A Comprehensive Review and Perspective on Natural Sources as Dipeptidyl Peptidase-4 Inhibitors for Management of Diabetes

Type 2 diabetes mellitus (T2DM) is an increasing global public health problem, and its prevalence is expected to rise in coming decades. Dipeptidyl peptidase-4 (DPP-4) is a therapeutic target for the management of T2DM, and its inhibitors prevent the degradation of glucose-dependent insulinotropic peptide and glucagon-like peptide 1, and thus, maintain their endogenous levels and lower blood glucose levels. Various medicinal plant extracts and isolated bioactive compounds exhibit DPP-4 inhibitory activity. In this review, we discussed different natural sources that have been shown to have anti-diabetic efficacy with a particular emphasis on DPP-4 inhibition. Furthermore, the effect of DPP-4 inhibition on pancreatic beta cell function, skeletal muscle function, and the glucose-lowering mechanisms were also discussed. We believe that scientists looking for novel compounds with therapeutic promise against T2DM will be able to develop antidiabetic drugs using these natural sources.


Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic condition marked by a prolonged hyperglycemic state caused by a combination of underlying defects, which include insulin tolerance in muscle and liver, and reduced insulin production by pancreatic beta cells [1,2]. T2DM is the most prevalent form of diabetes and accounts for about 91% of all cases, and the disease has been predicted to affect about 366 million people by 2030 [3]. T2DM is marked by enhanced blood glucose (BG) levels and microvascular and macrovascular complications that substantially enhance disease-associated morbidity and mortality, and epidemiological data show that people with diabetes are at slightly higher risk of developing various types of cancer, and musculoskeletal, cardiovascular, and psychiatric disorders [4][5][6][7]. Dipeptidyl peptidase-4 (DPP-4) has emerged as a target in T2DM, and as a result, its inhibitors are attracting increased research interest. DPP-4 accelerates the degradations of the incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) by removing a dipeptide from their N termini, resulting in altered glucose homeostasis. Interestingly, DPP4 knockout mice are resistant to diet-induced obesity and have better postprandial glucose balances than their counterparts [8,9].
Insulin controls the metabolism of carbohydrates, fats, and proteins [10], and thus, any defects in insulin synthesis or its activities cause severe metabolic complications. Insulin is produced by pancreatic beta cells, and as T2DM progresses, beta cell functions decline due to rising hyperglycemia and insulin resistance. This cellular decline can start early during the course of T2DM and worsens due to compensatory overload, which accelerates disease

DPP-4 Inhibition and Pancreatic Beta Cell Function
DPP-4 antagonists provide long-term, reliable, and effective treatments for T2DM that provide strong glycemic control. GLP-1 and GIP act on G-protein coupled receptors, which are expressed on pancreatic beta and alpha cells and in peripheral tissues, to lower glucose levels [20]. GLP-1 enhances insulin secretion, insulin gene transcription, and insulin biosynthesis by acting on pancreatic beta cells [36].
T2DM causes a gradual reduction in beta cell activity, and thus, to reverse insulin secretory defects, beta cell activity must be restored. Given that GLP-1 has been reported to induce the proliferation and inhibit the apoptosis of beta cells in rodents and to induce beta cell differentiation from human precursor cells [37,38], it seems safe to assume that DPP-4 inhibition enhances GLP-1 levels, and thus, improves beta cell mass and survival. Animal studies have shown that DPP-4 inhibitor promotes islet neogenesis, beta cell regeneration, and/or improved insulin biosynthesis, and thereby preserves or increases beta cell numbers [37,39]. Accordingly, histological analysis of pancreases after DPP-4 inhibitor treatment revealed elevated numbers of islets and beta cells [37].
DPP-4 inhibition has been shown to reduce T2DM-induced beta cell dysfunction and apoptosis in in vitro and in pre-clinical studies. DPP-4 is present in mouse and human islets, and inhibiting islet DPP-4 activity has been shown to have a direct stimulatory effect on GLP-1-dependent insulin secretion [40,41]. A similar effect was demonstrated in db/db diabetic mice after 2 weeks of des-F-sitagliptin treatment, which resulted in enhanced insulin exocytosis by beta cells [42]. In addition, DPP-4 inhibition has been related to beta cell mass and functional increases in several T2DM models [43,44], and the transcriptional activations of anti-apoptotic and pro-survival genes have also been linked to these positive effects in beta cells [45]. Furthermore, the DPP-4 inhibitor linagliptin has been shown to protect isolated human islets from gluco-and lipotoxicity [46], and interestingly, vildagliptin has been shown to have antioxidant properties, as evidenced by dose-dependent reductions in nitric oxide concentrations in serum and pancreatic homogenates of diabetic rats [47].

DPP-4 Inhibitors Improve Blood Glucose Response
DPP-4 inhibitors have been shown to decrease BG levels in T2DM patients by continuous glucose monitoring (CGM) [48][49][50]. In addition, various randomized controlled trials have shown by CGM that DPP-4 inhibitors suppress BG levels more efficiently than other agents such as sulfonylureas [51,52] or sodium glucose cotransporter 2 inhibitors [53][54][55] when used in combination with insulin administration [56,57]. DPP-4 inhibitors that enhance insulin secretion and decrease prandial glucagon levels have been shown to improve BG levels [49], and reductions in prandial glucagon levels are considered to underlie improvements in BG levels by DPP-4 inhibitors [58]. Vildagliptin was reported to lower postprandial glucagon levels and improve hyperglycemia in T2DM patients [58], and DPP-4 inhibitors were observed to increase the abilities of alpha and beta cells to detect and respond to hypoglycemia [59]. Furthermore, DPP-4 inhibitors can improve both hyperglycemia and hypoglycemia [60,61]. Moreover, when DPP-4 inhibitors block persistent glucagon oversecretion [58], glucagon responds normally to a drop in BG level and ameliorates hypoglycemia [59]. Overall, these studies show that DPP-4 inhibitors can improve hypoglycemia/hyperglycemia and BG levels in T2DM patients.

DPP-4 Inhibition and Skeletal Muscle Function
Skeletal muscles (SM) comprise the largest organ in the body, and thus, process the largest amounts of administered drugs [62]. Furthermore, muscle has recently been reported to release DPP-4 [63]. SM cell cultures were observed to release DPP-4 during differentiation [64], and DPP-4 activity in the bathing medium from intact SM was found to be enhanced by whey protein in situ [65]. DPP-4 inhibitors were also found to reduce SM loss in T2DM patients [66]. Recently, sitagliptin was reported to increased muscle mass and muscle/fat ratio in T2DM [67], and natural DPP-4 inhibitors such as chrysin and galangin were reported to promote SM cell proliferation [68,69]. Furthermore, in a diabetic animal model, myricetin administration reduced DDP-4 expression in muscle [70]. Overall, these studies revealed that DPP-4 inhibitors have a positive effect on SM, increasing SM cell proliferation and the muscle/fat ratio in diabetic patients.

DDP-4 Inhibitors from Natural Sources
Nature is a plentiful source of medicinal herbs, and several natural foods that contain bioactive components with health benefits are commonly used as herbal remedies for many life-threatening diseases [71][72][73]. Herbal remedies offered a valuable resource for pharmacological agents for diabetes even before insulin and other pharmacological drugs were discovered, and have become an increasingly important aspect of searches for curative and adjunctive treatments [74]. Below, we detail plants with extracts that inhibit DPP-4 and their corresponding IC 50 values; a summary is also provided as a list in Table 2.

Castanospermum austral
Castanospermum austral (also called black bean) is an herb that grows in Australian coastal regions and rainforests. C. australe seed extract inhibited DPP-4 with an IC 50 of 13.96 µg/mL, while the control compound diprotin A had an IC 50 of 1.543 µg/mL. In addition, in a T2DM animal model, C. australe seed extract lowered BG levels, prevented hyperinsulinemia, and increased glucose tolerance [80].

Eugenia jambolana and Pterocarpus marsupium
Eugenia jambolana is an evergreen, tropical, fruit-producing tree found in South Asia and South America, while Pterocarpus marsupium is native to India, Nepal, and Sri Lanka. Both P. marsupium and E. jambolana had potent inhibitory effects on DPP-4 with IC 50 values of 273.73 and 278.94 µg/mL, respectively [82].

Chenopodium quinoa Willd
Quinoa (Chenopodium quinoa Willd) is a flowering plant of the amaranth genus Amaranthaceae. Quinoa is a gluten-free grain that has a greater protein content than other grains including wheat, rice, maize, oat, and barley. Analysis of quinoa protein hydrolysate revealed potent DPP-4 inhibitory activity (IC 50 0.88 mg/mL) [83].

Allium sativum
Allium sativum (garlic), a member of the Alliaceae family, is widely used as a spice and as a treatment for a variety of diseases and physiological conditions [84]. Its bulb extract inhibits DPP-4 activity (IC 50 70.9 µg/mL) and enhances SM cell proliferation [85].

Pilea microphylla
Pilea microphylla (the gunpowder plant) is an annual herb found in Florida, Mexico, and tropical Central and Southern America. In vitro, P. microphylla inhibited DPP-4 with an IC 50 of 520.4 µg/mL. In addition, in an HFD/streptozotocin-induced diabetic rat, P. microphylla reduced plasma glucose and prevented beta cell destruction [86].

Mangifera indica
Mangifera indica (MI) is an ayurvedic herb that belongs to the Anacardiaceae family. MI leaf extract has been shown to have hypoglycemic properties [87]. The extract of its leaves was tested in vitro for DPP-4 inhibitory activity, and the results reveal an IC 50 of 182.7 µg/mL [88]. The main phytochemical in MI is mangiferin. In HFD/streptozotocininduced diabetic rat, lower serum DPP-4 levels were associated with improved insulin resistance and improved beta cell function [89].

Coreopsis lanceolata
Coreopsis lanceolata is a perennial herb of the Compositae family. A methanol extract of the flowers of C. lanceolata was found to inhibit DPP-4 activity by 87.2%. Among the various compounds isolated, compounds 2-4, 6, and 7 inhibited DPP-4 in a concentrationdependent manner, with IC 50 values ranging from 9.6 to 64.9 µM [91], which suggests that flowers of C. lanceolata and their active components have potential for the treatment of T2DM.

Melicope glabra
Melicope glabra is a tree of the Rutaceae family herb and an important source of flavonoids and coumarins. The plant is native to Sumatra, Peninsular Malaysia, Singapore, Java, and Borneo. The chloroform extract of the leaves of M. glabra effectively inhibited DPP-4 with an IC 50 of 169.40 µg/mL. Computational analysis showed that compounds (8) and (7) in this extract are potent DPP-4 inhibitors based on their binding affinities and extensive interactions with important DPP-4 residues [93]. The phytochemical profiles of these compounds indicated their potential as DPP-4 inhibitors.

Hibiscus rosa-sinensis
Hibiscus rosa-sinensis (HRS) is a tropical flowering plant that is common in Asia and is used in herbal medicine to treat a variety of ailments, such as cough, diarrhea, and diabetes. An ethanol extract of HRS significantly inhibited DPP-4 activity, increased insulin release, and thus, improved glucose tolerance in type 2 diabetic rats [94].

Annona squamosa
Annona squamosa, commonly called 'Ata', is a small tree that belongs to the Annonaceae family and is native to Bangladesh. This herb is well known for its various medicinal properties, which include antioxidant, anti-diabetic, and hepatoprotective effects [95]. Hot water extract of A. squamosa was recently reported to promote cellular glucose absorption and the secretion/action of insulin, and to suppress DPP-4 activity [96].

Emblica officinalis
Emblica officinalis, commonly known as Indian gooseberry (amla), is a member of the Phyllanthaceae family and used as a folk medicine to treat various diseases, including diabetes. Amla fruit extract inhibited DPP-4 (IC 50 3770 µg/mL) and alpha-glucosidases and exhibited antioxidant properties [97].

Berberis aristata
Berberis aristata belongs to the Berberidaceae family and is a shrub native to the Himalayas in India and Nepal. The roots of this plant have antibacterial, anti-inflammatory, analgesic, antioxidant, and hepatoprotective properties, and its crude bark extract inhibited DPP-4 activity with an IC 50 of 14.4 µg/mL [98].

Avena sativa
Avena sativa, also called the common oat, is a member of the Poaceae family and is widely cultivated in Western China as a staple food. A. sativa is considered a functional food due to its health-promoting properties. Oat flour was found to inhibit DPP-4 with an IC 50 of 0.99 mg/mL [99].

Camellia sinensis
Camellia sinensis is a member of the Theaceae family and native to China and Southeast Asia. The principal component of C. sinensis is caffeine, which acts as a secondary metabolite. Extract of C. sinensis leaves inhibited DPP-4 activity with an IC 50 value of 227 µg/mL [100].

Vitis thunbergii var. taiwaniana
The leaves and fruit of the small-leaf grape (Vitis thunbergii var. taiwaniana, VTT) are smaller than those of Vitis vinifera (standard grape). VTT is used in folk medicine to treat hepatitis, jaundice, diarrhea, and arthritis, and ethanol extracts of the stems and leaves of VTT inhibited DPP-4 activity by 26 and 11%, respectively. The VTT ethanol extracts treatment improved the impaired glucose tolerance of diet-induced obese animals [101].

Prunus amygdalus
Prunus amygdalus (also called badaam) is a member of the Rosacease family and is widely distributed in India, especially in the Himalayan region. P. amygdalus has several health benefits, which include antioxidative, lipid-reducing, anti-cancer, anti-inflammatory, and immunostimulatory effects. P. amygdalus extract inhibited DPP-4 activity with an IC 50 of 162.9 µg/mL [102].

Ferula assa-foetida
Ferula assa-foetida is an herbaceous plant of the Apiaceae family and has a number of medicinal properties. The ethanolic fraction of F. assa-foetida inhibited DPP-4 activity by 24.5% [103].

Helichrysum arenarium
Helichrysum arenarium is a perennial herbaceous plant of the Asteraceae family and is native to Europe. In European folk medicine, the medicinal properties of this plant are attributed to its choleretic, cholagogic, hepatoprotective, and detoxifying activities. H. arenarium methanol extract inhibited DPP-4 enzymatic activity with an IC 50 of 41.2 µg/mL [104].

Natural Phytochemicals
Natural products are an exceptionally rich resource for drug discovery, drug development, and clinical medicine [105]. A summary of natural compounds with DPP-4 inhibitory activity and their corresponding IC 50 values is provided in Table 3.

Alkaloids
Coptis chinensis (family: Ranunculaceae) is a goldthread species native to China. Its rhizomes contain the isoquinoline alkaloids berberine, palmatine, and coptisine and are used in traditional Chinese medicine. In vitro, berberine has a strong DPP-4 inhibitory effect with an IC 50 value of 13.3 µM [106]. Molecular docking results show 7-deoxy-6epi-castanospermine derived from C. australe inhibited DPP-4 with the same potency as berberine [80].

Flavonoids
The beneficial health effects of fruits and vegetables have been linked to their high flavonoid contents. DPP-4 activity was potently inhibited by anthocyanins isolated from blueberry/blackberry wine blends with an IC 50 of 0.07 µM [107]. Cyanidin 3-O-glucoside inhibited DPP-4 with an IC 50 of 125.1 µM [108].
Emodin obtained from Rheum palmatum inhibited DPP-4 in vitro with an IC 50 of 5.76 µM [109]. Citrus flavonoids have also been shown to have DPP-4 inhibitory activity; rutin was the most active inhibitor with an IC 50 of 485 µM [110]. Naringin is abundant in orange peel and has been shown to inhibit DPP-4 in vitro and in vivo and to enhance insulin levels, and thus, is considered an option for the low-cost treatment of diabetes [111].
Quercetin is a plant-derived flavonol that has been shown to regulate hyperglycemia and oxidative stress. Molecular docking studies showed that quercetin and galangin bind strongly with DPP-4 and inhibit its activity with IC 50 values of 4.02 and 40.13 µM, respectively [69,113]. Furthermore, isoquercetin from Apocynum cannabinum inhibited DPP-4 with an IC 50 of 96.8 µM [114].
Smilax china plants are found in tropical and temperate regions worldwide, especially in East Asia and North America. Its flavonoids kaempferol 7-O-α-L-rhamnoside, vitexin, and rutin were shown to inhibit DPP-4 with IC 50 values of 20.81, 33.12, and 32.93 µM, respectively [115].

VTT-derived hopeaphenol, (+)-vitisin A, and (−)-vitisin B inhibited DPP-4 with IC
Coumarins are heterocyclic molecules and have been associated with a variety of health benefits, which include antithrombotic, anti-inflammatory, and vasodilatory effects. Coumarins inhibited DPP-4 with an IC 50 of 54.83 nmol/mL [113]. In a molecular docking study, curcumin was found to bind strongly with DPP-4, and in vitro curcumin inhibited DPP-4 activity by up to 50% [119].
Macrotyloma uniflorum (horsegram) is a legume grown mainly in dry regions of Australia, Burma, India, and Sri Lanka and contains high concentrations of myricetin, which has been shown to have anti-hyperglycemic properties. Myricetin also inhibited DPP-4 (IC 50 of 4.8 µM), and thus, increased serum GLP-1 and insulin levels and ameliorated the manifestations of T2DM [70].

Glycosides
Foods contain a wide range of bioactive molecules, and their different scaffolds and functionalities make them the most important source of possible leads for drug discovery. Virtual screening of a polyphenol-rich food database for potential DPP-4 inhibitors resulted in the identification of chrysin, and an in vitro enzyme assay showed that chrysin inhibits DPP-4 in a concentration-dependent manner [120].

Bioactive Peptides
The herbaceous annual plant Phaseolus vulgaris (PV) is cultivated globally for its edible dry seeds or unripe berries. Protein fractions derived from PV were found to inhibit DPP-4 activity by 96.7%. In addition, bioactive peptides were isolated from a protein isolate of PV. EGLELLLLLLAG, AKSPLF, and FEELN peptides inhibited DPP-4 more effectively in silico with free binding energy values of −9.8, −9.6, and −9.5 kcal/mol, respectively, than the reference compound sitagliptin (−8.67 kcal/mol) [123]. In another study, protein digests and pure peptides derived from Mexican black and Brazilian Carioca beans inhibited DPP-4 with IC 50 values ranging from 0.03 to 0.87 mg dry weight/mL [124]. These studies suggest that peptides derived from bean protein isolates can inhibit DPP-4.
Oryza sativa L. (rice) bran protein hydrolysates inhibited DPP-4 enzyme with an IC 50 of 1.45 ± 0.13 mg/mL [125]. When Umamizyme G and Bioprase SP were used to defat and hydrolyze rice bran protein fractions, dipeptides digested with Umamizyme G inhibited DPP-4 with an IC 50 of 2.3 ± 0.1 mg/mL [126]. Proteins derived from Amaranthus hypochondriacus were subjected to simulated gastrointestinal digestion, and this resulted in the formation of bioactive peptides that suppressed DPP-4 in a concentrationdependent manner with an IC 50 of 1.1 mg/mL [127]. Glycine max (soybean) and Lupinus albus (lupin) protein hydrolysates contain bioactive peptides, and Soy 1 (IAVPTGVA) and Lup 1 (LTFPGSAED) effectively inhibited DPP-4 with IC 50 values of 106 and 228 µM, respectively [128]. Gastrointestinal digestion of proteins derived from Phalaris canariensis (canary) seeds inhibited DPP-4 by 43.5% [129], and enzymatic digestion of quinoa proteins with papain also resulted in DPP-4 inhibition with an IC 50 of 0.88 ± 0.05 mg/mL [83]. In a study that used quinoa protein to simulate duodenal digestion, the fraction collected after 120 min of digestion most inhibited DPP-4 with an IC 50 of 0.84 ± 0.07 mg protein/mL [130].
DPP-4 inhibitory activity was found in peptides isolated from tuna cooking juice hydrolyzed by the enzymes protease XXIII (PR) and orientase (OR). The amino acid sequences of three peptides isolated from PR and OR hydrolysates were PGVGGPLGPIGP-CYE (1412.7 Da), CAYEWQRPVDRIR (1690.8 Da), and PACGGFYISGRPG (1304.6 Da), and all three inhibited DPP-4 in a dose-dependent manner [131]. GPAE (372.4 Da) and GPGA (300.4 Da) peptide sequences obtained from Atlantic salmon skin gelatin also potently inhibited DPP-4 [132]. Several peptide sequences in an aqueous Palmaria palmata protein extract hydrolyzed with Corolase PP inhibited DPP-4, and three of these peptides (ILAP, LLAP, and MAGVDHI), when purified, potently inhibited DPP-4 with IC 50 values ranging from 43.40 to 159.37 µM [133]. In addition, peptides obtained from oats, buckwheat, and barley inhibited DPP-4 with IC 50 values from 0.13 to 8.15 mg/mL, and LQAFEPLR inhibited DPP4 in vitro with an IC 50 value of 103.5 µM [99]. The bioactive peptides that inhibit DPP-4 and their corresponding IC 50 values have been listed in Table 4.

Conclusions
At the molecular level, DPP4 inhibitors work by preventing the degradation of GIP and GLP1, and thus, preserve their endogenous levels and reduce BG levels. Nature has many herbal plants that have long been used to treat diabetes. In this review article, we detail the medicinal plants and their bioactive compounds that inhibit DPP-4 activity. Of these compounds, resveratrol, quercetin, and coumarins are highly effective DPP-4 inhibitors with IC 50 values in the nanomolar range. Other compounds (flavonoids and phenolics) have the additional benefit of being present in a variety of functional foods. DPP-4 inhibitors improve pancreatic beta cell function and enhance SM cell proliferation. Studies on natural DPP-4 inhibitors offer a powerful means of developing novel treatments for T2DM, and it is hoped this review will help researchers searching for safer, natural DPP-4 inhibitors for the treatment of diabetes.