Synthesized Peptides from Yam Dioscorin Hydrolysis in Silico Exhibit

Aug 8, 2016 - For plasma DPP-IV activity determination, 5 μL of plasma sample from each time point of the OGTT test was mixed with 95 μL of 100 mM T...
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Synthesized Peptides from Yam Dioscorin Hydrolysis in Silico Exhibit Dipeptidyl Peptidase-IV Inhibitory Activities and Oral Glucose Tolerance Improvements in Normal Mice Yin-Shiou Lin,† Chuan-Hsiao Han,§ Shyr-Yi Lin,*,‡,# and Wen-Chi Hou*,†,⊥ †

Graduate Institute of Pharmacognosy, College of Pharmacy, and ‡Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan § Department of Health and Creative Vegetarian Science, Fo Guang University, Yilan County 262, Taiwan # Department of Primary Care Medicine and ⊥Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei 110, Taiwan ABSTRACT: RRDY, RL, and DPF were the top 3 of 21 peptides for inhibitions against dipeptidyl peptidase-IV (DPP-IV) from the pepsin hydrolysis of yam dioscorin in silico and were further investigated in a proof-of-concept study in normal ICR mice for regulating glucose metabolism by the oral glucose tolerance test (OGTT). The sample or sitagliptin (positive control) was orally administered by a feeding gauge; 30 min later, the glucose loads (2.5 g/kg) were performed. RRDY, yam dioscorin, or sitagliptin preload, but not DPF, lowered the area under the curve (AUC0−120) of blood glucose and DPP-IV activity and elevated the AUC0−120 of blood insulin, which showed significant differences compared to control (P < 0.05 or 0.001). These results suggested that RRDY and yam dioscorin might be beneficial in glycemic control in normal mice and need further investigations in diabetic animal models. KEYWORDS: blood sugar, blood insulin, dipeptidyl peptidase-IV (DPP-IV), oral glucose tolerance test (OGTT), sitagliptin, yam dioscorin

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secretion.11 DPP-IV (a serine proteinase) selectively hydrolyzes X-Pro or X-Ala dipeptides from the N-terminus of the GLP-1 and produces an inactive one.11,12 Therefore, several studies focused on potential DPP-IV inhibitory compounds isolated from synthesized peptides and protein hydrolysates. For example, the Trp-containing dipeptides showed DPP-IV inhibitory activities, and the top four dipeptides were in the sequence WR > WK > WL > WP.13 κCasein showed the highest DPP-IV inhibitory potency index;14 ILAP, LLAP, and MAGVDHI were identified from Corolase PP hydrolysates of macroalga Palmaria palmata;15 and LPIIDI and APGPAGP were identified from silver carp protein hydrolysates.16 Fractions of different molecular masses were identified from yam dioscorin peptic hydrolysates;17 LP and IP were identified from Umamizyme G hydrolysates of defatted rice bran;18 and dairy ingredients (sodium caseinate, skim milk powders, and milk protein concentrates) were hydrolyzed by pepsin−pancreatin to generate DPP-IV inhibitory activities19 and peptic hydrolysates of whey protein.20 Synthesized WCKDDQNPHS, LAHKALCSEK, and LCSEKLDQWL from bovine α-lactalbumin were likewise identified.21 Potential DPP-IV inhibitory compounds were reported from isolated natural products, such as resveratrol tetramers of (−)-vitisin B22 and quinovic acid, quinovic acid glycoside, and lupeol.23

INTRODUCTION It has been estimated that 422 million adults worldwide suffered from diabetes in 2014 compared to 108 million in 1980, and the prevalence rose from 4.7 to 8.5% in the adult population as detailed in the first WHO Global report on diabetes.1 Diabetes caused 1.5 million deaths in 2012, and higher-than-optimal blood glucose caused an additional 2.2 million deaths by increasing the risks of cardiovascular and other diseases.1 In the report in 2011, it was estimated that there was an 8.3% world prevalence of diabetes (366 million adults) among adults (20−79 years old) in 2011, and that this would increase to 9.9% (551 million adults) by 2030.2 Of those with diabetes, 90−95% was categorized as type 2 diabetes mellitus (T2DM), a condition characterized by insulin resistance and relative insulin deficiency.3 Obesity and T2DM with insulin resistance4,5 are involved in metabolic syndrome observed in atherosclerosis and cardiovascular diseases.6,7 It was noted that newly diagnosed T2DM cases are about 80% also overweight.8 Several different drugs have been developed for oral T2DM treatments,9 among which dipeptidyl peptidase-IV (DPP-IV) inhibitors have been designed to improve glycemic control by inhibiting DPP-IV responsible for the breakdown of glucagonlike peptide-1 (GLP-1).8 Sitagliptin is the first approved drug for this purpose.10 GLP-1, a kind of incretin hormone, is secreted from the gut in response to ingested nutrients and stimulates glucose-dependent insulin secretions; however, these effects are quickly stopped by GLP-1 breakdown through DPPIV hydrolysis, and patients with T2DM show impaired or absent insulin secretion after oral glucose due to a lower GLP-1 © XXXX American Chemical Society

Received: May 29, 2016 Revised: July 27, 2016 Accepted: August 8, 2016

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DOI: 10.1021/acs.jafc.6b02403 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry The dried tuber slices of yams are a Traditional Chinese Medicine, and the fresh yam tuber in Taiwan is recognized as a vegetable. The storage protein of yam tuber, dioscorin, and/or its proteinase hydrolysates have been reported for different functional activities in vitro and/or in animal models.24 The pepsin hydrolysis of yam dioscorin in silico was reported to have antioxidant and antiradical,25,26 antiaging and antiglycation,27,28 and angiotensin-converting enzyme (ACE) inhibitory and blood pressure lowering activities.29 The identified octapeptide of LPQNIPPL from gouda-type cheese showed DPP-IV inhibitory activities and improved glucose metabolisms at a dose of 300 mg/kg in normal rats as shown by the oral glucose tolerance test (OGTT).30 The vildagliptin (50 mg), an approved DPP-IV inhibitor, was given 60 min before a drink containing a 25 g of whey protein preload, and 30 min later, a mashed potato meal was administered in a clinical trial. It was found that a protein preload could enhance the postprandial glucose-lowering efficacy of vildagliptin in metformin-treated T2DM patients.31 In a previous study, yam dioscorin intervention accompanying a high-fat feeding was shown to significantly (P < 0.05) improve impaired glucose tolerances compared to the high-fat group in obese rats by the OGTT, and the pepsin hydrolysate of yam dioscorin with fractions 95%, Hefei, China). Pepsin Hydrolysis in Silico of Yam Dioscorin. The pepsin hydrolysis of yam dioscorin in silico was performed according to a previous method using dioscorin A (UniProtKB/TrEMBL: Q9M519) and dioscorin B (UniProtKB/TrEMBL: Q9M501) as targets.29 Twenty-one peptides from mature protein were synthesized for DPP-IV inhibitory activity screenings, including KTCGNGME, PPCSE, PPCTE, KTCGY, KRIHF, RRDY, IHF, RSVF, PTNF, GISW, MGSF, VSIL, HSPA, DPF, RY, RF, NW, RL, GVI, GSL, and GPA, which all had been used before for inhibitions against ACE screenings.29 Inhibition against DPP-IV Activity by Synthesized Peptides. The inhibition against DPP-IV activity by synthesized peptides was performed following the previous study using Gly-Pro-p-nitroanilide as substrate.17,22 The DPP-IV enzyme was diluted to about 0.002 unit/ mL by 100 mM Tris buffer (pH 8.0) as a working solution. The absorbance at 405 nm was measured every 10 min for 60 min by using an ELISA reader (TECAN Sunrise microplate reader; Männedorf, Switzerland). The DPP-IV inhibition (%) was calculated as follows:

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RESULTS AND DISCUSSION

Inhibitions against DPP-IV Activity from Synthesized Peptides. The 21 peptides derived from mature protein pepsin hydrolysis in silico were synthesized for DPP-IV inhibitory activity assay. It was found that at the same concentration of 1 mM, seven peptides exhibited >20% DPP-IV inhibitory activity (Figure 1A) in the order RRDY (60.13 ± 1.73%) > RL (48.67 ± 0.15%) ≅ MGSF (46.57 ± 1.63%) > DPF (39.22 ± 4.00%) > GPA (32.93 ± 3.41%) > IHF (30.58 ± 0.97%) > KRIHF (20.57 ± 0.22%). The positive control of sitagliptin at 25 nM B

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residue at position 2 from the N-terminus of peptides or the Tyr residue in the C-terminus of peptides might act as DPP-IV inhibitors.36 RRDY, RL, and DPF in the present study with DPP-IV inhibitory activities might follow the above-mentioned rules and were used for OGTT test in normal mice. Glucose Metabolism Evaluations of Synthesized Peptide Preload in Normal ICR Mice by OGTT. It was reported that inhibitions against DPP-IV can lower postprandial blood glucose levels by prolonging active GLP-1 to stimulate glucose-dependent insulin secretion.11 Although several studies have focused on the isolation and purification of potential DPPIV inhibitory compounds,13−23 papers on further proof-ofconcept study in vivo for blood glucose regulations were limited. Uenishi et al.30 reported that the octapeptide of LPQNIPPL was identified from gouda-type cheese with DPPIV inhibitory activities and improved glucose metabolisms at a dose of 300 mg/kg in normal rats by OGTT. However, the plasma insulin levels in the peptide-treated group were not matched to DPP-IV inhibitory activities and showed levels similar to those of the control group. Therefore, RRDY, RL, and DPF were the top three potential DPP-IV inhibitory peptides in this study and were selected to investigate the proof-of-concept study in normal ICR mice for regulating glucose metabolisms by OGTT. Each peptide or distilled water (the control) was orally administered by feeding gauge 30 min before the glucose loads (2.5 g/kg of body weight), and blood was taken at intervals of 0, 15, 30, 60, 90, and 120 min for glucose determination (Figure 2). For RRDY preloads at a dose of 100 mg/kg (Figure 2A), the blood glucose levels (mg/dL) were lower and showed a significant difference compared to the control at 15 min (P < 0.01), 30 min (P < 0.01), 60 min (P < 0.05), 90 min (P < 0.05), and 120 min (P < 0.01); the AUC0−120 of the blood glucose levels of RRDY_100 were lower and significantly different compared to the control (P < 0.001), which meant RRDY at 100 mg/kg preload could reduce postprandial blood glucose levels compared to the control after administration of the glucose load (2.5 g/kg). The RRDY preload at 50 mg/kg was also investigated as shown in Figure 2B. The blood glucose levels (mg/dL) were lower and significantly different compared to the control at 60 min (P < 0.01) and 120 min (P < 0.05); the AUC0−120 of the blood glucose levels of RRDY_50 were lower and significantly different compared to the control (P < 0.05), which meant RRDY at 50 mg/kg preload could reduce postprandial blood glucose levels compared to the control after administration of the glucose load (2.5 g/kg). The RL preload at 100 mg/kg was investigated as shown in Figure 2C. The blood glucose levels (mg/dL) were lower and significantly different compared to the control at 60 min (P < 0.05) and 120 min (P < 0.01); the AUC0−120 of blood glucose levels of RL_100 were lower and significantly different compared to the control (P < 0.05), which meant RL at 100 mg/kg preload could reduce the postprandial blood glucose levels compared to the control after administration of the glucose load (2.5 g/kg). The DPF preload at 100 mg/kg was also investigated as shown in Figure 2D. The blood glucose levels (mg/dL) were higher and significantly different compared to the control at 90 min (P < 0.05); however, the AUC0−120 of the blood glucose levels of DPF_100 showed no significant difference compared to the control (P > 0.05), which meant DPF at 100 mg/kg preload could not reduce the postprandial blood glucose levels after administration of the glucose load (2.5 g/kg). In the proof-of-concept study in normal ICR mice for regulating glucose metabolisms

Figure 1. (A) Effects of 21 synthesized peptides at 1 mM on DPP-IV inhibitory activity. Peptides were derived from pepsin hydrolysis in silico of yam dioscorin. (B) Effects of 7 synthesized peptides (RRDY, IHF, KRIHF, RL, GPA, MGSF, and DPF) on DPP-IV inhibitory activities at concentrations of 0.25−5 mM.

showed DPP-IV inhibition (33.85 ± 1.86%)17 similar to that of 1 mM GPA. Therefore, the IC50 values of these seven synthesized peptides for DPP-IV inhibitory were determined and are shown in Figure 1B. The IC50 values of RRDY, RL, DPF, MGSF, GPA, IHF, and KRIHF were calculated as 0.93, 1.20, 1.54, 2.12, 2.87, 3.77, and 4.11 mM, respectively. Cheung et al.35 proposed that peptide with aromatic or branched-chain aliphatic amino acids close to the COOH-terminus exhibited ACE inhibitions. Therefore, the 23 peptides derived from yam dioscorin pepsin hydrolysis in silico were synthesized to test ACE inhibitory activity,29 and KTCGY, KRIHF, NW, and RRDY were shown to be the top four potential ACE inhibitory peptides; KTCGY and KRIHF also showed a vasodilating effect in phenylephrine-induced aortic ring tension and anti-hypertensive activity in spontaneously hypertensive rats. Except RRDY, the potent DPP-IV inhibitory peptides in the present study showed little correlation with ACE inhibitory activities.29 The Trp-containing dipeptides were reported to exhibit DPPIV inhibitory activities, and the top 4 of 27 dipeptides were in the sequence WR > WK > WL > WP; the reverse peptides showed no DPP-IV inhibitory activity except LW.13 The Pro C

DOI: 10.1021/acs.jafc.6b02403 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Effects of (A) RRDY (100 mg/kg), (B) RRDY (50 mg/kg), (C) RL (100 mg/kg), and (D) DPF (100 mg/kg) preload 30 min before glucose load (2.5 g/kg) on blood glucose levels in normal ICR mice by OGTT. The AUC0−120 of blood glucose was calculated for control and tested sample preload. The differences of blood glucose at the same time or AUC0−120 between tested sample preload and the control group were analyzed using Student’s t test, which was considered statistically significant when P < 0.05 (∗), P < 0.01 (∗∗), or P < 0.001 (∗∗∗).

(mg/dL) were lower and significantly different compared to the control at 60 min (P < 0.05), 90 min (P < 0.05), and 120 min (P < 0.001); the AUC0−120 of blood glucose levels of dioscorin_100 were lower and significantly different compared to the control (P < 0.05), which meant yam dioscorin at 100 mg/kg preload could reduce postprandial blood glucose levels after administration of the glucose load (2.5 g/kg). The positive control of the sitagliptin preload at 10 mg/kg was investigated as shown in Figure 3C. The blood glucose levels (mg/dL) were lower and significantly different compared to the control at 15 min (P < 0.001), 30 min (P < 0.001), 60 min (P < 0.01), and 90 min (P < 0.01); the AUC0−120 of blood glucose levels of sitagliptin_10 were lower and significantly different compared to the control (P < 0.001), which meant sitagliptin at 10 mg/kg preload could reduce postprandial blood glucose levels after administration of the glucose load (2.5 g/kg). BSA was available from commercial sources and selected for regulation of glucose metabolism in the comparison with yam dioscorin under the same concentration of 100 mg/kg. The identified tripeptide of PPL from pepsin hydrolysis of BSA in silico was reported to show DPP-IV inhibitory activities in vitro;37 however, BSA preload in the present study showed no capacities to reduce blood glucose levels after glucose loads (2.5 g/kg) by OGTT. It was proposed that some predicted DPP-IV inhibitory peptides derived from BSA might be further hydrolyzed to inactive ones in the gastrointestinal tract and lost

by OGTT, it was found that preloads of RRDY (50 or 100 mg/ kg) and RL (100 mg/kg), but not DPF (100 mg/kg), showed a lower AUC0−120 of blood glucose levels in vivo and were significantly different compared to those in the control (P < 0.001 or P < 0.05). Glucose Metabolism Evaluations of Bovine Serum Albumin and Dioscorin Preload in Normal ICR Mice by OGTT. The oral administration of yam tuber dioscorin or pepsin hydrolysates of yam dioscorin could lower blood pressure using spontaneously hypertensive rats as models.33 The oral administration of synthesized peptides from yam dioscorin pepsin hydrolysates in silico were shown to lower blood pressure using spontaneously hypertensive rats as models.29 Therefore, the starting material of the purified yam dioscorin preload (100 mg/kg) was used to monitor glucose metabolism in normal ICR mice by OGTT. BSA (100 mg/kg) was used for comparison, and the positive control of sitagliptin was used for comparisons. The BSA preload at 100 mg/kg was investigated, and the results are shown in Figure 3A. The blood glucose levels (mg/dL) were higher and significantly different compared to the control at 120 min (P < 0.05); the AUC0−120 of blood glucose levels of BSA_100 showed no significant difference compared to the control (P > 0.05), which meant BSA at 100 mg/kg preload could not reduce postprandial blood glucose levels after administration of the glucose load (2.5 g/ kg). The purified yam dioscorin preload at 100 mg/kg was investigated as shown in Figure 3B. The blood glucose levels D

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by OGTT. The changes of DPP-IV activity (Figures 4A−C) and insulin levels (Figures 4D−F) of RRDY, dioscorin, and sitagliptin preloa, and then glucose load (2.5 g/kg) were investigated as shown in Figure 4. For RRDY preloads at 100 mg/kg, the relative DPP-IV activity (Figure 4A) as lower and significantly different compared to the control at zero time (P < 0.05), 60 min (P < 0.05), and 120 min (P < 0.05); the blood insulin peak (Figure 4D) was found at 15 min after the glucose load, and the blood insulin levels (μg/L) were higher and showed a significant difference compared to the control at 30 min (P < 0.01) and 60 min (P < 0.01); the AUC0−120 of blood insulin levels of RRDY_100 were higher and showed a significant difference compared to the control (8.20 vs 7.78; P < 0.05), which meant RRDY at 100 mg/kg preload could inhibit DPP-IV activity and maintain high levels of lasting blood insulin compared to the control after administration of the glucose load (2.5 g/kg) to reduce postprandial blood glucose levels (Figure 2A). For the yam dioscorin preload at 100 mg/ kg, the relative DPP-IV activity (Figure 4B) was lower and significantly different compared to the control at 15 min (P < 0.01), 30 min (P < 0.05), 90 min (P < 0.01), and 120 min (P < 0.01); the blood insulin peak was also found at 15 min after administration of the glucose load, and the blood insulin levels (μg/L) were higher and significantly different compared to the blank at 30 min (P < 0.05), 60 min (P < 0.05), and 90 min (P < 0.01); the AUC 0−120 of the blood insulin levels of dioscorin_100 were higher and significantly different compared to the control (7.47 vs 5.67; P < 0.01), which meant dioscorin at 100 mg/kg preload could lower DPP-IV activity and keep high levels of lasting blood insulin compared to the control after administration of the glucose load (2.5 g/kg) to reduce postprandial blood glucose levels (Figure 3B). For the sitagliptin preload at 10 mg/kg, the relative DPP-IV activity (Figure 4C) was much lower and significantly different compared to the control at zero time (P < 0.001), 15 min (P < 0.001), 30 min (P < 0.001), 60 min (P < 0.001), 90 min (P < 0.01), and 120 min (P < 0.01); the blood insulin peak was also found at 15 min after administration of the glucose load, and the blood insulin levels (μg/L) were higher and significantly different compared to the control at 15 min (P < 0.01), 30 min (P < 0.01), and 120 min (P < 0.01); the AUC0−120 of the blood insulin levels of sitagliptin_10 were higher and significantly different compared to the control (5.67 vs 4.38; P < 0.05), which meant the positive control of sitagliptin at 10 mg/kg preload could lower DPP-IV activity and keep high levels of lasting blood insulin compared to the control after administration of the glucose load (2.5 g/kg) to reduce postprandial blood glucose levels (Figure 3C). Although the GLP-1 concentration was not determined in this study, it was found that the potential DPP-IV inhibitory peptides of RRDY preload could inhibit DPP-IV activity in turn to keep a constant GLP-1 concentration to stimulate glucosedependent insulin secretion, which was shown to reduce postprandial blood glucose levels. In Taiwan, fresh yam tubers are frequently used as vegetables,24 and dioscorin was the main tuber protein from different Dioscorea species estimated by the immune staining method.38 Reagan-Shaw et al.39 proposed the use of body surface area normalization to translate dose uses from animal to human studies. The human equivalent dose was 8.11 mg/kg translated from mouse experiments of yam dioscorin or RRDY (100 mg/kg), and a 60 kg adult may consume 500 mg of yam dioscorin or RRDY daily to achieve similar blood glucose regulatory benefits. This should be

Figure 3. Effects of (A) commercial bovine serum albumin (BSA, 100 mg/kg), (B) purified yam dioscorin (100 mg/kg), and (C) sitagliptin (10 mg/kg) preload 30 min before glucose load (2.5 g/kg) on blood glucose levels in normal ICR mice by OGTT. The AUC0−120 of blood glucose was calculated for control and tested sample preload. The differences of blood glucose at the same time or AUC0−120 between tested sample preload and the control group were analyzed using Student’s t test, which was considered statistically significant when P < 0.05 (∗), P < 0.01 (∗∗), or P < 0.001 (∗∗∗S).

the blood glucose regulatory function, which requires further investigations. Blood DPP-IV Activity and Insulin Levels of RRDY, Dioscorin, and Sitagliptin Preload in Normal ICR Mice E

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Figure 4. Effects of (A, D) RRDY (100 mg/kg), (B, E) yam dioscorin (100 mg/kg), and (C, F) sitagliptin (10 mg/kg) preloads 30 min before glucose load (2.5 g/kg) on relative DPP-IV activity (A−C) and blood insulin levels (D−F) in normal ICR mice by OGTT. The AUC0−120 of blood insulin was calculated for control and tested sample preload. The differences of relative DPP-IV activity and blood insulin at the same time or AUC0−120 between tested sample preload and the control group were analyzed using Student’s t test, which was considered statistically significant when P < 0.05 (∗), P < 0.01 (∗∗), or P < 0.001 (∗∗∗).

glucose load. These results suggested that the ingested RRDY and yam dioscorin were potential glycemic controls in normal mice in part by DPP-IV inhibitions. The yam dioscorin or RRDY peptide might be beneficial in the development of healthy (functional) foods for the improvement of glucose intolerance and/or T2DM and need further investigations in diabetic animal models.

investigated further. It was proposed that the ingested dioscorin might be better than one potential DPP-IV inhibitory peptide in glucose metabolism by OGTT. In conclusion, the current results demonstrated that the proof-of-concept study in normal ICR mice of RRDY and RL from in vitro DPP-IV inhibitory activity screenings showed improved glucose metabolism by OGTT, and RRDY preload at a dose of 100 mg/kg showed sustained higher levels of lasting blood insulin and in turn reduced postprandial blood glucose levels compared to the blank after administration of the glucose load (2.5 g/kg). The purified yam dioscorin preload was also shown to reduce postprandial blood glucose levels after the

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AUTHOR INFORMATION

Corresponding Authors

*(S.-Y.L.) Phone: +886-2-27361661, ext. 7100. E-mail: sylin@ tmu.edu.tw. F

DOI: 10.1021/acs.jafc.6b02403 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry *(W.-C.H.) Phone: +886-2-27361661, ext. 6160. Fax: +886-223780134. E-mail: [email protected].

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Funding

We express sincere thanks to the Ministry of Science and Technology, Republic of China (NSC 102-2324-B-038-004MY3), and to the Jin-lung-yuan Foundation (2014−2015) for financial support. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.6b02403 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b02403 J. Agric. Food Chem. XXXX, XXX, XXX−XXX