and triterpene oleanolic acid with IC 50 values of 4.4; 35; 32; and 83 µg/mL, respectively [54]. Other terpenes include crocin from Gardenia jasminoids with an IC 50 value of 28.63 µmol [54].

Polyphenolic Compounds-Fruits, Vegetables and Plants
The beneficial effect of polyphenols and their subgroups on lipid metabolism is extensively reported on literature. Flavonoids are the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants, mainly on leaves or shells, with the function of protecting them from harmful external influences [55]. Flavonoids can be classified, based on the degree of the oxidation of the C-ring, the hydroxylation pattern of the ring structure and the substitution of the C3-position, into: chalcones, dihydrochalcones, aurones, flavones, flavonols, dihydroflavonoles, flavanones, flavanols, anthocyanidins, leucoanthocyanidins, proanthocyanidins, bioflavonoids, and isoflavonoids [55]. On the other hand, phenolic acids occur as free fatty acids, esters, glycosides, or bound complexes, with antioxidant, anticarcinogenic, and antimicrobial activity, where hydroxycinnamic acids are phenolic compounds with major importance for secondary metabolism in plants occurring in fruits (apples, blueberries), vegetables (spinach, lettuce, potatoes), coffee, and cereals [55]. By-products rich in polyphenols from winemaking, were treated with pronase and viscozyme to improve the solubility of phenolics by Camargo and colleagues [48]. The study revealed that the inhibition of soluble phenolics against alpha-glucosidase and lipase increased from 75.6% ± 2.5% to 93.7 ± 0.5% and from 35.2 ± 0.2% to 45.5 ± 1.2%, respectively, in samples treated with pronase and from 84.5 ± 0.5% to 96.5 ± 2.9% and from 86.2 ± 0.3% to 94.3 ± 1.5%, respectively, with viscozyme [48]. Polyphenol-rich extracts of six common bean cultivars (Phaseolus vulgaris) showed inhibitory activity against alpha-amylase (IC 50 values ranged from 69 ± 1.9 to 126 ± 3.2 µg/mL and from 107.01 ± 4.5 to 184.20 ± 5.7 µg/mL before and after cooking), alpha-glucosidase (IC 50 values ranged from 39.3 ± 4.4 to 74.13 ± 6.9 µg/mL and from 51 ± 7.7 to 122.1 ± 5.2 µg/mL before and after cooking) and pancreatic lipase (IC 50 values ranged from 63.11 ± 7.5 to 103.2 ± 5.9 µg/mL and from 92 ± 6.3 to 128.5 ± 7.4 µg/mL before and after cooking) [38]. Ethanol and methanol extracts from Lovi (Botoko plum) from Flacourtia inermis, found in Sri Lanka exhibited inhibitory activity against alpha-glucosidase (IC 50 from 549 to 710 ppm), alpha-amylase (IC 50 from 1021 to 1949 ppm) and lipase (IC 50 from 1290 to 2096 ppm), where (S)-malic acid was characterized as the active principle for this inhibition effect [40]. Additionally, this fruit has great polyphenol (1.28 g gallic acid equivalents per 100 g of fresh fruit) and anthocyanin (108 mg cyaniding-3-glucoside equivalents per 100 g fresh fruit) content [40]. Acacia polyphenol extracted from the bark of the black wattle tree (Acacia mearnsii) was tested for its in-vitro inhibitory activity against lipase and glucosidase, as well as its effects on absorption of orally administered olive oil, glucose, sucrose, and starch solution in ICR mice [41]. The results showed that acacia polyphenol inhibited the activity of lipase (IC 50 = 0.95 mg/mL), maltase (IC 50 = 0.22 mg/mL) and sucrase (IC 50 = 0.60 mg/mL) and inhibited the rise plasma triglyceride concentration after olive oil loading, rise in plasma glucose concentration after maltose (more potent), glucose and sucrose loading [41]. Water extract of Juglans mandshurica strongly inhibited pancreatic lipase in-vitro (IC 50 = 2.3 mg/mL for 50% of inhibition) with further evaluation of this activity in isolated compounds reveling potent inhibitory effect of [42]. Simão and colleagues tested the inhibitory potential of an aqueous extract from the leaves of three cultivars of Psidium guajava (guava) on α-amylase, α-glucosidase, lipase and trypsin enzymes. These studies were done in the presence or absence of simulated gastric fluid and the content of phenolic compounds was determined [43]. The study revealed that all cultivars showed the same phenolic composition but in different proportions, where catechin was the major compound in Paluma cultivar, epigallocatechin gallate, and revesterol in Pedro Sato and syringic acid, o-coumaric acid, and quercetin for Século XXI [43]. The enzyme inhibition occured in major proportion for Século XXI cultivar against alpha-amylase before the exposure to gastric fluid (14410.60 ± 38 inhibited enzyme unit in µmol·min −1 ·g −1 ) and Paluma against alpha-glucosidase before (28.82 ± 0.02 inhibited enzyme unit in µmol·min −1 ·g −1 ) and after exposure (2.59 ± 0.06 inhibited enzyme unit in µmol·min −1 ·g −1 ) [43]. Pedro Sato revealed the highest inhibitory effect against lipase before (36.45 ± 0.68 inhibited enzyme unit in µmol·min −1 ·g −1 ) and after exposure (43.33 ± 1.80 inhibited enzyme unit in µmol·min −1 ·g −1 ) [43]. The three cultivars presented an inhibitory effect against trypsin activity before exposure, with significant reduction after exposure ranging from 84.88% for Paluma and 91.02% for Pedro Sato [43]. The investigators concluded that the inhibition of digestive enzymes could probably be explained by the presence of phenolic compounds in the cultivars aqueous extracts [43].
Condensed tannins called proanthocyanidins are found in the insoluble fraction of plant-derived foodstuffs (e.g., grape seed and skin, red wine, apples), where type-B are the more abundant and type-A are abundant in vegetables like peanut skin, avocados, and cranberries [49]. When the degree of polymerization of proanthocyanidins is >3, they are poorly absorbed in the intestine, reaching the colon as substrate for specific bacteria of the resident microbiota, contributing to the modulation of the composition of the colonic microbiota [49]. In the lumen of the small intestine, high molecular-weight condensed tannins may interfere with macronutrients, bile salts, mucosal alpha-glucosidase, and pancreatic enzymes (alpha-amylase, lipase and proteases) decreasing nutrient digestibility, being frequently considered as antinutrients [49]. In fact, grape extract exhibits inhibitory activity against lipase with an IC 50 value of 8.6 ± 1.1 mg/mL [49].

Alginates from Algae
Alginates are dietary fibers found in the cell walls of brown seaweed and certain bacteria and comprised of mannuronic and guluronic acid. Wilcox and colleagues found that alginate inhibited pancreatic lipase by a maximum of 72.2 ± 4.1% with synthetic substrate (DGGR) and 58 ± 9.7% with olive oil as substrate, where High-G alginates from Laminaria hyperborea seaweed has shown the most potent inhibitory activity than High-M alginates from Lessonia nigrescens, revealing that the alginate structure is related to the inhibition [56].

PTP1B Inhibition
The enzyme responsible for the reversal insulin receptor auto phosphorylation is a tyrosine phosphatase known as PTP1B (protein tyrosine phosphatase 1B), where inhibition results in a prolonged insulin signaling cascade, increasing insulin sensitivity [57]. However, low selectivity over the other protein tyrosine phosphatases, ubiquitously expressed, and poor cell permeability are two major challenges in the discovery of efficient inhibitors [58]. Nevertheless, there are potential PTP1B inhibitors from natural sources, some of these being described in Table 2.

Incretins and DPP4
Incretins are hormones produced by intestinal enteroendocrine cells on ingestion of glucose [2,11]. Incretins such as GIP (glucose-dependent insulinotropic polypeptide) and mainly GLP-1 (glucagon-like peptide-1) are responsible for the incretin effect, resulting from the observation that oral glucose is more effective in promoting insulin secretion than intravenous glucose [14]. Thus, 70% of postprandial insulin is secreted by pancreatic β-cells as a response to this incretin effect, being both this effect and physiological activity of the incretins reduced in T2DM [2,11,63]. GLP-1, in addition to insulin-tropic action in response to high concentrations of glucose, allows weight loss through the mediation of satiety and reduction of gastric emptying rate, being also responsible for the suppression of glucagon secretion by pancreatic α-cells in a glucose-dependent process, allowing the cessation of hepatic glucose secretion [14,63]. When plasma glucose levels return to normal, the inhibitory effect under alpha-cells ceases, preventing hypoglycemia [63]. GIP, like GLP-1, allows for the increase of the insulin secretion and inhibition of the glucagon discharge, emphasizing this by stimulating the secretion of glucagon during hypoglycemia [63]. GLP-1 and GIP are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP4) widely distributed throughout the body [64].

Pharmacological Approach
Active GLP-1 has a half-life of 1-2 min and its inhibition constitute an efficient pharmacological approach for T2DM treatment [4]. In fact, a new class of drugs has emerged, which allows the stimulation of endogenous insulin secretion, preventing the rapid degradation of incretin hormones, through the inhibition of DPP4 [65]. DPP4 inhibitors, or gliptins, are effective as monotherapy and in combination therapy, allowing the reduction of HbA1c without causing hypoglycemia or weight gain [11]. Gliptins can be classified as peptidomimetics (designed to mimic the N-terminal dipeptide that is cleaved by DPP4) such as Vildagliptin and Saxagliptin, or non-peptidomimetics such as Linagliptin and Sitagliptin [66]. The most widely antihyperglycemic agent prescribed worldwide is metformin, despite its association with vitamin B 12 deficiency and contraindication in patients with chronic kidney disease [4,5]. Another class of drugs based on the incretin effect refers to GLP-1 analogues, namely exenatide, which demonstrates statistically significant body weight reduction when compared to insulin/placebo [14]. Besides the existing pharmacological strategies, side effects and contraindications reveal the need for new natural therapies for inhibition for DPP4 [67]. In fact, the prolonged usage of these medications causes side effects such as pancreatitis, angioedema, infective disorders, pancreatic cancer, thyroid cancer, and severe joint pain [68], increasing interest of the scientific community for the development of natural inhibitors (Table 3). Gly-Pro-Ala-Glu IC 50  Pro-Ala-Cys-Gly-Gly-Phe-Tyr-Ile-Ser-Gly-Arg-Pro-Gly IC 50    Ile-Pro-Ala IC 50

µM
Ile-Pro-Ala-Val-Phe IC 50     Dietary proteins have been increasingly recognized as precursors of a variety of bioactive peptides, improving various aspects of human health [74]. These bioactive peptides are present in inactive forms in food, being activated once released from the proteins by enzymatic or acid hydrolysis, microbial fermentation or processing methods, and their biological activity is determined by their native amino acid composition and sequence [74]. In-silico analyses are useful to determine the frequency of the occurrence of bioactive peptides within a dietary protein (simulation of protein hydrolysis by bioinformatic tools to calculate a number of bioactive peptides found in a given dietary protein) and binding modes by docking analysis (simulate the binding and interactions between peptides and enzymes, like DPP4, in order to evaluate the inhibitory effects of the peptides) [74]. Peptides can inhibit DPP4 with competitive (Xaa-Pro, Pro-Xaa, Xaa-Ala, and food derived peptides with proline at their P 1 position), non-competitive and uncompetitive (N-terminal with tryptophan amino acid) and mixed-type modes of action, exerting their effect by binding either at the active site and/or outside the catalytic center of the enzyme [75]. The DPP4 inhibitory activity of bioactive peptides has been associated with some structural characteristics like length, isoelectric point, hydrophobicity, and net charge of the peptides, being the most predominant factor the specific amino acid sequence [75]. Other characteristics associated with potent inhibitory effect are: branched-chain amino acid or an aromatic residue with a polar group in the side-chain (tryptophan) at their N-terminal and/or a proline residue, were C-terminal amino acid also influences its potency since both are involved in the interaction with DPP4 [75].
DPP4 is known to act on substrates with proline or other small-uncharged residues such as serine and alanine at their penultimate amino acid position [75]. Because of high content in proline residues, collagen from fish and mammals has also attracted notable attention as a potential source of DPP4 inhibitory peptides [66,75]. To date, protein hydrolysates and bioactive peptides from cow's milk have been the most extensively investigated sources of DPP4 inhibitors. In fact, according to a study with in-silico approach, caseins from cow's milk (beta-casein with an occurrence frequency value of 0.249) and collagens from bovine meat and salmon (occurrence frequency values of 0.380 and 0.305, respectively) appeared to be the richest potential sources of DPP4 inhibitors, where Gly-Ala, Gly-Pro and Pro-Gly were the most frequently occurring sequences [69]. According to a review, peptides from Atlantic salmon skin gelatin (Gly-Pro-Gly-Ala and Gly-Pro-Ala-Glu sequence), tuna cooking juice (Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr and Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg and Pro-Ala-Cys-Gly-Gly-Phe-Tyr-Ile-Ser-Gly-Arg-Pro-Gly), Japanese rice bran, Native American amaranth, and Gouda cheese also present the DPP4 inhibitory effect [66].
Peptides which were not isolated from hydrolysates but are likely to occur in the sequence of dietary proteins, were synthetically produced and studied for their effect on DPP4 activity [75]. Diprotin A (tripeptide IPI) is the most potent peptide with DPP4 inhibitory activity (IC 50 ≈ 4 µM) and can be found in the sequence of k-casein. However, not as effective as diprotin A, the peptides WR, IPIQY, and WCKDDQNPHS found in the sequence of lactoferrin, k-casein, and α-lactalbumin, respectively, are among the most potent food protein-derived DPP4 inhibitors reported to date [75]. However, these peptides could not be released from dietary proteins during digestion or enzymatic proteases, like those isolated and identified in hydrolysates and thus unavailable for inhibitory effect of DPP4 [75]. Nongonierma and colleagues tested a selection of synthetic dipeptides and milk protein hydrolysates for their DPP4 inhibitory properties, and their superoxide and 2,2-diphenyl-1-picrylhydrazyl (DDPH) radical scavenging activities [70]. The study revealed superoxide and DPPH scavenging activity and the DDP4 inhibitory effect, by dipeptide Trp-Val (EC 50 39.75 ± 0.01 mg/mL; EC 50 0.07 ± 0.01 mg/mL; IC 50 0.020 ± 0.001 mg/mL respectively) and lactoferrin hydrolysate LFH1 (EC 50 0.10 ± 0.01 mg/mL; EC 50 1.15 ± 0.40 mg/mL; IC 50 1.088 ± 0.106 mg/mL). However, the dipeptide Ala-Leu had bigger superoxide scavenging activity (EC 50 1.74 ± 0.01 mg/mL) than Trp-Val, which was the dipeptide with greater DPPH scavenging activity [70]. Besides LHF1, casein hydrolysate (CasH2) also showed a potent inhibitory effect on DPP4 (IC 50 0.882 ± 0.057 mg/mL) [70].
Polyphenolic Compounds: Fruits, Vegetables, and Plants The potential of phenolic compounds in the treatment and prevention of obesity is due to their thermogenic effects, which corresponds to the ability to oxidize fat and decrease intestinal absorption of fats and carbohydrates, resulting in the inhibition of digestive enzymes with consequent weight loss [43]. Beneficial health effects of fruits and vegetables in the diet have been attributed to their high phenolic content, such as flavonoids. With the purpose of inhibit DPP4 and PTPB1, Bower and coworkers studied the ability of greenhouse-grown and commercially purchased Greek oregano (Origanum vulgare), marjoram (Origanum majorana), rosemary (Rosmarinus officinalis), and Mexican oregano (Lippia graveolens) [57]. Greenhouse herbs were richer in polyphenols than the commercial ones. Mexican oregano and marjoram were the best inhibitors of PTP1B (32.4-40.9% at 500 µM) with cirsimaritin, naringenin, hispidulin, eriodictyol, and carnosol in their composition according to LC-ESI-MS method [57]. According to computational modeling, the last three phytochemicals have the best binding affinities for DPP4, but biochemically the best inhibitors of DPP4 were cirsimaritin (IC 50 = 0.43 ± 0.07 µM), hispidulin (IC 50 = 0.49 ± 0.06 µM) and naringenin (IC 50 = 2.50 ± 0.29 µM), found in rosemary and Mexican oregano extracts [57]. Fan and colleagues investigated the DPP4 inhibitory effect of well-characterized anthocyanins isolated from berry wine blends, and twenty-seven other phenolic compounds commonly found in citrus, berry, grape and soybean using luminescence assay and computational modeling (for the most potent compounds) [71]. Malvidin-3-galactoside and cyaniding-3-glucoside were the main anthocyanins present in blueberry wine, while delphinidin-3-arabinoside was predominant in the blackberry wine [71]. Anthocyanins from blueberry-blackberry wine blends (IC 50 = 0.07 ± 0.02 to > 300 µM) and phenolics resveratrol (IC 50 = 0.6 ± 0.4 nM), luteolin (IC 50 = 0.12 ± 0.01 µM), apigenin (IC 50 = 0.14 ± 0.02 µM) and flavone (IC 50 = 0.17 ± 0.01 µM) exhibit the most strongly inhibiting activity, where phenolics present IC 50 values lower than diprotin A (IC 50 = 4.21 ± 2.01 µM) [71]. According to computational modeling, resveratrol and flavone were competitive inhibitors and luteolin and apigenin docked in a noncompetitive manner [71].

Polyphenolic Compounds: Seaweed
Seaweed (also called algae) are simple unicellular (microalgae) or multicellular organisms (macroalgae), with rudimentary conductive tissues, presenting a high range of morphological and reproductive level variation that allows their division into different phyla and classes [76]. The presence of chlorophyll, production of the same carbohydrates, proteins and metabolic pathways, render algae biochemically similar to plants, differing in the absence of embryo and multicellular envelope around sporangia and gametangia in algae (except freshwather gren algae, charophytes) [76]. Marine macroalgae, are rich in bioactive compounds in the form of polyphenols, carotenoids, vitamins, phycobilins, phycocyanins, and polysaccharides, known for their benefits to human health [1,76]. An in-vitro assay revealed that ethanolic precipitates of Sargassum binderi, Padina sulcata, and Turbinaria conoides had inhibitory activity against DPP4 (IC 50 = 2.194; 2.306 and 3.594 mg/mL, respectively) [64]. Additionally, the same study evaluated the viability of pGIP/neo STC-1 cells by measurement of cell membrane integrity by means of the Tryptan blue exclusion assay [64]. The evaluation revealed that water extracts of S. binderi, P. sulcata, and T. conoides allowed for the stimulation of GIP secretion of 5.46; 4.92 and 5 pM GIP per million cells per hour at 2.5; 10; 2.5 mg/mL, respectively [64]. Furthermore, the butanol fraction of S. binderi and P. sulcata allows for the stimulation of GIP secretion of 56.38 and 40.67 pM, respectively, GIP per million cells per hour at 5 mg/mL [64]. Another study revealed that methanol extract of brown seaweeds Sargassum wightii and Sargassum polycystum has an inhibitory effect against DPP4 (IC 50 = 38.27 µg/mL and IC 50 = 36.94 µg/mL, respectively) and acetone extract with moderate antioxidant activity (43% and 22%) at a concentration of 1000 µg/mL according to DPPH free radical scavenging activity method [65]. The same group of investigators found that methanol extract of brown seaweed Turbinaria conoides possesses inhibitory activity against DPP4 (55.4%, IC 50 = 55.2 µg/mL) at a concentration of 80 µg/mL and significant scavenging ability on DPPH (65%) at a concentration of 1000 µg/mL of acetone extract [72].
Mangiferin is a glucosyl xanthone and is the major phytochemical in Mangifera indica (family of Anacardiaceae). It has strong antioxidant, antilipid peroxidation, immunomodulation, antidiabetic cardiotonic, hypotensive, wound healing, antihyperlipidemic, antiatherogenic, and antidegenerative properties [68]. Suman and colleagues studied the influence of mangiferin and synthetic drugs (metformin and vildagliptin) using two control groups of adult Wistar rats (normal control and diabetic control feed for 10 weeks with distilled water and a high-fat diet, respectively) [68]. The other subjects where fed for 10 weeks with a high-fat diet and subsequent streptozotocin (STZ)-induced T2DM (40 mg/kg) after 3 weeks, followed by the administration of metformin, vildagliptin or mangiferin from the fifth week to tenth week daily [68]. The study revealed a high DPP4 inhibitory effect of mangiferin (89 ± 8%) when compared with synthetic drugs (90 ± 7% for VIL and 84 ± 8% for sitagliptin) according to ELISA kit (Enzyme-Linked Immunosorbent Assay) [68]. Mangiferin permitted the reduction in blood glucose, HbA1c levels and MDA levels (marker of lipid peroxidation in organs like liver, heart and kidney) [68]. Additionally, this xanthone improves insulin sensitivity and C-peptide levels, showing a favorable effect on inflammatory markers hs-CRP [68]. Total cholesterol (p < 0.001), triglycerides (p < 0.001), LDL (p < 0.01), and atherogenic index (p < 0.01) were significantly reduced and HDL was increased (p < 0.01) in mangiferin and standard drugs treated groups [68]. Other marine sources with health benefits have been studied, such as sponges and anemones, where aqueous extracts of sponge Xetospongia muta and sea anemones Bunodosoma granulifera and Bartholomea annulata present inhibitory activity against DPP4 (0.82; 2.26 and 1.78 U/mg, respectively) [73].

Alpha-Amylase and/or Alpha-Glucosidase Inhibition
Pancreatic alpha-amylase allows the hydrolysis of carbohydrates through the breakdown of α-1,4-glycosidic bonds, forming linear and branched oligosaccharides, which are subsequently converted to glucose [12,13,65]. Such a conversion is catalyzed by the intestinal alpha-glucosidase, allowing its absorption into the bloodstream [12,13]. The inhibition of both enzymes, will allow to reduce the postprandial hyperglycemia by delayed digestion of carbohydrates and intestinal absorption of glucose [12,13,77].

Pharmacological Approach
Acarbose, miglitol and voglibose are examples of inhibitors, where the former shows an excessive inhibitory activity of pancreatic alpha-amylase with consequent abnormal bacterial fermentation of carbohydrates in the colon, resulting in side effects such as flatulence, bloating, and possible diarrhea [77]. As a result of several side effects and high costs of pharmacological control of pathology, the scientific community is constantly in the search for natural sources with inhibitory effect against alpha-amylase and alpha-glucosidase, being summarized in Tables 4 and 5, respectively, and described below.

Naturally-Occurring Alpha-Amylase and/or Alpha-Glucosidase Inhibitors
Peptides Protein hydrolysate from Chinese giant salamander (Andrias davidianus) was evaluated for its potential inhibitory activity against alpha-amylase and alpha-glucosidase, with further purification and identification of antidiabetic peptides [78]. The peptides amino acid sequences were Cys-Ser-Ser-Val, Tyr-Ser-Phe-Arg, Ser-Ala-Ala-Pro, Pro-Gly-Gly-Pro, and Leu-Gly-Gly-Gly-Asn with alpha-amylase IC 50 [78]. Garza and colleagues also reported the inhibition of alpha-amylase by valoneaic acid dilactone obtained from banaba (Lagerstroemia speciosa), ethanol extract of chestnut astringent skin, and a purified compound isolated from white beans (Phaseolus vulgaris) [22].

Conclusions
T2DM is a serious worldwide disease, occuring mainly because of an unhealthy lifestyle. Its prevention and control are achieved with changes in lifestyle, sometimes coupled with medication with several side effects and high costs associated. As a result, the search for new possible anti-hyperglycemic and anti-diabetic agents from natural sources without, or with less, side effects and at a low cost for the patient, has attracted interest from the scientific community, as shown in this review. The natural sources include extracts from plants, fruits, vegetables and specially from marine macro or microorganisms with bioactive compounds. Indeed, marine organisms such as seaweed from edible species have several compounds with modes of action involving specific mechanisms that can be employed in T2DM treatment. The bioactive compounds include polysaccharides and dietary fibers, fatty acids (MUFA and PUFA), and phenolic compounds. Taking into account that the development of T2DM takes time, the glycemic control can be prevented with the intake of healthy foods like seaweed for glycemic control as preventive measures. However, further research in the future is required to fully understand the anti-diabetic mechanisms of this type of food in the prevention and management of this pathology.