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Inhibition of pancreatin-induced digestion of cooked rice starch by adzuki (Vigana angularis) bean flavonoids and possibility of decrease in the inhibitory effects in the stomach Sachiko Harota, and Umeo Takahama J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05442 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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Journal of Agricultural and Food Chemistry

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Inhibition of pancreatin-induced digestion of cooked rice starch by adzuki (Vigana

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angularis) bean flavonoids and possibility of decrease in the inhibitory effects in the

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stomach

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Sachiko Hirota and Umeo Takahama*

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Department of Health and Nutrition Care, Faculty of Allied Health Sciences, University

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of East Asia, Shimonoseki 751-8503, Japan

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*Corresponding author

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Umeo Takahama

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Department of Health and Nutrition Care, Faculty of Applied Health Sciences, University

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of East Asia, Shimonoseki 751-8503, Japan

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Fax: +81-93-582-1131

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E-mail address: [email protected]

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Key words: Flavonoid/starch complex, nitrite, quercetin, starch digestion, stomach,

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vignacyanidin

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ACS Paragon Plus Environment

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ABSTRACT

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Flavonoids of adzuki bean bind to starch when cooked with rice. The purpose of the

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present study is to show that adzuki flavonoids can suppress pancreatin-induced digestion

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of cooked rice starch. Diethyl ether extract of water boiled with adzuki bean inhibited the

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starch digestion, and quercetin and a cyanidin-catechin conjugate (vignacyanidin) but not

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taxifolin in the extract contributed to the inhibition. The order of their inhibitory effects

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(taxifolin < quercetin < vignacyanidin) suggested that the effects increased with the

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increase in their hydrophobicity. Diethyl ether extract also inhibited the starch digestion

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of cooked rice pre-incubated in artificial gastric juice, and the inhibition was decreased

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by nitrite. The decrease was due to nitrite-induced consumption of quercetin and

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vignacyanidin. Taking the above results into account, mechanisms of quercetin- and

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vignacyanidin-dependent inhibition of starch digestion and possibility of the decrease in

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their inhibitory effects by nitrite in the stomach are discussed.

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INTRODUCTION Adzuki bean, Vigna angularis (Willd.) Ohwi & H.Ohashi, is used to prepare Japanese

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and Chinese sweets, sekihan (glutinous rice cooked with adzuki bean), and adzuki-meshi

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(non-glutinous rice cooked with adzuki bean). When Japanese and Chinese sweets are

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prepared, adzuki ann, which is paste of adzuki bean, is used. The color of the paste is

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purplish red, suggesting the binding of reddish pigments in the seed coat to adzuki bean

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starch. When sekihan or adzuki-meshi is prepared, the pigments are transferred to rice,

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making the color purplish red. Purplish cyanidin-catechin conjugates, vignacyanidin and its

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isomers, which are contained in seed coat of adzuki bean, may participate in coloring rice

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purplish red1−3. In addition, other flavonoids such as quercetin, its glycosides, taxifolin, and

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catechins in adzuki bean may also be transferred from the seed coat to rice4−7. The transfer

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of adzuki flavonoids from adzuki seed to non-glutinous rice during preparation of

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adzuki-meshi has been reported.3

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It has been reported that flavonoids and lipids bind to starch8−12, and that the binding

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results in the inhibition of pancreatin- or α-amylase-catalyzed starch digestion2,9,10,12−15.

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Since kaempferol, quercetin, and vignacyanidin suppressed α-amylase-catalyzed starch

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digestion by binding to soluble starch2,14,15, we postulated that flavonoids contained in ann,

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sekihann, and adzuki-meshi might make slower α-amylase-catalyzed digestion of their

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starch in the intestine. By the ingestion of the above foods, however, they are stay in the

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stomach for 2−3 hr, so that flavonoids such as quercetin and vignacyanidin in the foods can

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react with nitrite, which is a component of mixed whole saliva and a food additive, in the

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stomach2,3,16. Then, the suppressive effects of the above flavonoids on starch digestion in

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the intestine are supposed to be affected by their reactions with nitrite in the stomach.

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The present study deals with i) preparation of adzuki extract from water boiled with

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adzuki bean, ii) suppression of pancreatin-induced cooked rice starch digestion by adzuki

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extract, iii) elucidation of the components contributed to the suppression, and iv) effects of

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nitrite treatments of adzuki extract in artificial gastric juice on the pancreatin-induced


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starch digestion. The results obtained suggest that i) quercetin and vignacyanidin but not

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taxifolin in adzuki extract can suppress the starch digestion, that ii) efficiency of the

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suppression is dependent on hydrophobicity of the flavonoids, and that iii) the suppressive

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effects of quercetin and vignacyanidin can be reduced by nitrite in the stomach. Taking the

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above results into account, we discuss mechanisms of the inhibition of starch digestion by

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adzuki extract, and propose that the reactions of the components in adzuki extract with

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nitrite in the stomach should be taken into account to estimate their inhibitory effects in the

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intestine.

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MATERIALS AND METHODS Reagents and Ingredients. Quercetin and taxifoloin were obtained from

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Sigma-Aldrich Japan (Tokyo). Maltose, 4-hydroxybenzhydrazide, and pancreatin from

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hog pancreas were from Wako Pure Chemical Ind. (Osaka, Japan). Vignacyanidin was

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prepared as reported previously with slight modification3. The structure of isolated

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vignacyanidin has been determined by mass spectra and NMR spectra1.

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Dry adzuki bean (Vigna angularis (Willd.) Ohwi & H.Ohashi cv. erimo) cultivated in

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Memuro, Hokkaido (Japan), was provided from Japan Pulse Foundation. Polished

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non-glutinous rice (Oryza sativa L. cv. koshihikari) cultivated at Shikano in Shunan City,

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Yamaguchi Prefecture (Japan), was obtained from a local market.

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Preparation of Solutions. The above flavonoids were dissolved in dimethyl

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sulfoxide (DMSO) at a concentration of 10 mM, and the solutions were kept at −20°C.

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Pancreatin (5 mg mL−1) dissolved in water was prepared every day and kept at 0°C

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during experiments. The solution to quantify reducing sugars was prepared by mixing

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solutions I and II (1:9, v/v) just before the experiments17. Solution I contained 0.33 M

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4-hydroxybenzhydrazide and 0.6 M HCl, and solution II contained 0.042 M sodium

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citrate, 0.007 M CaCl2, and 0.5 M NaOH. Potassium iodide (1.5 g) was dissolved in 12.5

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mL of water. Iodine (0.635 g) was added to the solution, and volume of the mixture of


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potassium iodide and iodine was adjusted to 50 mL with water. The iodine solution

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(equivalent to 100 mM iodine) was kept in the dark. Artificial gastric juice contained 34

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mM NaCl and 80 mM HCl, and the pH was adjusted to 1.5 by NaOH.

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Preparation of Adzuki Extract. Two hundred mL of water was added to 100 g of

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adzuki bean and then boiled for 1 min. After discarding the water, 200 mL of water was

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added to the remaining adzuki bean, and heated for 10 min under mild boiling conditions

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to obtain water extract (about 150 ml) of adzuki bean. This procedure was repeated twice.

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The water extracts were combined and the pH was adjusted to 2 with HCl. The acidified

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extract was centrifuged at 10,000g for 5 min, and the precipitate was extracted with

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methanol. The methanol solution was combined with the supernatant to extract with 100

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mL of ethyl acetate three times. The ethyl acetate extracts were combined, and washed

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with 100 mL of 0.1% HCl three times.

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Ethyl acetate was removed from the ethyl acetate extract in vacuo, and the purplish

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red residue was dissolved in 200 mL of diethyl ether. After washing the diethyl ether

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solution with 100 mL of 0.1% HCl three times, diethyl ether was evaporated in vacuo.

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The residue was dissolved in 2 mL of methanol to obtain methanol soluble fraction.

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Methanol of the fraction was removed in vacuo, and the remaining was dissolved in 0.5

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mL of DMSO to keep at −20°C. The DMSO solution was used as adzuki extract in this

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study.

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The adzuki extract were analyzed by HPLC using a Shim-pack CLC-ODS column

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(15 cm × 6 mm i.d.) combined with a pump (LS-10AS) and spectrophotometric detector

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with a photodiode array (SPD-M10vp) (Shimadzu, Kyoto, Japan). The mobile phase was

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mixtures of methanol and 0.2% (v/v) formic acid, and the flow rate was 1 mL min−1. The

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concentration of methanol (v/v) was increased stepwise. Details are shown in Fig. 1.

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Preparation of Cooked rice and its Homogenization. Non-glutinous rice (360 mL

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equivalent to about 320 g) was cooked with 500 mL of water using an electric rice-cooker,

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and used as cooked rice. The cooked rice was divided into every 3 g, wrapped with


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clingfilm, and keep at −20°C. When used for experiments, the frozen cooked rice (3 g)

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was thawed with a microwave oven, and homogenized for 1 min at 13,500 rpm in 27 mL

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of 0.1 M sodium phosphate (pH 7.0) with 0.15 M NaCl, using an Ultra-Turrux T25 (Ika

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Werke).

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Binding of Adzuki Flavonoids to Cooked Rice. The above diethyl ether-extract

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dissolved in DMSO (0.1 mL) was added to 1 mL of a cooked rice homogenate (0.1 g of

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rice/mL). The homogenate was incubated for 5 min at 37°C, and then centrifuged at

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12,000g for 10 min. The supernatant was extracted with 2 mL of ethyl acetate. After

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removing ethyl acetate in vacuo, the residue was dissolved in 0.4 mL of 50% methanol in

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0.2% formic acid. The precipitate was extracted with a mixture of 1 mL of ethyl acetate

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and 4 mL of methanol. After removing the solvents in vacuo, the residue was dissolved in

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0.4 mL of 50% methanol in 0.2% formic acid. An aliquot of each sample (0.2 mL) was

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analyzed by HPLC. Areas of separated components were compared between the

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supernatants and the precipitates, and the results are expressed as the ratios as a function

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of retention time.

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Estimation of Starch Digestion of Cooked Rice. The above cooked rice

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homogenate (0.1 g mL−1) was diluted to 0.01 g mL−1 with the buffer solution used for

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homogenization. The diluted homogenate (1 mL) was pre-incubated with adzuki extract

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or flavonoids for 10 min at 37°C, and then incubated further by adding pancreatin (0.05

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mg mL−1). After the incubation for defined periods in the presence of pancreatin, 0.05 mL

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of the homogenate was added to 3 mL of solution prepared to quantify reducing sugars

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(see above), and incubated in boiling water for 6 min 14,17. The yellow solution obtained

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was cooled in iced water, and centrifuged at about 12,000g for 5 min to record the

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absorbance of the supernatant at 410 nm with a spectrophotometer (UV-2550)

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(Shimadzu). Light path of the measuring bean was 4 mm. Reducing sugars formed were

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estimated using standard maltose, because maltose was produced by α-amylase-catalyzed

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digestion of starch.


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When starch digestion was estimated by the formation of starch/iodine complexes,

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0.1 mL of the diluted homogenate, which had been incubated with pancreatin (0.05 mg

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mL−1) for defined periods, was added to the mixture of 0.1 mL of iodine solution and 0.9

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mL of 0.1 M sodium phosphate (pH 7.0) with 0.15 M NaCl, and absorption spectra of

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starch/iodine complexes were recorded from 500 to 850 nm using a spectrophotometer

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(UV-2450) equipped with an integrating sphere assembly (ISR-240A) (Shimadzu). The

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digestion of starch was estimated by the decrease in absorbance of starch/iodine

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complexes at 550 nm. Light path of the measuring bean was 4 mm.

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Starch Digestion of Cooked Rice Treated with Artificial Gastric Juice. Cooked

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rice homogenate (0.1 mL) prepared as above was mixed with 0.9 mL of artificial gastric

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juice to obtain 0.01 g of rice mL−1. The acidic homogenate (1 mL), the pH of which was

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about 2, was pre-incubated for 10 min at 37°C with 20 µL of adzuki extract, 0.6 mM

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quercetin, or 0.2 mM vignacyanidin in the presence and absence of 1 mM sodium nitrite,

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and then 1.0 mL of 0.1 M Na2HPO4 with 0.3 M NaCl was added to the pre-incubated

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homogenate. The neutralized homogenate (2 mL), the pH of which was about 6.8, was

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incubated further with pancreatin (0.05 mg mL−1) at 37°C. After the incubation, 0.1 mL

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of the homogenates was used to estimate the concentrations of reducing sugars and

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starch/iodine complexes.

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Presentation of Data. Adzuki bean extract was prepared three times, and each

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extract was used for pancreatin-dependent rice starch digestion. Typical data or mean

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with SDs were presented as figures.

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RESULTS AND DISCUSSION

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Components in Adzuki Extract. Adzuki bean and adzuki-meshi contain following

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flavonoids and their contents increase in the order vignacyanidin < quercetin ≈ quercetin

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7-O-glucoside ≈ (−)-epicatechin < taxifolin < rutin < (+)-catechin3. In these flavonoids,

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taxifolin (peak I) and quercetin (peak II) were detected as major components in adzuki


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extract used in this study, but the contents of catechins and quercetin glycosides were

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much lower than those of taxifolin and quercetin (Fig. 1, HPLC at 280 nm). The lower

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contents were supposed to be due to their lower solubility to diethyl ether used to prepare

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adzuki extract. These flavonoids were identified by comparing retention times,

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UV-visible absorption spectra, and mass spectra with the standard compounds3. The

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concentrations of taxifolin and quercetin in the extract were estimated to be 13.6 ± 0.9

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and 4.2 ± 0.3 mM (n = 3), respectively. Vignacyanidin (peak III) (Fig. 1, HPLC at 560

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nm) was also identified by comparing the retention time, UV-visible absorption spectrum,

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and mass spectrum with standard vignacyanidin prepared as shown in Materials and

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Methods. The absorption spectra and mass spectra of two components eluted before and

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after vignacyanidin were identical with that of vignacyanidin, suggesting that the

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compounds were isomers of vignacyanidin1,3. The concentration of vignacyanidin plus

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two isomers was estimated to be 1.8 ± 0.6 mM (n = 3) by postulating that the molar

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absorption coefficients of the isomers were the same as that of vignacyanidin. Although

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UV absorption maxima of several unidentified components (peaks 1−4) were given in Fig.

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1, these were not concerned in the followings.

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Adzuki Extract-Dependent Inhibition of Reducing Sugar Formation. Adzuki

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extract inhibited pancreatin-induced reducing sugar formation in cooked rice homogenate,

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and about 50% inhibition was observed at 30 µL of adzuki extract mL−1 (Fig. 2A). The

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volume of DMSO slightly enhanced the reducing sugar formation (data not shown). The

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concentrations of taxifolin, quercetin, and vignacyanidins in the reaction mixture, which

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contained 30 µL of adzuki extract mL−1, were calculated to be about 0.41, 0.13, and 0.054

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mM, respectively, from their average concentrations in adzuki extract (see above).

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Flavonoid-Dependent Inhibition of Reducing Sugar Formation. Because the

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concentration of taxifolin, quercetin, and vignacyanidins were much higher than those of

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other identified flavonoids in adzuki extract, effects of these flavonoids on

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pancreatin-induced reducing sugar formation were studied. Fig. 2B indicates that i)


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taxifolin did not suppress, and quercetin and vignacyanidins suppressed the starch

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digestion by about 15 and 90%, respectively, at 0.3 mM, and that ii) the concentrations of

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taxifolin (about 0.41 mM) could not, but quercetin (about 0.13 mM) and vignacyanidins

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(about 0.054 mM) in 30 µL of adzuki extract mL−1 could suppress the reducing sugar

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formation by about 5 and 18%, respectively.

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The latter result suggests that quercetin and vignacyanidins in adzuki extract partly

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contributed to adzuki extract-dependent suppression of reducing sugar formation, since

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30 µL of adzuki extract mL−1 inhibited the starch digestion by about 50% (Fig. 2A). The

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former result suggests that the rice starch digestion might become slower by forming

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flavonoids/starch complexes by hydrophobic interactions, because the inhibitory activity

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of flavonoids increased with the increase in their hydrophobicity, which was presumed

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from their retention times in Fig. 1. This postulation is supported by the result that the

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binding ability of the above flavonoids to cooked rice increased in the order taxifolin
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