Dipeptidyl Peptidase IV Inhibitory Peptides Derived from Oat (Avena

Article pubs.acs.org/JAFC

Dipeptidyl Peptidase IV Inhibitory Peptides Derived from Oat (Avena sativa L.), Buckwheat (Fagopyrum esculentum), and Highland Barley (Hordeum vulgare trif urcatum (L.) Trofim) Proteins Feng Wang,† Guoyong Yu,† Yanyan Zhang,‡ Bolin Zhang,† and Junfeng Fan*,† †

Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Key Laboratory of Forest Food Process and Safety, Beijing Forestry University, Beijing 100083, China ‡ Food Science and Engineering College, Beijing University of Agriculture, Beijing 102206, China S Supporting Information *

ABSTRACT: Peptides released from oat, buckwheat, and highland barley proteins were examined for their in vitro inhibitory effects on dipeptidyl peptidase IV (DPP4), an enzyme that deactivates incretin hormones involved in insulin secretion. All of the hydrolysates exhibited DPP4 inhibitory activities, with IC50 values ranging from 0.13 mg/mL (oat glutelin alcalase digestion) to 8.15 mg/mL (highland barley albumin tryptic digestion). The lowest IC50 values in gastrointestinal, alcalase, and tryptic digestions were 0.99 mg/mL (oat flour), 0.13 mg/mL (oat glutelin), and 1.83 mg/mL (highland barley glutelin). In all, 35 peptides of more than seven residues were identified in the tryptic hydrolysates of oat globulin using liquid chromatography− mass spectroscopy. Peptides LQAFEPLR and EFLLAGNNK were synthesized and their DPP4 inhibitory activities determined. LQAFEPLR showed high in vitro DPP4 inhibitory activity with an IC50 value of 103.5 μM. KEYWORDS: oat, buckwheat, highland barley, trypsin, diabetes, incretin, globulin

1. INTRODUCTION Type 2 diabetes (T2D) is a chronic disease characterized by several pathophysiologic defects, including insulin resistance, excess hepatic glucose production, and progressive pancreatic cell dysfunction.1 Incretins, including glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are peptide hormones released into plasma from K cells in the duodenum and L cells in the intestine mucosa, respectively.2 The concentration of active endogenous GLP-1 and GIP after the ingestion of the meal increases from 2- to 3-fold, which enhance approximately 60% of the meal-induced insulin secretion from β-cells of the islets of langerhans into the bloodstream in a glucosedependent manner and play an important role in the regulation of glucose homeostasis.3,4 However, the incretins are endogenous physiological substrates of dipeptidyl peptidase IV (DPP4, EC 3.4.14.5) and can be rapidly inactivated by DPP4 in approximately 2−7 min. People with T2D have a significant decrease in the incretin response, thereby influencing multiple aspects of glucose homeostasis and leading to the abnormalization of blood glucose levels.3,5 Therefore, the inhibition of DPP4 has been exploited as a novel incretin-based therapy to lower hyperglycemia by extending the half-life of incretins in diabetic patients, especially those with T2D.6,7 In recent years, various DPP4 inhibitors, also termed gliptins (sitagliptin, vildagliptin, and saxagliptin), have been used as fasting and postprandial serum glucose-lowering agents.8 However, these drugs can lead to some undesired side effects, such as angioedema, pancreatitis, and infective disorders.9 Multiple studies have highlighted the possibility of using food-derived bioactive substances, including polyphenols,10 alkaloids,11 and food-derived peptides,6−9,12−14 as natural sources of DPP4 inhibitors. China is the one of the largest producers of cereals in the world. Cereals, including oat (Avena sativa L.), © XXXX American Chemical Society

buckwheat (Fagopyrum esculentum), and highland barley (Hordeum vulgare trif urcatum (L.) Trofim), are generally cultivated in Western China as staple foods and are currently viewed as functional foods because of their beneficial healthpromoting characteristics. These cereals are a good source of soluble fiber, essential amino acids, unsaturated fatty acids (oleic, linoleic, and linolenic acids), vitamins (B1 and B2), minerals (phosphorus and iron), and phytochemicals (avenanthramides).15−17 In addition, people have always been interested in the highly viscous β-glucan fraction in oats and highland barley because of their ability to lower blood cholesterol levels and the rate of intestinal absorption of glucose.17,18 Buckwheat is also a health-supporting food that is believed to prevent coronary disease and diabetes mellitus.19 It has been shown that oat, buckwheat, and highland barley have high protein contents ranging from 12.4 to 24.5%, from 9.19 to 17%, and from 7.68 to 17.52%, respectively.20−22 These proteins are generally composed of albumin, globulins, and glutelin. However, there is little information about the nutritional and biological activities regarding these storage proteins. We evaluated the DPP4 inhibitory activities of peptides encrypted in storage proteins from oat, buckwheat, and highland barley seeds. For this purpose, the main proteins from oat, buckwheat, and highland barley seeds were extracted and digested using three digestion models, including a gastrointestinal digestion, an intestinal protease trypsin digestion, and a microbiological protease alcalase digestion. Protein hydrolysates from the three Received: August 17, 2015 Revised: October 13, 2015 Accepted: October 15, 2015

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DOI: 10.1021/acs.jafc.5b04016 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Tris buffer (pH 8.0) for 14 h at 37 °C. The reaction was stopped by freezing. The enzyme was then removed by ultracentrifugation using a 10 kDa molecular mass cutoff filter (Sartorius Vivaflow 50). Filtrates were then collected, and protein concentrations were determined using the Lowry-based DC Protein Assay using BSA as a standard. The aforementioned OGb tryptic hydrolysate (14 h) was ultrafiltered into two fractions with molecular masses (MMs) of 3−10 and 10 units/mg; 88% sequence homology with the human form), Gly Pro-pNA, trypsin (4−6 units/mg; from porcine pancreas), pancreatin, which digests no 96.6%) and LQAFEPLR (>99.0%) were synthesized by ChinaPeptides Co. Ltd. (Shanghai, China). All other reagents were of analytical grade. 2.2. Storage Protein Extraction. The main protein fractions, including oat globulin (OGb), glutelin (OGt), buckwheat albumin (BAb), glutelin (BGt), highland barley albumin (HAb), and glutelin (HGt), were extracted according to a previously reported method.20 Each sample (100 g of powder) was mixed with distilled water (400 mL). Mixtures were allowed to stand with intermittent stirring for 1.5 h, sonicated for 0.5 h, and centrifuged at 8000g for 20 min to obtain a supernatant (S1) and a precipitant (R1). S1 was precipitated by the addition of 1 M HCl to pH 4.1 with subsequent centrifugation at 8000g for 20 min, and the precipitant was lyophilized. The obtained powder was termed the albumin fraction (BAb; HAb). After being mixed with 5% NaCl (400 mL), R1 was stirred for 1.5 h, sonicated for 0.5 h, and centrifuged at 8000g for 20 min. The obtained supernatant and precipitant were termed S2 and R2, respectively. S2 was precipitated by the addition of 1 M HCl to pH 4.3 with subsequent centrifugation at 8000g for 20 min, and the lyophilized precipitant was termed globulin (OGb). R2 was mixed with 0.1 M NaOH (400 mL), stirred for 1.5 h, sonicated for 0.5 h, and centrifuged at 8000g for 20 min. The obtained supernatant was termed S3. S3 was precipitated by the addition of 1 M HCl to pH 4.8 with subsequent centrifugation at 8000g for 20 min. The obtained precipitate was freeze-dried, and glutelin (OGt, BGt, and HGt) fractions were recovered. Protein amounts were determined using the Bradford Protein Assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard. All of the fractions were stored at −20 °C until they were used. 2.3. Gastrointestinal Digestion Simulation in Vitro. An in vitro gastrointestinal digestion model was established as suggested previously,9 with slight modifications. Briefly, 3 g of sample (oat, buckwheat, or highland barley powder) was resuspended in 60 mL of 0.03 M NaCl (pH 2.0). The suspensions were heated in a water bath at 80 °C for 5 min to inactivate bacteria and proteases and allowed to cool to room temperature. Porcine pepsin, previously dissolved in 0.03 M NaCl (pH 2.0), was added to each suspension at a 1:20 (w/w) enzyme:substrate ratio. Samples were digested at a constant pH for 3 h at 37 °C. The pH was then adjusted to 7.5. A mixture of trypsin and pancreatin was prepared [1:1 (w/w) trypsin:pancreatin ratio in 0.1 N NaHCO3], added to the digestive solution, and incubated at a constant pH for an additional 3 h [1:20 (w/w) enzyme:substrate ratio for both enzymes, trypsin and pancreatin]. Digestion was stopped by heating the suspensions at 75 °C for 20 min. Hydrolysates were centrifuged at 13000g for 30 min. Protein concentrations were determined by the Lowry-based DC Protein Assay (Bio-Rad) using BSA as a standard and samples stored at −20 °C until they were used. 2.4. Tryptic Digestion. The OGb, OGt, BAb, BGt, HAb, and HGt fractions were subjected to tryptic digestion. Trypsin was used at a trypsin:protein ratio of 1:1 (w/w).9 Digestions were conducted in 100 mM B


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Table 1. OGb Tryptic Peptide Fractions (MM < 3 kDa, 14 h) from Oat 11S Globulin, Oat 12S Seed Storage Globulin 2, and Others protein origin

molecular mass (Da)

peptide sequencea

11S globulin LQAFEPLR50−57 SQAGITEYFDEQNEQFR61−77 QKEFLLAGNNK200−210 EFLLAGNNK202−210 EFLLAGNNKR202−211 IQSQNDQR242−249 NIENPQHADTYNPR339−352 VQVVNNNGQTVFNDILR406−422 TNPNSMVSHIAGK451−463 ALPIDVLANAYR469−480 GEEFGAFTPK494−503

972.53 2060.91 1260.68 1004.53 1160.63 987.47 1667.77 1952.02 1354.66 1314.73 1081.51

LQAFEPLR50−57 SQAGIIEYFDEQNEQFR61−77 QKEFLLAGNNK200−210 EFLLAGNNK202−210 EFLLAGNNKR202−211 IQSQNDQR242−249 NFPTLNLVQMSATR355−368 VQVVNNHGQTVFNDILR398−414 TNPNSMVSQIAGK443−455 ALPVDVLANAYR461−472 GEEFDAFTPK486−495

972.53 2060.91 1260.68 1004.53 1160.63 987.47 1590.82 1952.02 1354.66 1300.71 1139.51

ALPVDVLANAYR GQESGVFTPK FTQTSFQPYPEGEDESSLINK

1300.71 1048.52 2416.11

SFPAFATTGSTDVR AIGVDLLR WSPSSTDK

1455.7 855.52 906.41

FGVPMGYGGPHAAFLATSQEYKR DAHKHGVKVVMATDLLALTTLR

2483.21 2548.26

ISIMSNNITELSFSPKCK

2170.94

GAKVILSSHLGRPK

1621.81

12S seed storage globulin 2

avenin

endochitinase

victorin binding protein

NCBI accession number

theoretical mass (Da)

27.7

Q38780

59368.89

21.43

P14812

58639.24

20.9

P27919

24213.94

15

P86181

21714.65

4.36

NBS-LRR type disease resistance protein 3-phosphoglycerate kinase gliadin-like avenin QLAQIPR

Q38766

111328.8

10.6

O51526

51380.56

16.28

Q38768

9402.26

3.87

L0L6J7

20425.98

1.77

F5B4I6

58103.58

4.55

F4MJY0

22802.29

4.5 4.55

F2Q9W3 P80356

25716.39 25258.57

2.73

P48684

52901.33

1.24

P06594

5.62

G1JSL4

824.49

development-related protein kinase FEIQLYKTR

1196.66

LEQIPEQLR QLAQIPEQVR

1124.62 1180.66

QLEQIPEQLR

1252.68

avenin protein (fragment)

avenin-3 ribulose bisphosphate carboxylase large chain ASVGFQAGVKDYK

1448.67

HAINNPLSGMLYSR

1651.75

ALVAVDVNNPEGTK

1425.75

phytochrome A type 3 peroxygenase 1 a

coverage (%)

124913.3 28089.39

The subscript numbers represent peptide positions in the globulin.

C


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Figure 1. Tandem mass spectra of the (A) LQAFEPLR and (B) EFLLAGNNK characterized sequences. After the sequences had been interpreted and the database searched, the peptides were identified as 11S globulin (50−57) and 12S seed storage globulin 2 (202−210), respectively. The peptide sequences are shown with the fragment ions observed in the spectra. Fragment ions are labeled according to the nomenclature proposed by Roepstorff and Fohlman.26 Only b and y product ions are labeled for the sake of clarity.

Oat flour hydrolysates exhibited a high degree of DPP4 inhibition. 3.2. DPP4 Inhibitory Activity of Tryptic Hydrolysates from Cereal Proteins. Comparisons of the inhibitory activities of tryptic hydrolysates (14 h) of OGb, OGt, BAb, BGt, HAb, and HGt are shown in Supporting Information 1B. All of the samples showed concentration-dependent inhibition of DPP4. Note that the peptides obtained from glutelins showed degrees of DPP4 inhibition greater than those of the peptides obtained from albumins and globulin (p < 0.05). The HAb hydrolysates exhibited the weakest inhibition (IC50, 8.15 mg/mL), while the

3. RESULTS 3.1. In Vitro Gastrointestinal Digestion of Cereal Flours. Using the aforementioned simulated in vitro gastrointestinal digestion model, flours from oat, buckwheat, and highland barley were hydrolyzed and the DPP4 inhibitory activities of the flour hydrolysates evaluated (Supporting Information 1A). Among these three flours, the one that exhibited the lowest level of inhibition upon DPP4 with an increasing dose was highland barley flour (IC50, 3.91 mg/mL), whereas oat flour showed the highest level of inhibition (IC50, 0.99 mg/mL). Buckwheat flour exhibited 50% inhibition at a concentration of 1.98 mg/mL. D


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the C-terminal residue following tryptic digestion, and most were 7−14 amino acid residues in length (Table 1). Peptides from 11S and 12S globulins covered 49.13%. LQAFEPLR, IQSQNDQR, QKEFLLAGNNK, EFLLAGNNK, EFLLAGNNKR, VQVVNNNGQTVFNDILR, and SQAGITEYFDEQNEQFR were identified in both the 11S and 12S proteins. As an example, Figure 1 shows the MS/MS spectrum of molecular ions (the peptide EFLLAGNNK and LQAFEPLR) found in the oat 11S and 12S globulin fractions. For the sake of clarity, only the b and y fragment ions, resulting from the cleavage of a peptide bond, are labeled, following the nomenclature of Roepstorff.26 Specifically, the b ions are those in which the charge remained on the N-terminal portion of the peptide. The y ions are those in which the charge was retained on the C-terminal portion of the peptide.26 Overall, the most abundant ions in the MS/MS spectra of these peptide fragments were b and y ions. The previously described general rules concerning peptide fragmentation were used to make peptide assignments. 3.5. DPP4 Inhibitory Activity of Oat Pepetides LQAFEPLR and EFLLAGNNK. Thirty-five peptides were computationally docked with crystal structure 1X70 using Autodock Vina to identify bioactive peptides. The results of computational modeling showed that nine of these peptides were able to occupy the active site of the enzyme with low binding energies (Supporting Information 3). In general, lower binding energies indicate stronger inhibitory potencies. Among the nine peptides, two short peptides, LQAFEPLR and EFLLAGNNK, with lower binding energies (Supporting Information 3) were synthesized and their DPP4 inhibitory activities determined (Figure 2).

HGt hydrolysates exhibited the strongest inhibition (IC50, 1.83 mg/mL). The other protein hydrolysates exhibited IC50 values of 2.04 mg/mL (OGb), 2.76 mg/mL (OGt), 5.36 mg/mL (BAb), and 2.44 mg/mL (BGt). These results indicate that the source protein of the peptides affects their DPP4 inhibitory activity. The hydrolysis time was also found to affect DPP4 inhibitory activity. The activities of tryptic hydrolysates of OGb with hydrolysis times of 1, 2, 4, 8, and 14 h are compared in Supporting Information 1C. The degree of inhibition increased with hydrolysis time. The 1 h hydrolysates exhibited the lowest degree of inhibition (IC50, 5.61 mg/mL), whereas the 14 h hydrolysates exhibited the highest degree of inhibition (IC50, 2.04 mg/mL). A long hydrolysis typically produced a greater number of peptides in the resulting hydrolysate. 3.3. DPP4 Inhibitory Activity of Alcalase Hydrolysates from Cereal Protein. To determine the efficacy of an industrialized microbiological protease to produce hydrolysates with potent DPP4 inhibition, alcalase was used to hydrolyze OGb, OGt, BAb, BGt, HAb, and HGt. Hydrolysates obtained at hydrolysis times of 0.5, 1.0, 1.5, and 2.0 h are compared in Supporting Information 2A−F. As shown in Supporting Information 2, all of the protein hydrolysates exhibited DPP4 inhibitory activity, and the degree of inhibition increased with digestion time up to 1.5 h. For OGb and OGt, the highest degree of inhibition occurred after digestion for 2 h, resulting in IC50 values of 0.84 and 0.13 mg/mL, respectively. For BGt, HGt, BAb, and HAb, the greatest degree of inhibition occurred at 1.5 h, resulting in IC50 values of 0.98, 0.85, 1.49, and 1.98 mg/mL, respectively. Further digestion resulted in significant decreases in activity. These results indicated that 1.5 h was a suitable hydrolysis time for future studies or industrial production schemes. At concentrations of 4 mg/mL, most of the globulin or albumin digestions exhibited 60% inhibition. This indicates that glutelins release DPP4 inhibitory peptides more easily than do albumins. Among the glutelin digestates, OGt hydrolysates showed the highest degree of inhibition on DPP4 (>60%) even at low concentrations (0.5 mg/mL). Relative to tryptic digestion, alcalase was more potent in the hydrolysis of cereal proteins with regard to the release of bioactive peptides. 3.4. Characterization of Peptides in OGb. Because OGb is the main storage protein of oats, its amino acid sequence has been clearly expounded. Therefore, the peptides within the OGb tryptic hydrolysates were identified in this study. To determine the peptide composition of OGb digestates, the 14 h hydrolysates of oat globulin were divided into two fractions by ultrafiltration. The first fraction contained peptides with MMs between 3 and 10 kDa. The second fraction contained peptides with MMs of

Dipeptidyl Peptidase IV Inhibitory Peptides Derived from Oat (Avena

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