Persimmon tannin decreased the glycemic response through

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Persimmon tannin decreased the glycemic response through decreasing the digestibility of starch and inhibiting #amylase, #-glucosidase and intestinal glucose uptake Kaikai li, fen Yao, Jing Du, Xiangyi Deng, and Chun-mei Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05833 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 3, 2018

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Journal of Agricultural and Food Chemistry

Persimmon tannin decreased the glycemic response through decreasing the digestibility of starch and inhibiting α-amylase, α-glucosidase and intestinal glucose uptake § § Kaikai Lia , Fen Yaoa , Jing Dua, Xiangyi Denga, Chunmei Lia, b*

a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan,

China, 430070 b

Key Laboratory of Environment Correlative Food Science (Huazhong Agricultural

University), Ministry of Education §

These authors contribute to this paper equally.

*Corresponding author: Chunmei Li (Tel: 86-27-87282966; Fax: 86-27-87282966; E-mail: [email protected])

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ACS Paragon Plus Environment


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ABSTRACT: Regulation of postprandial blood glucose levels is an effective

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therapeutic proposal for Type 2 diabetes treatment. In this study, the effect of

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persimmon tannin on starch digestion with different amylose levels was investigated

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both in vitro and in vivo. Oral administration of persimmon tannin-starch complexes

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significantly suppressed the increase of blood glucose levels and the area under the

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curve (AUE) in a dose-dependent manner compared with starch treatment alone in an

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in vivo rat model. Further study proved that persimmon tannin could not only interact

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with starch directly, but also inhibit α-amylase and α-glucosidase strongly with IC50

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values of 0.35 mg/mL and 0.24 mg/mL, separately. In addition, 20 µg/mL of

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persimmon tannin significantly decreased glucose uptake and transport in Caco-2

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cells model. Overall, our data suggested that persimmon tannin may alleviate

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postprandial hyperglycemia through limiting the digestion of starch as well as

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inhibiting the uptake and transport of glucose.

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Kew Words:

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Persimmon tannin; Blood glucose levels; Starch digestibility; α-amylase and

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α-glucosidase; Glucose uptake and transport

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Introduction

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Diabetes is characterized by chronic hyperglycemia, which leads to an increased risk

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of retinopathy, cardiovascular disease, nephropathy, and metabolic syndrome.1-2

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Diabetes has received increasing attention due to the rising prevalence (415 million in

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2016 over the world) suffering from this disease and its related disease. Postprandial

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hyperglycemia is the main risk factor in the development of Type II diabetes.

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Therefore, the most effective therapeutic proposal for Type 2 diabetes treatment is to

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regulate post-prandial carbohydrate absorption and delay glucose uptake. Starch is

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the main dietary carbohydrate and also a major contributor for blood glucose.

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Therefore, inhibiting digestion of starch and reducing glucose uptake in small

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intestine are effective strategies to control the blood glucose in diabetic patients.3-4

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The natural products, especially those from food materials, have been proved with

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multi-health benefits on human. Therefore, applying these natural products to inhibit

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the starch digestion and glucose uptake may be an important strategy in the

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management of hyperglycemia linked to type II diabetes. Evidence from a number of

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in vitro and in vivo studies indicated that the polyphenols, such as tea catechins,5

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showed inhibitory effect on starch digestion and adsorption through inhibiting the

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digestive enzymes (α-amylase and α-glucosidase) and glucose transporter proteins

37

(GLUTs).6

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Tannins is a kind of procyanidin polymers resulting from the polymerization of

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flavan-3-ol units, which widely distributed in food materials, such as sorghum, cocoa,

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grape seed, persimmon, and other fruits and vegetables.7-9 Influences of tannin from 3



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sorghum on starch digestion have been well documented in previous studies.10-12

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Beside its inhibitory activity on the digestive enzymes, tannin also showed strong

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interaction with starch, thus reducing the starch digestibility,11, 13-14 which worked

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together for its inhibitory potential on the digestion and adsorption of carbohydrates.

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The ability of tannin on inhibiting the digestive enzymes was structure dependent.14 It

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was reported that tannins with galloylated subunits and A type linkage showed more

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potent inhibiting effect on the digestive enzymes than that with B type linkage.15

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Persimmon tannin had unique structures compared with tannins from other fruits: it

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was highly polymerized (with mDP (degree of polymerization) of 26) and

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3-O-galloylated (72%); and it had both A-type and B-type interflavan linkages 7 (Fig.

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1). In addition, as we previous studies, the content of tannins was very high, about 2%

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- 4% in persimmon fresh fruit. We also have taken a survey which showed that in

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many area, especially in main production area of persimmon, lots of people eat more

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than 2 fruits (about 500 g fresh fruits) every day, without any adverse effect

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(unpublished data). There were about 10 - 20 g tannins in 500 g fresh fruits, which

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may be enough for persimmon tannin to play its health benefits. Therefore, we

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proposed that persimmon tannin might be used as a potential health supplement for

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management of postprandial glucose. In addition, starches which with different

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amylose content showed different digestibility properties, and direct interactions

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between tannins and starch were influenced not only by the structure of phenolic

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compounds, but also by the conformational flexibility and amylose content of starch.

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Therefore, in this study, we systematically evaluated the effects of persimmon tannin 4


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on the digestibility of three kinds of starch (high amylose starch, intermediate

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amylose starch, lower amylose starch) both in vivo and in vitro, and the influence of

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persimmon tannins on digestive enzymes and glucose uptake and transport were also

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included.

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5



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Materials and methods

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Materials.

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Different kinds of starch including high amylose corn starch (HAC, amylose content

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= 77.8%), intermediate amylose corn starch (IAC, amylose content = 56.7%) and low

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amylose corn starch (LAC, amylose content = 7.9%) were purchased from Henan

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Dayuan Food additive company (Henan, China). The amylose contents of three kinds

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of starch were determined using the colorimetric method of the iodine complexes and

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a wavelength of 600 nm was used for measurement of the amylose content of

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starches as previous described.16 α-amylase (50 unit/mg) from porcine pancreatic and

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α-glucosidase (26.5 unit/mg) from saccharomyces cerevisiae was purchased from

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Sigma-Aldrich (St. Louis, MO USA). All other reagents were of analytical grade and

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from Sinopharm Chemical reagent factory (Shanghai, China).

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Sample preparation

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Persimmon tannin was extracted and purified from the astringent persimmon

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(Diospyros kaki Thunb., GongChengYueShi). Briefly, 200 g persimmon fruit were

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extracted with 2 L HCl/methanol (1%, v/v) at 80 °C for 40 min for three times. The

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concentrated extract solution was applied into a glass column packed with AB-8

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macroporous resin (Tianjin, China). After absorption, the column was firstly eluted

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with deionized water to remove sugar and other soluble impurity. After that, 10%

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ethanol/water (v/v) was used to wash low molecular weight phenolic compounds. At

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last, 95% ethanol/water (v/v) was used to elute the target tannin. After eluting, solvent

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was removed using a rotary evaporator under vacuum at 35 °C, and then the extracts 6


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were freeze-dried and the purified persimmon tannins were stored at -20 °C until used.

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The

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thiolysis-HPLC-ESI-MS and NMR methods as previously reported.7 The mean

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degree of polymerization was estimated to be 26, and epigallocatechin-3-O-gallate

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(EGCG) and epicatechin-3-O- gallate (ECG) as the main extender units. The detail

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information about the structural composition of persimmon tannin was shown in Fig.

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1 and S. Tab. 1.

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Postprandial Glycemic Response Measurement.

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Eight-week-old SD rats were purchased from Laboratory Animal Center of Huazhong

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Agricultural University (Wuhan, China). Animals were allowed free access to pellet

structure

of

persimmon

tannin

was characterized by

MALDI-TOF,

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chow and water ad libitum.

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(22 ± 1 °C) and humidity (55 ± 10%) controlled room with a 12 h light/dark cycle

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(07:00 a.m.-19:00 p.m). After acclimation for 1 week, rats were randomly assigned to

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12 groups (eight rats per group) with equal mean body weight. Different kinds of

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starch (5% w/v in distilled water) with persimmon tannins (0%, 5%, 10%, 15% of

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starch) were cooked (LAR in boiling water, IAR and HAR at 120 °C) for 20 min.

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Then starch tannin complex samples (0.5 g/kg BW) were administrated orally after an

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overnight fasting. The doses of persimmon tannins equaled as 0, 25, 50 and 75 mg/kg

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bodyweight. Blood samples from the lateral tail vein were collected at 0, 30, 45, 60,

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90, 120 min after the administration of starch-tannin complexes, and the fasting

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glucose were measured using a glucose analyzer (Roche diagnostics, Germany) to

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obtain the glycemic index and the area under the glycemic curve were calculated. All

Ninety-six SD rats were maintained at a temperature

7



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procedures were approved by the Experimental Animal Review Committee of

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Huazhong Agricultural University of China.

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Assays of the inhibition of persimmon tannin on activities of α-amylase and

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α-glucosidase

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α-amylase activity assay

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The α-amylase activity was assessed using the method previously reported and

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acarbose was included as a positive control.17 Briefly, 100 µL of enzyme solution (25

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U/mL in 20 mM sodium phosphate buffer at pH 6.9) was incubated at 37 °C for 10

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min with 100 µL of different concentrations of persimmon tannin (the final

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concentrations of tannins were 0.1-0.35 mg/mL). Then, 100 µL of starch solution (1%,

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w/v) was added and the mixture was incubated at 37 °C for another 10 min. The

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reaction was terminated by adding 400 µL of dinitrosalicylic acid color reagent.

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Subsequently, all samples were heated in boiled water for 10 min. When the reaction

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mixture was cooled to room temperature, the samples were diluted and the

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absorbance was measured at 540 nm. The activity of α-amylase was calculated as

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follows: % Activity = (Asample- Ablank)/(Acontrol -Ablank) * 100

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The kinetics assay of α-Amylase inhibitory activity of persimmon tannin was also

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investigated as the above method except that the final concentrations of tannins were

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0.2 and 0.25 mg/mL, and the starch solution were 0.5%, 1% and 2% (w/v). The

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reaction samples were collected every 5 mins, then added with DNS, the absorbance

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was measured at 540 nm. A Lineweaver-Burk plot between 1/[substrate] (mg/mL)

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and 1/[V] (reaction rate) was used to examine the action type of persimmon tannin on 8


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α-amylase inhibitory activity.

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α-glucosidase activity assay

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Briefly, 20 µL of 20 mM sodium phosphate buffer and 20 µL of persimmon tannin

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solution were mixed in a 96-well microplate (the final concentrations of tannins were

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0.1-0.30 mg/mL). Then, α-glucosidase solution (20 U/mL) was added and the mixture

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was incubated at 37 °C for 10 min. Subsequently, 20 µL of 2.5 mmol/L

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p-nitrophenyl-α-D-glucopyranoside (PNPG) solutions were added to each well and

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incubated for another 30 min. The reaction was stopped by 80 µL of 0.2 mol/L

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sodium carbonate. The absorbance was measured at 405 nm. Acarbose was applied as

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a positive control. The activity of α-glucosidase was calculated as follows: % Activity

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= (Asample- Ablank)/(Acontrol -Ablank) * 100.

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The kinetics assay of α-glucosidase inhibitory activity of persimmon tannin was also

146

investigated as the above method except that the final concentrations of tannins were

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0.2 and 0.25 mg/mL, and the PNPG solution were 0.5 - 5 mm/L. The absorbance was

148

measured at 405 nm every 5 mins. A Lineweaver-Burk plot between 1/[substrate]

149

(mm/L) and 1/[V] (reaction rate) was used to examine the action type of persimmon

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tannin on α- glucosidase inhibitory activity.

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In vitro starch digestibility and interaction of tannin with starch

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In vitro starch digestibility

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The in vitro digestion of starch samples was performed according to a modified

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Englyst’s method.18 Starch (1.0 g) with different concentration of persimmon tannin

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(0%, 5%, 10%, 15%) were dissolved in 50 mL sodium acetate buffet and cooked 9



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(LAR in boiling water, IAR and HAR at 120 °C) for 20 min, then cooled to 37 °C.

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After take, 3 mL starch-tannins samples and 3 mL pre-incubated enzyme solution

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(α-amylase and α-glucosidase mixed in a proportion of 120 : 80 U/mL) were mixed

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kept at 37 °C for up to 120 min. Then 0.3 mL reaction mixtures was collected and put

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into a plastic tube containing 2.7 mL of ethanol at 0, 20, 40, 60, and 120 min,

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respectively. All reactions were carried out in four replicates. The solutions were

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vortexed and then centrifuged at 3000 rpm for 20 min at room temperature. The

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supernatant was used to analyze the content glucose using DNS colorimetry.

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Interaction between persimmon tannins and starch

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The interaction between persimmon tannin and different kinds of starch was studied

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using iodine-binding analysis as described previously.19 Briefly, 25 µL of persimmon

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tannin was added to 0.9 mL of soluble starch suspension (the final proportion of

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persimmon tannins were 0%, 5%, 10%, 15% to starch). After vertexing, 0.1 mL of

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iodine solution was added to the suspension. Immediately after the addition of iodine

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solution, measurements of absorption spectra were started. The absorption spectrum

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of the starch-iodine complex was measured from 500 to 900 nm using a UV-1800

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spectrophotometer (Shimadzu, Tokyo, Japan).

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Changes of polyphenol content before and after persimmon tannin cooked with

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starch

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Changes in the polyphenol content before/after cooking were evaluated to

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demonstrate the interactions between persimmon tannin and starch molecules by

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Barros’s method with some modification.13 Solutions of starch (LAR, IAR, HAR, 10% 10


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w/v in distilled water) -persimmon tannin (10% starch basis) complexes were shook

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for 1 h at 120 rpm. Then, the mixture was cooked (LAR in boiling water, IAR and

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HAR at 120 °C) for 20 min. The control (persimmon tannin alone) was also cooked.

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All samples before and after cooking were freeze-dried and then extracted with

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methanol. The extractable phenols content was measured using Folin-Ciocalteu

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method.13

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Inhibition of Glucose adsorption and Transport through Caco-2 Human Intestinal

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Cell Monolayers by tannin

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Cell culture and MTT assay

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Caco-2 cells were maintained in DMEM supplemented with 10% FBS, 1% NEAA, 1%

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HEPES, 1% pen/strep and 0.1% gentamicin. MTT method was used to determine the

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influence of persimmon tannin on cell viability. Briefly, Caco-2 cells were seeded at 4

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× 104 cells/well in a 96-well plate and incubated overnight. The cells were treated

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with different concentrations of tannins (0 - 100 µg/mL) for 48 h. After the incubation,

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20 µL of MTT solution (5 mg/mL) was added and then incubated for another 4 h.

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After that, the medium was removed and 200 µL of DMSO was added to each well.

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The absorbance was measured at 570 nm. Influence of persimmon tannin on the cell

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viability was calculated as: (absorbance of treated well/absorbance of control well) ×

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100%.

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Evaluation of Caco-2 cell monolayer

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Cells were seeded at 1.5 × 105 cells/well in a Millicell 12-well plate and incubated

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under a humidified atmosphere of 95% CO2 at 37 °C. At 3, 5, 9, 15, 21 days, the 11



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transepithelial electrical resistance (TEER) value was measured with a Millicell®

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ERS-2 voltammeter (Millipore corporation, USA) in order to evaluate the monolayer

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integrity of the Caco-2 cell monolayer.20 To ensure the establishment of Caco-2 cell

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monolayer model, in this study, we also determined the permeability of

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apical-to-basolateral of lucifer yellow, which is a paracellular transport marker.

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Adsorption and Transport Experiments

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After 21 days of culture, the complete medium was removed and the monolayer was

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incubated in glucose-free DMEM for 2 h preceding treatment, then washed three

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times and balanced with HBSS (prior warmed to 37 °C) for 30 min. Test media for

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initial experiments was prepared by solubilizing glucose (0.55 mM), and tannin in

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DMAO (0.01%) (10 - 60 µg/mL). For the AP-BL permeability (absorptive transport

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study), the HBSS was removed and replaced with 0.4 mL of samples (dissolved in

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HBSS, pH 7.4) on the apical side (AP) and 1.6 mL of fresh HBSS (pH 7.4, 37 °C) on

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the basolateral chamber of the transwell insert. After 60 min of incubation, cell

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membranes were washed with ice cold PBS and collected, followed by lysing with 1%

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triton-100 solution on ice, and then centrifuged at 14000 rpm for 15 min at 4 °C.

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Supernatants were collected to determine the glucose and protein content.21

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Basolateral and apical media were also collected to determine the glucose content

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using a glucose Assay kit (Sigma Aldrich, USA). All treatments were performed in

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quadruplicate.

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Statistical analysis

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All data were presented as Means ± standard deviation (Means ± SD). Comparisons 12


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between groups were carried out using one-way ANOVA of SPSS 19.0 followed by

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Tukey’s multiple-range test. p-value < 0.05 was considered statistically significant.

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13



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Results and discussion

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Postprandial Glycemic Response to Starches in the Presence of Persimmon tannin

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In order to explore the inhibitory activity of persimmon tannin on the starch

228

digestibility in vivo, we firstly investigated the influences of the persimmon tannin on

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the postprandial glycemic response of three kinds of starch (LAC, IAC, HAC). The

230

results were showed in Fig. 2. For LAC, the peak time of blood glucose was delayed

231

from 30 min to 45 min by addition of different concentration of persimmon tannin

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and the blood glucose levels of persimmon tannin-starch treated rats were

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significantly lower than that of the control rats (p < 0.05). For IAC and HAC, the

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peak time was not changed, however the blood glucose level was significantly lower

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than that of the control, especially for HAC groups. Furthermore, we found that the

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addition of 10% and 15% persimmon tannin significantly (p < 0.05) decreased the

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areas under blood glucose curve (AUC) compared to the control group. For LAC,

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IAC, HAC, addition 15% of persimmon tannin resulted in decrease of the AUC by

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6.61%, 9.05%, 11.33%, respectively (Fig. 2) compared to control group. This result

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suggested that persimmon tannin could reduce the postprandial glycemic response

241

and this potential was also related with the amylose level of starch.

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Effect of Persimmon Tannin on In Vitro Starch Digestibility

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The postprandial glycemic response to starch depends on both the rapid release of

244

glucose from starch digestion and the glucose absorption in the small intestine.

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Therefore, we firstly evaluated the inhibitory effect of persimmon tannin on the in

246

vitro digestibility of starch with different amylose levels (Fig. 3). The enzymatic 14


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hydrolysis rate of LAC, IAC, HAC alone at 120 min were 31.18% (Fig. 3A), 43.44%

248

(Fig. 3B), 65.07% (Fig. 3C) respectively. Generally, the digestibility of starch

249

decreased with the increase of amylose content.22 However, some high amylose

250

starches such as corn starch, tomato starch corn, showed a high enzyme-catalyzed

251

degradation. This may be explained by that the digestion of starch was not only

252

influenced by the amylose levels, but also by the molecular weight and particular size

253

of starch.23 There was also an interesting phenomenon that HAS showed the highest

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digestibility in vitro, However, in the animal study, LAS treatment rats showed a

255

higher blood glucose level (Fig. 2). There were maybe two main reasons, firstly in the

256

in vitro study, the gelatinizing process was different between LAS, IAS and HAS

257

(LAR in boiling water, IAR and HAR at 120 °C), which could produce higher

258

digestibility of HAS in vitro. Secondly, the digest process and mechanism of starch in

259

vivo may be different with that in vitro. This also need to take a further study to

260

confirm our hypothesis.

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As shown in Fig. 3, the digestibility of the three kinds of starches was inhibited by

262

the addition of persimmon tannin, and increases of tannin concentration led to a

263

decrease in starch digestibility. Among the three concentrations tested, 15%

264

persimmon tannin showed the highest inhibitory on the digestibility of HAC

265

(32.24%), compared with that of LAC (22.64%) and IAC (22.84%). This result

266

indicated that the inhibition of persimmon tannin on starch digestibility might

267

contribute to its reducing effect on the postprandial glycemic response to starches in

268

the above animal models (Fig. 2 C and F). Quek and Henry (2015) found that 7% 15



269

(w/v) of red grape polyphenol could reduce the in vitro digestibility of white rice.24

270

Sorghum tannins (10%) and Baobab tannins (4.07% per g CHO) were also reported

271

to decrease the digestibility of starch.13 Our results were in line with previous studies.

272

It was found that monomeric sorghum polyphenols had limited effect on the starch

273

digestibility at equivalent levels of tannins.12 These findings indicated that the

274

polyphenols, especially polymeric polyphenols could slow down the digestibility of

275

starch. This result was also consisted with the in vivo animal study which indicated

276

that persimmon tannin showed a strong hypoglycemic effect.

277

Inhibitory activities of Persimmon Tannin on α-amylase and α-glucosidase

278

The above results indicated that persimmon tannin could decrease the blood glucose

279

levels by inhibition of the digestibility of starch. α-amylase and α-glucosidase are the

280

main digestive enzymes involved in the hydrolysis of dietary starch. Inhibition of

281

a-amylase and a-glucosidase was believed to be one of the most effective approaches

282

for postprandial glycemic control and diabetes care, and it is also one of the most

283

important ways of polyphenols to exert their glycemic index (GI) reducing effect in

284

vivo. Therefore, we further investigated the inhibitory effects of persimmon tannin on

285

the activities of α-amylase and α-glucosidase. As shown in Fig. 4, similar as the

286

positive control (acarbose), which is a classic starch digesting enzyme inhibitor,

287

persimmon tannin exerted strong inhibition on α-amylase in a dose-dependent manner.

288

The IC50 of persimmon tannin and acarbose were 0.3452 mg/mL and 0.2005 mg/mL,

289

separately, indicating that although less potent than acarbose, persimmon tannin was

290

a strong α-amylase inhibitor. Moreover, the IC50 of persimmon tannin and acarbose 16


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on α-glucosidase were 0.2391 and 0.2445 mg/mL, separately, indicating that

292

persimmon tannin showed similar potential on inhibiting α-glucosidase compared

293

with acarbose. The Lineweaver-Burk plots of the persimmon tannin were given in Fig.

294

5 A and B. The results suggested that persimmon tannin had a mixed-type inhibition

295

(competitive and non-competitive) against α-amylase with the Ki value of 0.32

296

mg/mL. For α-glucosidase, it had an intersection at the y axis which indicated their

297

inhibitory types were competitive on α-glucosidase with the Ki value of 0.62 mmol/L.

298

These results suggested that persimmon tannin was a potent inhibitor for both

299

α-amylase and α-glucosidase. Our results were in line with those findings on tannins

300

from sorghum and Eugenia jambolana seeds.25-26

301

Although the detailed inhibitory mechanisms of polyphenols on digesting enzymes

302

were not fully understood, previous studies had determined several key structural

303

features needed for monomeric flavonoids to inhibit α-amylase and α-glucosidase

304

activity.27 For example, galloylated catechins showed stronger α-amylase and

305

α-glucosidase inhibitory activities than non-galloylated catechins and the number of

306

the hydroxyl groups on the B ring was associated with this inhibitory activity.4 De

307

Freitas and Mateus (2011) also found that procyanidins with a nonhydrolyzable

308

oligomeric structure may occupy the substrate binding pocket of α-amylase, thereby

309

competitively inhibiting the enzyme. Furthermore, it was also proved that galloylated

310

procyanidin dimers of grape seeds had particular “closed” conformations that

311

reportedly enhance the interactions with α-amylase, resulting in a strong inhibitory

312

activity.28 17



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313

It was reported that the inhibitory activity of condensed tannins on α-amylase was

314

dependent on the DP (degree of polymerization). Gu et al (2011) found that monomic

315

cocoa polyphenols showed little α-amylase inhibition activity, whereas polyphenols

316

with a DP < 5 exerted a 15% of inhibition and procyanidins with a DP ranging from 5

317

- 10 inhibited α-amylase by 17 - 45.5% at 100 µM.29 High molecular weight sorghum

318

proanthocyanidins were also reported to have more potent inhibitory effect on

319

α-amylase than low molecular weight ones.30 In addition, Eisuke Kato et al (2017)

320

also found that the presence of (gallo)catechin in the extension unit of procyanidins

321

may also have a great contribution on its strong inhibitory effect on α-amylase.31 As

322

we previously reported, persimmon tannin had unique structure compared with

323

tannins from other fruits: it is highly polymerized (m DP 26) and 3-O-galloylated

324

(72%); and it has both A-type and B-type interflavan linkages.7 This could partly

325

explain the strong inhibitory activities of persimmon tannin on α-amylase and

326

α-glucosidase. However, for the reason that persimmon tannin used in this study was

327

a heteropolymer which with different DP values, therefore, it is very necessary to take

328

a further purify study, and then to elucidate the structure/function relationships and

329

also the influence of DP on the inhibitory activities.

330

Interaction of Persimmon Tannin with Starch

331

Direct binding to starch granules is one way of polyphenols to reduce the digestibility

332

of starch. Previous studies have proved that sorghum tannin could directly interact

333

with starch molecules to reduce the starch digestibility.11,

334

persimmon tannin could directly interact with the starch to affect the digestibility of 18


13

In order to clarify if

Page 19 of 39


335

starch, we used a spectroscopic method to investigate whether persimmon tannin

336

could bind with starch. The ∆A curve of the sample was obtained through subtracting

337

the absorption curve of persimmon tannin + iodine from the absorption curve of

338

(starch solution + persimmon tannin) + iodine. Samples absence of persimmon tannin

339

were used as the control. From Fig. S1, the results indicated that persimmon tannin

340

suppressed the formation of the starch-iodine complex and the suppression effects

341

increased with increasing concentration. For LAC, ∆A spectra of LAC-iodine

342

complex was 0.35 at 560 nm (Fig. S1A). However, the LAC-iodine ∆A spectra

343

decreased to 0.28 when 15% persimmon tannin was added (S. Fig. 1A). Similarly, for

344

IAC and HAC, ∆A spectra were 0.38, 0.50 respectively at 620 nm (S. Fig. 1 B, C).

345

When 15% of persimmon tannin was added, the starch-iodine ∆A spectra decreased

346

to 0.32 and 0.43, respectively (S. Fig. 1 B, C). These results indicated that persimmon

347

tannin could bind with starch directly and suppress the formation of starch-iodine

348

complex, thus suppressing the formation of the starch-iodine complexes. Our result

349

was similar with previous studies.19, 32

350

The binding of persimmon tannin and starch were further investigated by measuring

351

the content of extractable polyphenols of starch-phenolic extract mixtures. As shown

352

in Tab. 1, The polyphenol contents of starch-tannin mixture before cooking were

353

365.30 ± 25.95, 111.52 ± 5.77, and 62.28 ± 3.39 mg GAE/g in LAC, IAC and HAC,

354

respectively, which were significantly lower than that of the control (593.30 ± 24.04

355

mg GAE/g). These results indicated that persimmon tannin might bind with IAC and

356

HAC starch strongly and resulted in a decrease of extractable phenols. The result also 19



357

indicated that persimmon tannin showed a stronger interaction with amylose

358

compared to amylopectin. Previous research had demonstrated that polyphenols

359

could adsorb on raw starches, and this adsorption was dependent on both the starch

360

properties and the structure of polyphenols. Generally, larger molecular weight

361

tannins provide more hydroxyl groups for hydrogen bonding and also contain more

362

hydrophobic domains, thus resulting in stronger interaction with starch.12-13

363

Condensed tannins could not only be adsorbed on the starch surface but also enter

364

into the hydrophobic pocket of the amylose to form inclusion complexes. The strong

365

bind of persimmon tannin with HAC could probably be explained by the following

366

reasons: firstly, compared with highly branched amylopectin, the linear nature of

367

amylose made its hydrophobic core more accessible to persimmon tannin in solution.

368

Secondly, the steric hindrance of the amylopectin side chains would likely interfere

369

with its ability to efficiently interact with the persimmon tannins even though it could

370

also provide some hydrophobic sites.13 This finding was also consisted with the in

371

vitro starch digestibility results that persimmon tannin showed the highest inhibitory

372

on the digestibility of HAC.

373

In order to investigate the specific interactions of persimmon tannin with gelatinize

374

starch, mixtures of starch with tannin were cooked. As shown in Tab. 1, little change

375

was observed in polyphenol content when persimmon tannin was cooked alone at

376

100 °C or 121 °C for 20 min. When persimmon tannin-starch complexes were cooked,

377

great decrease in the extractable tannins was observed in these persimmon

378

tannin-starch complexes. After cooking at 100 °C for 20 min, the polyphenol contents 20


Page 20 of 39

Page 21 of 39


379

were 98.77 ± 2.70 and 69.50 ± 1.30 mg GAE/g in LAC-tannin complex and

380

IAC-tannin complexes, respectively (Tab. 1). HAC with persimmon tannin (121 °C

381

for 20 min) showed a lower polyphenols content (41.03 ± 2.69 mg GAE/g) compared

382

to those in LAC and IAC. Our result was also consisted with these findings about the

383

sorghum proanthocyanidins.13 The decrease in the extractable tannins after cooking

384

indicated that persimmon tannin chemically interacted with gelatinized starch

385

molecules. The increased swelling and opening of starch chains after cooking seemed

386

to enable the tannins molecular to bind to specific sites of starch via hydrogen bonds

387

and hydrophobic interactions.13 Barros et al also demonstrated that the molecular

388

weight of PA played a major role in inhibiting starch digestibility. Polymeric PAs bind

389

more strongly to starch, mainly to amylose, and generated more RS. For example, the

390

percentage of PAs bound to amylose increased from 45% (PAs with degree of

391

polymerization (DP) = 6) to 94% (polymeric PAs, DP > 10). The results demonstrate

392

that the higher DP of PAs, the stronger binding to amylose.33 As we described before,

393

persimmon tannin is highly polymerized (mDP 26) and 3-O-galloylated (72%), which

394

provided more hydroxyl groups for hydrogen bonding and also contain more

395

hydrophobic domains that would promote the stronger interactions with gelatinized

396

starch. The binding potency of persimmon tannin with starch was in line with its

397

inhibition on the digestibility of starch. Taken together, persimmon tannins not only

398

could directly inhibit the enzyme activity of α-amylase and α-glucosidase, but also

399

bind with starch, resulting in a decrease of starch digestibility.

400

Inhibitory effect of persimmon tannins on the glucose uptake in Caco-2 cells 21



401

After the starch was digested, it was turned to simple sugars, and then transferred to

402

the blood. Therefore, inhibition of the glucose uptake and transport also played an

403

important role in controlling blood glucose level and preventing hyperglycemia.

404

Glucose is absorbed in the small intestine via the SGLT 1and GLUT 2 expressed on

405

the apical side of the intestinal epithelial cells.34 Caco-2 cell line, which was derived

406

from human colon adenocarcinoma, was a good intestinal absorption model for

407

studying permeability and transport characteristics of drugs.35 Caco-2 cells were also

408

widely used to evaluate the intestinal adsorption and transportation of glucose.36 As

409

the results showed in Fig. 3, even though 15% persimmon tannin showed the strong

410

inhibitory on the digestibility of HAC, the enzymatic hydrolysis rate of HAC with 15%

411

persimmon tannin was still very high, however, treatment with HAC with 15%

412

persimmon tannin showed a significant decrease of the blood glucose levels (Fig. 2).

413

These results indicated that except persimmon tannins could decrease the digestibility

414

of starch, the inhibition of absorption rather than digestion maybe the key mechanism

415

for its activity to reduce the postprandial glycemic response. Therefore, in this study,

416

the Caco-2 cell model was applied to investigate the influences of persimmon tannins

417

on the adsorption and transportation of glucose. As shown in Fig. 6 A, persimmon

418

tannin showed no cell-toxicity when the concentration was below 60 µg/ml.

419

Therefore, 10 - 60 µg/mL were selected for the further study.

420

Cell monolayer integrity was controlled by measurement of transepithelial electrical

421

resistance (TEER) and evaluation of cell permeability to Lucifer yellow. TEER of

422

Caco-2 cell monolayer was increased with prolonged incubation time and was often 22


Page 22 of 39

Page 23 of 39


423

used after 21 days of culture on polycarbonate inserts. In this study, the TEER value

424

was about 764 ± 9 Ω/cm2 after 21 days of culture and the apparent permeability

425

coefficient (Papp) for Lucifer yellow was 2.67 × 10-7 cm/s, indicating it was tight

426

enough for the transport experiments.37 As shown in Fig. 6, as a positive control, 10

427

µg/mL of phlorizin, could significantly inhibit the uptake (65.89%) and transport of

428

glucose (62.20%). Adding persimmon tannin to the apical side also led to a

429

dose-dependent inhibition of glucose uptake. 20 µg/mL of persimmon tannins could

430

significantly inhibit the uptake of glucose by 26.36% (p < 0.05). For the transport of

431

glucose, persimmon tannin also showed significantly inhibitory activities in a

432

dose-dependent manner. These results indicated that, except for interacting with

433

starch to decrease its digestibility and directly inhibit activities of α-amylase and

434

α-glucosidase, inhibition of glucose uptake and transport might also be one of the

435

mechanisms of persimmon tannin on decreasing blood glucose level.

436

Previous research found that polyphenols could decrease the glucose uptake through

437

inhibiting the expression and activities of glucose transporters, such as SGLT1,

438

GLUT2 and GLUT5.4, 20 Manzano and Williamson (2010) found that polyphenols

439

from strawberry and apple could inhibit glucose transport from the intestine lumen

440

into cells and also the GLUT 2-facilitated exit on the basolateral side.20 Welsch et al.

441

(1989) found that tannic acids could reduce glucose uptake through favoring the

442

dissipation of the Na+ electrochemical gradient brush border membrane vesicles

443

isolated from rat small intestine.38 Kobayashi et al. (2000) found the galloyl ester

444

group may be essential for the inhibitory activity of epicatechin gallate, which could 23



445

bind to the glucose transporter and inhibit the glucose transport 4. Tamura et al. (2015)

446

also found that inhibition activity on glucose transport of oligomeric polyphenols

447

from peanut skin was increased as the DP increased.39 All of these findings suggested

448

that the inhibitory activity of tannins on glucose uptake was also related with their

449

chemical structure, such as degree of polymerization and content of galloylated

450

catechins. Although condense tannins could hardly be adsorbed in the intestine, it

451

may regulate the glucose uptake through directly interaction with the related

452

receptors on the cell membrane of the intestinal epithelial cells, and then inhibiting

453

the expression and activities of glucose transporters as such as SGLT and GLUT.

454

Studies on the detailed mechanisms of persimmon tannin on the glucose uptake and

455

the specific acceptors of persimmon tannin on the cell membrane are needed in the

456

further study.

457 458

Supporting Information

459

(1) Interactions between persimmon tannins and different kinds of starch. ∆A = A

460

(soluble starch + persimmon tannins +iodine) – A (persimmon tannins + iodine);

461

LAC (A), IAC (B), HAC (C) (Fig. S1);

462

(2) Structural Composition of Persimmon Tannin Determined by Thiolysis-HPLC

463

Analysis. (Tab. S1)

464 465

Conflicts of Interest

466

The authors have declared no conflict of interest.

467

24


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468

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Mol. Nutr. Food Res., 2010, 54(12), 1773-1780.

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and Rose, D. J. Effects of sorghum [Sorghum bicolor (L.) Moench] tannins on

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α-amylase activity and in vitro digestibility of starch in raw and processed flours.

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(37) Wang, X. D.; Meng, M. X.; Gao, L. B. Permeation of astilbin and taxifolin in Caco-2 cell and their effects on the P-gp. Int. J. Pharm, 2009, 378, 18.

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membrane vesicles. J. Nutr., 1989, 1698-1704.

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Inhibitory Effect of Oligomeric Polyphenols from Peanut-skin on Sugar

587

Digestion Enzymes and Glucose Transport. Food Sci. Technol. Res, 2015, 21 (1),

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111-115.

589 590

Funding sources

591

This work was supported by the Natural Science Foundation of China (81403160 and

592

31701712), Hubei Provincial Natural Science Foundation of China (2017CFB197)

593

and the Fundamental Research Funds for the Central Universities (2662016QD035).

594

30


Page 30 of 39

Page 31 of 39


Figure Captions: 595

Fig. 1 Structure of Persimmon tannins

596

Fig. 2 Influence of Persimmon Tannin on the Postprandial Glycemic Response by

597

Orally Administration of Different Kinds of Starch. A, B and C: The blood glucose

598

levels of the rats treated with LAC(A), IAC(B), HAC(C) and different doses of

599

persimmon tannin. D, E and F: the area under the curve (AUE) of the rats treated

600

with LAC(D), IAC(E), HAC(F) and different doses of persimmon tannin. Data were

601

analyzed by ANOVA and post-hoc Dunnett's test. Different small letters on the bars

602

indicated significant difference at p < 0.05.

603

Fig. 3 In Vitro Starch Digestibility Patterns of the Different Kinds of Starch with the

604

Persimmon Tannins, A: LAC; B: IAC; C: HAC.

605

Fig. 4 Effects of Persimmon Tannins on the Enzyme Activities. A: α-amylase B:

606

α-glucosidase. Acarbose was selected as a positive control.

607

Fig. 5 Lineweaver-Burk Plots of Persimmon Tannin on The Digestive Enzyme. A:

608

α-amylase B: α-glucosidase.

609

Fig. 6 Influences of Persimmon Tannins on The Glucose Uptake and Transport in a

610

Caco-2 Monolayer Model. A: The influence of persimmon tannins on the cell

611

viability of Caco-2 cells; B: Effect of persimmon tannin on glucose uptake in Caco-2

612

monolayer model; C: Impact of persimmon tannin on glucose transport across Caco-2

613

cell monolayers over 60 min. Different small letters on the bars indicate significant

614

difference at p < 0.05.

615 31



616

Page 32 of 39

Table 1. Changes of Phenol Content (mg GAE/g) Before and After Starch Cooked with Persimmon Tannin. Total phenol content(mg/g) Before cooking

After cooking

Persimmon tannin (100 ℃ 20 min)

593.30 ± 24.04 A a

591.20 ± 44.92 A a

Persimmon tannin (121 ℃ 20 min)

593.30 ± 24.04 A a

571.00 ± 20.92 A a

LAC + Persimmon tannin (100 ℃ 20 min)

365.30 ± 25.95 b

98.77 ± 2.70 b

IAC + Persimmon tannin (121 ℃ 20 min)

111.52 ± 5.77 c

69.50 ± 1.30 c

HAC + Persimmon tannin (121 ℃ 20 min)

62.28 ± 3.39 d

41.03 ± 2.69 d

Data were expressed as means ± S.D. Data were analyzed by ANOVA and post-hoc Bonferroni test. The same letter in a row (capital letters) or column (lower-case letters) are not significantly different

32


Page 33 of 39


Fig. 1

33



Fig. 2

34


Page 34 of 39

Page 35 of 39


F 35



Fig. 4

36


Page 36 of 39

Page 37 of 39


Fig. 5

37



Fig. 6 38


Page 38 of 39

Page 39 of 39


Table of Contents Graphic (TOC)

39


Persimmon tannin decreased the glycemic response through

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