Young apple polyphenols postpone starch digestion in vitro and in vivo

Journal of Functional Foods 56 (2019) 127–135

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Young apple polyphenols postpone starch digestion in vitro and in vivo a

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Dan Li , Lijun Sun , Yongli Yang , Zichao Wang , Xi Yang , Ting Zhao , Tian Gong , Li Zou , ⁎ Yurong Guoa, a b

College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, China Shaanxi Product Quality Supervision and Inspection Research Institute, Xi’an 710000, Shaanxi Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Young apple polyphenols α-amylase α-glucosidase Postprandial blood glucose Insulin level

Type II diabetes is largely related with hyperglycemia that can be controlled through inhibition the activities of carbohydrate-hydrolyzing enzymes by dietary polyphenols. This study aims to explore the effects of young apple polyphenols (YAP) on starch digestion in vitro and in vivo by acute and 1-week intervention administrations using mice. It was found that YAP was able to inhibit the starch digestive enzymes including α-amylase and α-glucosidase, in which tannic acid and chlorogenic acid showed inhibition on α-amylase, and tannic acid and phlorizin showed inhibition on α-glucosidase. In addition, the levels of postprandial blood glucose and insulin were lowered by around 10% at a peak for the mice fed with combination of starch and YAP than that only fed with starch, which was observed for acute and 1-week administrations. Taken together, YAP may have potentials as a functional food in assisting prevention and alleviation of type II diabetes disease.

1. Introduction Diabetes is a metabolic disease that occurs increasingly with obesity and aging (Gutierrez-Rodelo, Roura-Guiberna, & Olivares-Reyes, 2017). Its incidence increases year by year, especially type II diabetes that is defined as a non-insulin dependent and adult-onset disease (Gavin et al., 1997; Zinman, 2015). Postprandial hyperglycemia has been considered as an important and independent factor in the development of type II diabetes, as well as in some cardiovascular complications (Cavalot et al., 2006). Karetnikova, Gruzdeva, Uchasova, Osokina, and Barbarash (2016) have demonstrated that there is a positive correlation between blood glucose level and mortality in patients suffered from myocardial infarction as well. The level of postprandial hyperglycemia can be controlled by stimulating the secretion of insulin, inhibiting the glucose transporters, or delaying the digestion of starchy foods through inhibiting the activities of starch-hydrolyzing enzymes, such as α-amylase and α-glucosidase (Anderson & Polansky, 2002; Krentz & Bailey, 2005; Manzano & Williamson, 2010). Currently, several medicines have been used for treating diabetes. For example, sulfonylurea and biguanide are used to control postprandial blood glucose level, and metformin is used to stimulate the secretion of insulin. However, these commercial medicines may cause several side effects, including flatulence and diarrhea (Nathan et al., 2008). Therefore, it is necessary to explore some safe and ideal components and foods, especially from nature sources, for the

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prevention of postprandial hyperglycemia. Dietary therapy has been recognized as a promising way in preventing and treating type II diabetes. Natural products are in the key position for this, as many plant extracts have been explored for the antihyperglycemic effect (Hossain et al., 2002; Manzano & Williamson, 2010; Montagut et al., 2012; Thent, Seong Lin, Das, & Zakaria, 2012). Compared with synthetic drugs, natural plant ingredients are more available for consumers due to available originality and lowcost. Fruits and vegetables are considered as main dietary sources of phenolic compounds for human beings (Elosta, Ghous, & Ahmed, 2012). For example, polyphenols and phenolic acids from strawberry and apple could decrease glucose uptake (Manzano & Williamson, 2010). Polyphenolic profile extracts from freeze-dried black raspberrie could antioxidant and anti-diabetes (Xiao, Guo, Bi, & Zhao, 2017). Procyanidin extracted from grape-seed could activate the insulin receptor and improve the insulin signaling pathway (Montagut et al., 2012). Mulberry anthocyanin extract regulated glucose metabolism by promotion of glycogen synthesis and reduction of gluconeogenesis in human HepG2 cells (Yan, Zhang, Zhang, & Zheng, 2016). Apple is the pomaceous fruit of apple tree, species Malusdomestica in the rose family (Rosaceae). It has been reported that the content of total extractable polyphenols existing in fresh apple ranges from 230 to 360 mg/100 g (Podsedek, Wilska-Jeska, Anders, & Markowski, 2000), and the content of polyphenols in young apples is approximately ten times as that in fresh apples (Zheng, Kim, & Chung, 2012). In China, in

Corresponding authors. E-mail addresses: [email protected] (L. Sun), [email protected] (Y. Guo).

https://doi.org/10.1016/j.jff.2019.03.009 Received 18 September 2018; Received in revised form 28 January 2019; Accepted 8 March 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

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order to increase yield and improve quality of apple, there are about 1.6 million tons of young apples thinned and abandoned each year after blossom. Therefore, it is valuable to recycle these young apples and develop their potential values. In previous studies, the main components, antioxidant properties, preservative effects and in vitro inhibitory activity against α-amylase of young apple polyphenols (YAP) have been reported (Sun et al., 2016; Sun, Guo, Fu, Li, & Li, 2013; Sun, Sun, Thavaraj, Yang, & Guo, 2017). However, the effect of YAP on the activity of α-glucosidase that hydrolyzes the initial hydrolyzing product of starch by α-amylase, and the effect of YAP on starch digestion in vivo need to be further investigated to synthetically elucidate its anti-hyperglycemic effect. The effects of the YAP on α-amylase and α-glucosidase in vitro, as well as on postprandial blood glucose and insulin levels in vivo through acute and 1week intervention administrations were evaluated.

(methanol) and solvent B (0.1% formic acid in water) in a step gradient way was performed as follows: 0–3 min, 100% B; 3–19 min, 100–60% B; 19–31 min, 60–40% B; 31–32 min, 40–100% B; 32–41 min, 100% B. HPLC-ESI-ToF-MS/MS analysis was performed on an Agilent® 6460 QQQ system with an Agilent® 1260 HPLC (Agilent Technologies, USA) and an Agilent® 6550 iFunnel Q-TOF (Agilent Technologies, USA). The chromatographic separation conditions were the same as the above. Mass conditions were set as follows: negative ionization mode mass spectra: m/z 0–1200; nitrogen: 350 °C, 8 L/min; spray voltage: 4 kV; octopole RF amplitude: 150 Vpp; skim 1 voltag: 47.7 V; skim 2 voltage: 6.0 V; capillary exit: 127.3 V; cap exit offset: 79.6 V. In addition, Seg: drying gas: 280 °C, 13 L/min; sheath gas: 350 °C, 12 L/min; nebulizer: 20 psig. Eept: VCap: 4 kV; nozzle voltage: 1 kV. MS TOF: fragmentor: 350 V; skimmer: 0 V; OCT TRF Vpp: 750 V.

2. Materials and methods

2.5. Quantification of phenolic compounds

2.1. Materials

The contents of phenolic compounds in YAP were quantified by HPLC at 280 nm. The chromatographic separation conditions were used as the description in 2.4. Standard solutions were detected for constructing calibration curves. The calibration curves were established by plotting the peak areas versus their concentrations. The limit of detection (LOD) and quantification (LOQ) were evaluated at the concentrations that generated peaks with signal-to-noise values (S/N) of 3 and 10, respectively.

Two thinned young apple varieties, including brachyplast Fuji (BF) and Pink Lady (PL) were collected 30 days (April 2017) after blooming in Liquan, Shaanxi province, China. The harvest season of “BF and PL” is in October every year. YAP were separated and extracted from the thinned young apples using macroporous resin according to our previously reported method (Sun et al., 2013). The young apples were ground into particles by a grinder, and extracted with 70% ethanol at 65 °C for 3 h. Then, the extracting solution was filtered using Buchner funnel and concentrated to remove the ethanol in a rotary evaporator at 65 °C, and YAP was purified with X-5 resin using ethanol elution. Then, YAP solution was concentrated to remove the ethanol in a rotary evaporator and freeze-dried to obtain YAP powder. Porcine pancreatic αamylase (Product 10080) and Baker’s yeast α-glucosidase (Product G0660) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Phlorizin, chlorogenic acid, tannic acid (the structures of three polyphenols shown in Fig. 1) and p-Nitrophenyl-α-D-glucopyranoside (pNPG) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). All the other reagents used were of the highest grade commercially available.

2.6. Inhibition of enzymes in vitro 2.6.1. α-Amylase inhibition assay Inhibition of α-amylase in vitro by five phenolic compounds (YAP from BF, YAP from PL, phlorizin, chlorogenic acid, tannic acid) was examined according to one previous method (Sun, Warren, Netzel, & Gidley, 2016) with some modifications. Normal maize starch (20 mg/ mL) was dissolved in PBS buffer (pH 6.9), followed being cooked at 90 °C for 20 min, and then diluted to 10 mg/mL with PBS buffer when it was cooled to room temperature. Polyphenols were dissolved in PBS buffer (pH 6.9) with a series of concentration (concentrations presented in Fig. 3A), which were chosen according to the sample solubility and inhibitory activity of enzyme. Then, 50 μL of polyphenol solution was incubated with 50 μL of 7.65 U/mL α-amylase solution at 4 °C for 15 min, and the mixture of 50 μL of PBS and 50 μL of α-amylase solution were used as blank. After that, the polyphenol-enzyme mixture and the diluted starch of 4 mL were thoroughly mixed and digested at 37 °C. At 0, 4, 8, and 12 min, the reaction solution was thoroughly mixed and 300 μL of reaction solution was transferred into 1.5 mL centrifuge tubes containing 300 μL of 0.3 M Na2CO3 solution to stop the reaction (Tahir, Ellis, & Butterworth, 2010). Then, the mixture in the centrifuge tubes was centrifuged at 12,000g for 5 min, and 100 μL of the supernatant was withdrawn into 1.5 mL centrifuge tubes. The sugar content in the supernatant was determined using the reported method (Sun et al., 2016). An assay time of 5 min was taken as the standard after heating in boiling water, because the rate of production of reducing sugar was linear up to this point. The absorbance values were examined by UV–VIS spectrophotometer (Shimadzu®, Japan) at 410 nm can be converted to the concentration of sugar (maltose equivalents) through the obtained maltose standard curve, and the inhibition of α-amylase was calculated as follows:

2.2. Total polyphenols, tannins content The content of total polyphenols was determined by Folin-phenol method used gallic acid as the standard and tannins was determined by colorimetric method used tannic acid as the standard (Muthukumaran et al., 2016). 2.3. Preparation the standard solution and sample solution The standard solutions were prepared freshly. Each standard was weighed accurately, and dissolved in methanol-water (80:20, v/v) as the stock solution. Then, stock solution containing 9 reference standards was diluted to 6 appropriate concentrations with methanol-water (80:20, v/v) defined as standard solutions. 60 mg YAP was dissolved in methanol-water (80:20, v/v) in 100 mL volumetric flask as the sample solution. 2.4. Identification of phenolic compounds The phenolic compounds in young apples were identified by high performance liquid chromatography (HPLC) equipped with a Thermo®UV–Vis detector and Agilent®C18 column (250 × 4.6 mm I.D., 5 μm) (California, U.S.A.). YAP was dissolved in methanol-water (80:20, v/v), and analyzed at a flow rate of 1.0 mL/min. The column oven was maintained at 35 °C. The injection volume was 4 μL, and the detection wavelength was 280 nm. Mobile Phase with solvent A

Inhibition (%) =

V1 − V 2 × 100% V1

(1)

where V1 was defined as the initial reaction velocity without polyphenols and V2 was defined as the initial reaction velocity with different polyphenols. The IC50 values for different polyphenols were calculated as follows (Epand et al., 2007): 128

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Fig. 1. Structures of phenolic compounds in YAP. Phlorizin (A), chlorogenic acid (B) and tannic acid (C).

IC50 ⎞ Inhibition (%) = Imax ⎛1 − [I ] + IC50 ⎠ ⎝ ⎜

2.7. In vivo studies

⎟

(2) 2.7.1. Animals and treatments Male C57 BL/6J mice (300–350 g body mass) were purchased from The Fourth Army Medical University (Xi’an, China). All the experimental mice were housed individually in stainless steel cages under standard laboratory conditions at 25 °C with 12 h of light and dark cycles, 40–70% humidity and free access to diet and water. All the experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. After 1-week acclimatization, the mice were subjected to study the acute hypoglycemic effect of four phenolic compounds (YAP from BF, phlorizin, chlorogenic acid, tannic acid). The mice were divided into five groups (n = 8) according to the body weight. Each group of mice was fasted for 18 h with free access to sterile water prior to treatments. The fasted mice were given maize starch (5 g/kg b.w., i.g.) alone or with YAP (150 mg/kg b.w., i.g.), phlorizin (150 mg/kg b.w., i.g.), chlorogenic acid (150 mg/kg b.w., i.g.), or tannic acid (150 mg/kg b.w., i.g.). Blood glucose was measured at 0, 30, 60, 90 and 120 min after feeding using a blood glucose meter, blood from tail vein was into tubes containing EDTA centrifuged at 3200g for 15 min at 4 °C and plasma was obtained and stored at −20 °C for plasma insulin analysis (Forester, Gu, & Lambert, 2012; Sun et al., 2017). Area under the curve (AUC) was calculated using the trapezoidal rule. In addition, the 1-week intervention hypoglycemic effect of polyphenols was studied as well. For this study, the mice were divided into five groups (n = 8), and were given YAP (150 mg/kg b.w., i.g.), phlorizin (150 mg/kg b.w., i.g.), chlorogenic acid (150 mg/kg b.w., i.g.), tannic acid (150 mg/kg b.w., i.g.) or saline for 6 days continuously, during which all the mice were allowed to free access to water and foods. At the 7th day, all the mice were treated with maize starch (5 g/kg b.w., i.g.), and the blood glucose was measured at 0, 30,

where [I ] is the inhibitor concentration, and Imax is the maximum percentage inhibition, %. IC50 values were calculated using a log (inhibitor) vs. normalized response curve.

2.6.2. α-Glucosidase inhibition assay α-Glucosidase inhibitory activity by five phenolic compounds (YAP from BF, YAP from PL, phlorizin, chlorogenic acid, tannic acid) was assayed in vitro using one previously reported method (Apostolidis & Lee, 2010) with some modifications. Polyphenols were dissolved in PBS buffer (pH 6.9) with a series of concentration (concentrations presented in Fig. 3B), which were chosen according to the sample solubility and inhibitory activity of enzyme. Then, 200 μL of polyphenol solution were incubated with 200 μL of 4 U/mL α-glucosidase solution and 4 mL of PBS at 4 °C for 15 min, 200 μL of PBS with 200 μL of 4 U/mL α-glucosidase solution and 4 mL of PBS were used as blank. After that, the mixture and 500 μL of 10 mM pNPG solution in PBS were thoroughly mixed and incubated at 37 °C. At 0, 3, 6, and 9 min after the addition of pNPG, the reaction solution was thoroughly mixed and 500 μL of solution was transferred into 1.5 mL centrifuge tubes containing 500 μL of 0.3 M Na2CO3 solution to stop the reaction. Then, the absorbance values were examined by UV–VIS spectrophotometer (Shimadzu®, Japan) at 400 nm. In addition, the inhibitory activity of phlorizin on α-glucosidase has been reported (Cho et al., 2013). Thus, phlorizin was used as positive control. The inhibition of α-glucosidase was calculated as the formula (1), and the IC50 values for different polyphenols were calculated as the formula (2).

129

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60, 90 and 120 min after feeding using blood glucose meter, blood from tail vein was into tubes containing EDTA centrifuged at 3200g for 15 min at 4 °C and plasma was obtained and stored at −20 °C for plasma insulin analysis (Forester et al., 2012; Sun et al., 2017). Area under the curve (AUC) was calculated using the trapezoidal rule. 2.7.2. Plasma insulin measurement The plasma insulin level was determined using the Rat/Mouse Insulin ELISA Kit from Shanghai Xitang Biotechnology Co., Ltd. (Shanghai, China), according to the manufacturer’s instructions using Thermo Scientific Multiskan Spectrum (Waltham, MA, USA). 2.8. Statistical analysis The data were presented as the means ± SEM in triplicate experiments and evaluated by one-way analysis of variance (ANOVA) using SPSS 18.0 Statistics (SPSS Inc., Chicago, IL, USA). Statistical analyses were performed using the Student’s t-test. The inhibition ratio and IC50 value of enzyme activity were calculated using related equation, area under the curve (AUC) value was calculated using the trapezoidal rule, and charts were drawn with the software Origin® 8.0. * Denotes the difference was statistically significant from control group. The differences between the groups were defined as significantly at p < 0.05. 3. Results and discussion Fig. 3. Inhibition of α-amylase (A) and α-glucosidase (B) by different concentrations of YAP from BF, YAP from PL, phlorizin, chlorogenic acid and tannic acid. All the curves are fitted based on Eq. (2) used for calculating IC50 values.

3.1. Phenolic compounds in YAP The chromatographic fingerprints of polyphenols in the samples were initially analyzed using HPLC at 280 nm and presented in Fig. 2. Based on the chromatograms, BF and PL shared the same fingerprints with 15 peaks were detected. Notably, peaks 6 and 15 had a big advantage in peak area and can be considered as the main phenolic

x10 3

Phlorizin

167.0420

5 4.8 4.6 4.4

15

4.2 4 3.8 3.6 273.0858 1

3.4 3.2 3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 125.0300

1.2 1 0.8 0.6 0.4 0.2 0 60

70

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110

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130

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190

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240

250 260 270 280 290 300 Counts vs. Mass-to-Charge (m/z)

310

320

330

340

350

360

370

380

390

400

410

420

430

440

450

460

470

480

490

x10 3

Chlorogenic acid

191.0627 1

3.2 3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1

353.0999

0.8 0.6 0.4 0.2 0 60

70

80

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100

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120

130

140

1 7

10

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220 230 240 250 260 Counts vs. Mass-to-Charge (m/z)

2 3 4 5

270

280

290

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310

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330

340

350

360

370

380

390

400

410

420

6 7 8

9

10

14 11 12 13

18

14

22

Retention Time (min) Fig. 2. HPLC chromatogram of phenolic compounds in YAP, and the HPLC-ESI-ToF-MS/MS ion peaks of phlorizin and chlorogenic acid. 1, unknown (10.9 min); 2, procyanidin B1(12.7 min); 3, epigallocatechin (12.9 min); 4, catechin (13.1 min); 5, procyanidin B2 (13.8 min); 6, 5-caffeoylquinic acid (14.4 min); 7, 4-P-coumaroylquinic acid (14.8 min); 8, epicatechin (15.4 min); 9, caffeic acid (16.0 min); 10, procyanidin C1 (16.2 min); 11, quercetin-3-O-glucoside (17.1 min); 12, quercetin-3-O-arabinoside (17.5 min); 13, quercetin-3-O-rhamnoside (18.6 min); 14, quercetin-3-O-galactoside (18.9 min); 15, phlorizin (20.1 min). 130

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Table 1 The phenolic composition in YAP. Peak

Rt (min)

λmax

[M−H]−(M/Z)

MS2 (M/Z)

Tentative identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

10.9 12.7 12.9 13.1 13.8 14.4 14.8 15.4 16 16.2 17.1 17.5 18.6 18.9 20.1

275 289 280 279 279 325 311 279 279 280 350 350 350 350 285

375.3424 577.1495 304.9231 289.0804 577.1477 353.0999 337.1032 289.0807 179.0838 865.2327 463.1130 433.0891 447.1054 463.2692 435.1413

327.8766(15),165.3526(100),121.7322(17) 425.8675(100), 451.1127(24), 407.3068(21), 289.0808(18) 254.3750(16), 203.9231(41), 126.0866(100) 290.0871(100), 244.7619(18) 425.6721(100), 451.1418(24), 407.3068(21), 289.0807(18) 191.0627(100) 173.3215(100), 163.5241(21) 289.0808(100), 245.5672(51), 205.3311(11), 179.2671(9) 179.2671(100), 87.1531(51) 713.6215(85), 575.3286(51), 425.8674(21), 407.3086(41), 287.3261(16) 301.3068(100), 300.5836(61) 365.1418(41), 271.0981(58) 301.3144(100), 300.5836(58) 301.3068(100), 300.4174(62) 273.0858(100), 167.0420(14),125.0300(5)

Unknown Procyanidin B1 Epigallocatechin Catechin Procyanidin B2 5-Caffeoylquinic acid 4-P-coumaroylquinic acid Epicatechin Caffeic acid Procyanidin C1 Quercetin-3-O-glucoside Quercetin-3-O-arabinoside Quercetin-3-O-rhamnoside Quercetin-3-O-galactoside Phlorizin

the YAP was second to that of phlorizin with 112.51 and 76.48 μg/mg from BF and PL, respectively. Other phenolic compounds existed in YAP only in a small proportion (Table 2). For examples, epigallocatechin had the content of 40.23 and 90.21 μg/mg in YAP from BF and PL, respectively, followed by epicatechin with the content of 32.67 and 18.52 μg/mg, quercetin-3-O-galactoside was quantified as 37.52 and 16.00 μg/mg, and quercetin-3-O-rhamnoside was 18.54 and 39.18 μg/ mg in YAP from BF and PL, respectively. Besides, procyanidin B1, B2 and C1, catechin, quercetin-3-O-glucoside, quercetin-3-O-arabinoside, 4-p-coumaroylquinic acid and caffeic acid also existed in YAP, but the contents were very low.

compounds existing in YAP. They were then identified using HPLC-ESIToF-MS/MS in a negative mode by comparing the detail MS data (Table 1) with published data (Habib, Plata, Meudec, Cheynier, & Ibrahim, 2014; Pires et al., 2018). Peak 6 exhibited the molecular ion of M− = 353.0999 and showed the characteristic fragment ion at 191.0627, therefore, it could be tentatively identified as 5-caffeoylquinic acid. Peak 15, presenting a molecular ion of M− = 435.0858, might correspond to a phloretin containing a glucoside unit, owing to the characteristic fragments at m/z 273.0858, 167.0420 and 125.0300. So peak 15 was tentatively identified as phlorizin (phloretin 2′-glucoside). For comprehensive understanding phenolic composition in the samples, the remaining peaks were also identified through comparison of the detailed MS data with published data, which were shown in Table 1. These phenolic compounds were quantified by HPLC at 280 nm using external standard method. The calibration curves were linear in the concentration ranges of standard samples, which were verified by Correlations (R2) obtained from the data of six standard samples, and a signal-to-noise ratio (S/N) of 3 and 10 was considered as LOD and LOQ, respectively (Table 2). The total content of polyphenols was determined by Folin-phenol method. The contents of individual and total polyphenols were summarized in Table 2. Notably, phlorizin was the dominant phenolic compounds in YAP with the content of 161.50 and 229.72 μg/mg from BF and PL, respectively. It has been reported that the content of phlorizin in ripe apples and corresponding apple juices are only in a small proportion (Malec et al., 2014). Hence, phlorizin mainly exists in young apples. The content of 5-caffeoylquinic acid in

3.2. Inhibition of α-amylase and α-glucosidase in vitro It has been reported that type II diabetes may be managed by inhibiting the activities of carbohydrate-hydrolyzing enzymes, through which the increase of postprandial blood glucose is supposed to be retarded. In this study, we investigated the inhibition of polyphenols in young apples on two key starch digestion enzymes, including α-amylase and α-glucosidase (Fig. 3). As shown in Table 1, phlorizin and chlorogenic acid are two main phenolic compounds in YAP. Although YAP contained no tannic acid through MS analysis, tannic acid was used as the equivalent standard of total tannins as suggested previously (Muthukumaran et al., 2016). Therefore, the inhibitory activities against the digestive enzymes were investigated for YAP, phlorizin, chlorogenic acid and tannic acid in this study. The results showed that the activities of both the enzymes decreased as the concentrations of

Table 2 Calibration and quantitation of phenolic compounds. Standard Compounds

Procyanidin B2

Quercetin

Epigallocatechin Catechin Chlorogenic acid 4- hydroxybenzoic acid Epicatechin Caffeic acid Phlorizin Tannic acid Gallic acid

Compounds

Procyanidin B1 Procyanidin B2 Procyanidin C1 Quercetin-3-O-glucoside Quercetin-3-O-arabinoside Quercetin-3-O-rhamnoside Quercetin-3-O-galactoside Epigallocatechin Catechin 5-Caffeoylquinic acid 4-P-coumaroylquinic acid Epicatechin Caffeic acid Phlorizin Total Tannins Total polyphenols

Calibration curves

Correlations (R2)

Linearity range (ug/ml)

LOQ (ug/mL)

LOD (ug/mL)

Y = 5.6111X + 0.1702

0.9995

1.00–50.00

0.43

0.13

Y = 5.2574X + 0.3201

0.9997

1.00–50.00

0.28

0.08

Y = 1.8038X + 0.0456 Y = 7.8438X + 0.0506 Y = 8.1265X + 0.0391 Y = 8.0656X + 0.1713 Y = 3.1856X + 0.1632 Y = 8.1422X + 0.0387 Y = 7.4572X + 1.327 Y = 0.0137X + 0.0363 Y = 0.4383 X + 0.1114

0.9998 0.9999 0.9998 0.9997 0.9996 0.9999 0.9995 0.9993 0.9991

10.00–200.00 1.00–50.00 10.00–200.00 1.00–50.00 1.00–50.00 1.00–50.00 10.00–300.00 0.00–500.00 0.00–500.00

1.08 0.91 0.75 0.35 0.46 0.68 0.10 – –

0.32 0.27 0.23 0.11 0.14 0.20 0.03 – –

131

Contents (ug/mg) BP

PL

3.23 12.18 9.82 8.74 9.29 18.54 37.52 40.23 1.67 112.51 9.23 32.67 3.82 161.50 383.22 489.59

2.21 7.53 25.67 7.71 5.17 39.18 16.00 50.21 3.51 116.48 5.81 18.52 7.79 229.72 403.81 454.06

5.1 ± 0.4 10.9 ± 0.6 18.3 ± 1.0 25.6 ± 1.2 5.1 ± 0.4 10.6 ± 0.6 17.1 ± 0.9* 23.8 ± 1.1* 5.0 ± 0.5 10.3 ± 0.7 16.3 ± 0.7* 22.5 ± 0.9* 5.3 ± 0.6 11.5 ± 0.7 19.4 ± 1.5 26.7 ± 1.7 3702.0 ± 146.1 7617.5 ± 202.3* 11602.7 ± 287.9* 15497.7 ± 321.1* 3759.3 ± 110.2 7947.2 ± 163.9 12226.8 ± 241.4 16211.7 ± 310.9 3756.9 ± 109.6 7862.1 ± 197.5* 12069.0 ± 265.9* 16096.8 ± 301.3 3841.2 ± 116.3 8184.6 ± 257.9 12551.4 ± 301.9 16529.4 ± 355.7 (B) 0–30 0–60 0–90 0–120

3677.2 ± 151.7* 7620.9 ± 209.8* 11700.0 ± 289.9* 15657.0 ± 331.1*

5.0 ± 0.5 10.2 ± 0.7* 17.0 ± 0.8* 24.9 ± 1.2* 5.0 ± 0.4 11.2 ± 0.7 19.3 ± 1.0 27.4 ± 1.3 5.1 ± 0.3 10.5 ± 0.7* 17.5 ± 0.9* 25.6 ± 1.3* 4.9 ± 0.4* 10.1 ± 0.6* 17.0 ± 1.0* 25.0 ± 1.2* 5.3 ± 0.5 12.1 ± 0.8 21.0 ± 1.1 29.4 ± 1.3 3595.5 ± 137.1* 7300.8 ± 1 88.2* 11059.7 ± 219.5* 14708.0 ± 242.3* 3729.9 ± 102.3 7941.9 ± 179.5* 12208.2 ± 218.4* 16010.6 ± 287.3 3758.1 ± 99.7 7862.2 ± 174.3* 12058.8 ± 196.8* 16017.8 ± 246.3 3661.7 ± 138.6* 7472.6 ± 199.2* 11291.9 ± 283.5* 14973.2 ± 301.2* 3822.2 ± 108.2 8334.8 ± 236.3 12807.8 ± 269.5 16561.8 ± 304.7 (A) 0–30 0–60 0–90 0–120

Control

Phlorizin

YAP

Chlorogenic acid

132

Tannic acid

polyphenols increased. The inhibitory activities against α-amylase were in the order of YAP from BF > YAP from PL > tannic acid > chlorogenic acid, as suggested by the IC50 values in Table 3. However, phlorizin showed almost no inhibitory effect. Besides, it was found that the inhibition on α-glucosidase was in the order of tannic acid > YAP from BF > YAP from PL > phlorizin (Table 3). Interestingly, chlorogenic acid showed almost no inhibitory effect on this enzyme.Table 4. α-Amylase and α-glucosidase are two key enzymes in hydrolyzing dietary starch, through which starch is hydrolyzed into maltose and other oligosaccharides by α-amylase via breaking the α-1,4-glycosidic bonds, and then degraded into glucose by α-glucosidase located in the brush-border surface membrane of intestinal cells. Glucose is further absorbed by intestinal epithelial cell, leading to the increase in blood glucose level. It has been reported that formation of hydrogen bonding between the hydroxyl groups of polyphenols and amino acid residues at the active site of α-amylase contributes to the inhibition of the enzyme (Lo Piparo et al., 2008). Tannic acid is composed of ten galloyl moieties (Fig. 1C), and each galloyl group provides three hydroxyl groups. In addition, the carbonyl group existing in the galloyl group is conjugated to the benzene ring, which has been certified to stabilize the binding interactions between polyphenols and amino acid residues at the active site of α-amylase (Lo Piparo et al., 2008). Therefore, tannic acid showed relatively strong inhibition on α-amylase. Chlorogenic acid contains two neighboring hydroxyl groups existing in the catechol ring (Fig. 1B), which has been reported to strengthen the inhibitory activity of chlorogenic acid family on α-amylase (Narita & Inouye, 2011). Besides, the conjugated structure formed by C]C double bonds and carbonyl groups also exists in chlorogenic acid molecule (Lo Piparo et al., 2008). Hence, chlorogenic acid exhibited considerable inhibitory activity on the enzyme as well. For α-glucosidase, previous studies have demonstrated that polyphenols with more hydroxyl groups can more effectively inhibit the activity of α-glucosidase (Kim, Nam, Kurihara, & Kim, 2008), which may explain the relatively high inhibitory activity of tannic acid against the enzyme. Besides, in addition to polyphenolic hydroxyl groups, there might be an inhibitor-enzyme binding site blocking the substrate-enzyme binding (Sun et al., 2016), or interactions between polyphenols and maize starch preventing the substrateenzyme binging (Xu, Leng, Wang, & Zhang, 2012), both of which may result in the inhibition of α-glucosidase activity by the polyphenols in thinned young apples, and two hundreds more potent than phlorizin. Interestingly, phlorizin has the inhibitory activity against α-glucosidase but not against α-amylase, while chlorogenic acid has the inhibitory activity against α-amylase but not against α-glucosidase. As YAP contains both the phenolic compounds, it can develop the inhibitory activities against both the enzymes. In addition, procyanidin, catechin, epicatechin and caffeic acid have been identified to inhibit the activity of both starch-hydrolyzing enzymes (Bellesia & Tagliazucchi, 2014; Chiou, Sung, Huang, & Lin, 2017; Kawakami, Aketa, Nakanami, Iizuka, & Hirayama, 2010; Loo & Huang, 2007; Sun et al., 2016; Zhang, Seeram, Lee, Feng, & Heber, 2008). What’s more, flavonols also have been reported to inhibit the activity of α-amylase with a low level of IC50 values (Lo Piparo et al., 2008). Therefore, YAP showed considerable inhibition on starch-hydrolyzing enzymes in vitro may be due to synergistic effect of phenolic compounds existing in the young apples,

Chlorogenic acid

Table 4 Effects of YAP, phlorizin, chlorogenic acid and tannic acid on postprandial blood glucose and insulin IAUC in mice for the acute (A) and one-week intervention (B) administrations.

“–” represents that the polyphenols showed almost no inhibition on the enzymes.

YAP

0.0072 0.0102 0.0009 – 2.1916

Phlorizin

0.15 0.22 0.86 8.62 –

Control

YAP from BF YAP from PL Tannic acid Chlorogenic acid Phlorizin

Insulin IAUC (ng/mL)

α-glucosidase (mg/mL)

Glucose IAUC (mg/dL)

α-amylase (mg/mL)

Time (min)

Sample

Tannic acid

Table 3 IC50 values of five phenolic compounds against α-amylase and α-glucosidase.

5.1 ± 0.6 10.8 ± 0.7 16.9 ± 1.0* 23.0 ± 1.1*

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acute administration. Co-administration of phenolic compounds in young apples with maize starch significantly reduced the increase in the levels of blood glucose and insulin after meal, obviously suppressing the peak blood glucose level at 60 min and the peak insulin level at 90 min with the dosage of 150 mg/kg compared with the control group (p < 0.05). The decreasing effects of the four phenolic compounds were in the order of tannic acid > phlorizin > YAP > chlorogenic acid (Fig. 4A and B). YAP decreased the blood glucose level by 13.3% at 60 min and the insulin level by 16.2% at 90 min compared with control group, which were verified by iAUC calculated over a time period of 60 and 90 min, although there were no difference in the total amount of glucose absorption calculated as area under the curve (Table 4). In addition, for the 1-week intervention experiment, it was found that after 6 days phenolic compound administration continuously, at the 7th day the increase of postprandial blood glucose and insulin levels after giving maize starch by gavage were also decreased, and the peak blood glucose at 60 min and the peak insulin at 90 min were suppressed by the polyphenols with the dosage of 150 mg/kg compared to the control group (p < 0.05, except for chlorogenic acid). The decreasing effects of the four polyphenols were in the order of tannic acid > phlorizin > YAP > chlorogenic acid (Fig. 4C and 4D), and YAP decreased the blood glucose level by 7.3% at 60 min and the insulin level by 18.5% at 90 min compared with control group, and similar iAUC were shown in Table 4. The acute in vivo results indicate

and which indicate that YAP may have potentials in inhibiting starch digestion in vivo, and can be suggested by the measurement of blood glucose and insulin levels in mice after meal. Thus, the in vivo effects of YAP and pure polyphenols on the levels of postprandial blood glucose and insulin were performed in our study for both acute and 1-week intervention experiments. 3.3. Effects on blood glucose and insulin levels Hyperglycemia that has been recognized as a critical factor in the development of type II diabetes, and a series of long-term complications such as retinopathy, cardiovascular and renal diseases may be caused by persistent high blood glucose levels as well (Rios, Francini, & Schinella, 2015). Insulin is a protein hormone secreted by pancreatic βcells when it is stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine and glucagon, and it is the only hormone that is used to reduce blood sugar. The incidence of type II diabetes disease is increasing by years, and its development has been reported to be potentially prevented or delayed by controlling postprandial blood glucose and insulin levels (Wein & Wolffram, 2014). Therefore, we studied the effects of YAP, phlorizin, chlorogenic acid and tannic acid on the two index levels in normal mice by acute and 1week intervention experiments. Fig. 4A and B describe the levels of blood glucose and insulin for the

Fig. 4. Effects of YAP, phlorizin, chlorogenic acid and tannic acid on postprandial blood glucose (A) and insulin levels (B) in mice for the acute administration; Mice were treated with YAP, phlorizin, chlorogenic acid or tannic acid (150 mg/kg, i.g.) in combination with maize starch (5 g/kg, i.g.). Effects of YAP, phlorizin, chlorogenic acid and tannic acid on postprandial blood glucose (C) and insulin levels (D) in mice for the 1-week intervention administration. Mice were pretreated with YAP, phlorizin, chlorogenic acid or tannic acid (150 mg/kg/day, i.g.) for 6 days continuously, during which all the mice were allowed to free access to water and foods. At day 7, all the mice were treated with maize starch (5 g/kg, i.g.), blood glucose and insulin levels were measured. Results are shown as means ± SD (n = 8), *p < 0.05. 133

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stimulating insulin secretion and improving the function of β-cell (Rowley et al., 2017). To support the anti-hyperglycemic activity of YAP, the effects of YAP on glucose transporters, such as SGLT1 and GLUT2, and insulin secretion need to be further studied. In addition, the therapeutic effects of YAP on type II diabetes that can be performed through establishment of diabetic mice model are in progress as well.

that YAP, phlorizin, tannic acid and chlorogenic acid all could develop the inhibitory activities against the hydrolyzing enzymes in vivo, delaying the starch digestion and thereby suppressing the increase in postprandial blood glucose and insulin levels. Besides, the 1-week intervention results suggest that long term intake of polyphenols in young apples can also help control postprandial blood sugar level as well. The level of insulin is closely related to the level of blood glucose. The secretion of insulin can be divided into two classes, one is for maintaining fasting blood glucose in a normal range and is called basic insulin, and another one is for decreasing postprandial blood glucose to a normal range and is called postprandial insulin. The secretion of postprandial insulin aims at controlling the extent and duration of postprandial hyperglycemia at a level that closes to the fasting state at any time. Although it has been reported that polyphenols in some plants could inhibit increase in blood glucose level due to promotion of insulin secretion, such as tea polyphenols, persimmon-leaf polyphenols and apple-leaf extracts (Anderson & Polansky, 2002; Kawakami et al., 2010; Shirosaki, Koyama, & Yazawa, 2012), the insulin secretion was found to be inhibited in our study. This results from the inhibited release of glucose from starch by polyphenols in young apples (Fig. 3) and thus decreased the need for insulin secretion. Additionally, after starch digestion, the glucose in intestinal lumen is transported into blood through glucose transporters in enterocyte, such as sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). It has been reported that some polyphenols from strawberry and persimmon were able to inhibit the expression of SGLT1 and GLUT2, thereby to retard the absorption of glucose (Hossain et al., 2002; Manzano & Williamson, 2010). Notably, phlorizin has been reported to be an inhibitor of SGLT1 (Schulze et al., 2014). Therefore, the decrease of insulin secretion may be attributed to the inhibition of carbohydratehydrolyzing enzymes and SGLT1, and further experiments are necessary to verify this hypothesis. Type II diabetes is closely associated with elevated postprandial hyperglycemia that can be controlled by different mechanisms including retarding the starch digestion, inhibiting glucose transporter in the small intestine, such as SGLT1 and GLUT2, suppressing hepatic glucose production, increasing insulin sensitivity, and modulating intracellular signaling pathways related to glucose homeostasis. Some synthetic medicines have been employed to relieve type II diabetes disease regarding retarding starch digestion, including acarbose, miglitol and voglibose. However, because of undesirable side effects, they are not suggested to be used frequently (Hanefeld, 1998). In this study, we have demonstrated that YAP can delay the increase of postprandial blood glucose and insulin levels effectively through delaying starch digestion. Every year, million tons of thinned young apples are discarded in grove after fruit and flower thinning process (Zheng et al., 2012). Thus, YAP is possibly with potential to be developed as a functional food in relieving and preventing type II diabetes symptom. The main ingredients of YAP are phlorizin, chlorogenic acid and tannins. In this study, the levels of postprandial blood glucose and insulin were found to be decreased by YAP through the potential inhibition of α-amylase by chlorogenic acid and tannins, inhibition of α-glucosidase by phlorizin and tannins, as well as the inhibition of SGLT1 by phlorizin as reported (Schulze et al., 2014). Besides, other phenlic compounds in YAP also play a role in controlling the hyperglycemia. It has been reported that quercetin-3-O-rhamnoside and 5-caffeoylquinic acid could anti-hyperglycemia by inhibiting glucose transporter GLUT2 (Bondonno, Bondonno, Ward, Hodgson, & Croft, 2017). Procyanidins extracted from grape-seed could decrease hyperglycaemia by stimulating glucose uptake in insulin-sensitive cell lines (Montagut et al., 2012). Epicatechin and caffeic acid could strengthen the insulin signaling by activating the key proteins of the insulin pathways and regulating the glucose production through PI3K/AKT and 5′-AMP-activated protein kinase, alleviate insulin resistance (Cordero-Herrera, Angeles Mart, Bravo, Goya, & Ramos, 2013; Da, Szu, & James Swi, 2009). Catechin can be used to improve the type II diabetes via

4. Conclusions The polyphenols in young apples were identified by HPLC-ESI-ToFMS/MS and quantified by HPLC in this study. Chlorogenic acid, phlorizin and tannins are the main phenolic compounds in young apple polyphenols. Then, the effects of YAP on starch digestion in vitro and in vivo were investigated. It was found that YAP, tannic acid and chlorogenic acid could inhibit the activity of α-amylase in vitro to some extent, while phlorizin had no inhibition on the enzyme. Besides, YAP, tannic acid and phlorizin were able to inhibit α-glucosidase in vitro, while chlorogenic acid had no inhibitory activity against the enzyme. Furthermore, YAP was found to retard the increase of postprandial blood glucose and insulin levels in mice for both the acute and 1-week intervention trials. This may be due to the inhibition of both α-amylase and α-glucosidase by YAP in vitro. The retarding effect on starch digestion indicates that YAP may be applied as an alternative to the prescribed medicines in relieving postprandial hyperglycemia and thus type II diabetes disease. Ethics statements All the experiments were performed in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. Conflict of interest The authors claim no conflict of interest in this work. Acknowledgements This study is supported by China Agriculture Research System (CARS-27) from Ministry of Agriculture of People’s Republic of China. References Anderson, R. A., & Polansky, M. M. (2002). Tea enhances insulin activity. Journal of Agricultural and Food Chemistry, 50(24), 7182–7186. Apostolidis, E., & Lee, C. M. (2010). In vitro potential of Ascophyllum nodosum phenolic antioxidant-mediated alpha-glucosidase and alpha-amylase inhibition. Journal of Food Science, 75(3), H97–102. Bellesia, A., & Tagliazucchi, D. (2014). Cocoa brew inhibits in vitro α-glucosidase activity: The role of polyphenols and high molecular weight compounds. Food Research International, 63, 439–445. Bondonno, N. P., Bondonno, C. P., Ward, N. C., Hodgson, J. M., & Croft, K. D. (2017). The cardiovascular health benefits of apples: Whole fruit vs. isolated compounds. Trends In Food Science & Technology, 69, 243–256. Cavalot, F., Petrelli, A., Traversa, M., Bonomo, K., Fiora, E., Conti, M., ... Trovati, M. (2006). Postprandial blood glucose is a stronger predictor of cardiovascular events than fasting blood glucose in type 2 diabetes mellitus, particularly in women: Lessons from the San Luigi Gonzaga Diabetes Study. The Journal of Clinical Endocrinology and Metabolism, 91(3), 813–819. Chiou, S. Y., Sung, J. M., Huang, P. W., & Lin, S. D. (2017). Antioxidant, antidiabetic, and antihypertensive properties of Echinacea purpurea flower extract and caffeic acid derivatives using in vitro models. Journal of Medical Food, 20(2), 171–179. Cho, J. Y., Lee, K. D., Park, S. Y., Jeong, W. C., Moon, J. H., & Ham, K. S. (2013). Isolation and identification of α-glucosidase inhibitors from the stem bark of the nutgall tree. Journal of the Korean Society for Applied Biological Chemistry, 56, 547–552. Cordero-Herrera, I., Angeles Mart, M., Bravo, L., Goya, I., & Ramos, S. (2013). Cocoa flavonoids improve insulin signaling and modulate glucose production via AKT and AMPK in HepG2 cells. Molecular Nutrition and Food Research, 57(6), 974–985. Elosta, A., Ghous, T., & Ahmed, N. (2012). Natural products as anti-glycation agents: Possible therapeutic potential for diabetic complications. Current Diabetes Reviews, 8(2), 92–108.

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