Article pubs.acs.org/JAFC
Slow Digestion Property of Octenyl Succinic Anhydride Modified Waxy Maize Starch in the Presence of Tea Polyphenols Shanli Peng,† Lei Xue,† Xue Leng,† Ruobing Yang,† Genyi Zhang,*,† and Bruce R. Hamaker⊥ †
State Key Laboratory of Food Science and Technology School of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, People’s Republic of China ⊥ Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, West Lafayette, Indiana 47906, United States ABSTRACT: The in vivo slow digestion property of octenyl succinic anhydride modified waxy corn starch (OSA-starch) in the presence of tea polyphenols (TPLs) was studied. Using a mouse model, the experimental results showed an extended and moderate postprandial glycemic response with a delayed and significantly decreased blood glucose peak of OSA-starch after cocooking with TPLs (5% starch weight base). Further studies revealed an increased hydrodynamic radius of OSA-starch molecules indicating an interaction between OSA-starch and TPLs. Additionally, decreased gelatinization temperature and enthalpy and reduced viscosity and emulsifiability of OSA-starch support their possible complexation to form a spherical OSAstarch−TPLs (OSAT) complex. The moderate and extended postprandial glycemic response is likely caused by decreased activity of mucosal α-glucosidase, which is noncompetitively inhibited by tea catechins released from the complex during digestion. Meanwhile, a significant decrease of malondialdehyde (MDA) and increased DPPH free radical scavenging activity in small intestine tissue demonstrated the antioxidative functional property of the OSAT complex. Thus, the complex of OSAT, acting as a functional carbohydrate material, not only leads to a flattened and prolonged glycemic response but also reduces the oxidative stress, which might be beneficial to health. KEYWORDS: OSA-starch, tea polyphenols, postprandial glycemic response, α-glucosidase, functional carbohydrate
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anhydride (OSA),14,15 is an amphiphilic derivative of starch that can stabilize water-in-oil (w/o) and oil-in-water (o/w) emulsions.12,16 Therefore, OSA-starch has been widely applied in different delivery systems as a carrier material for various bioactive compounds.16,17 Tea polyphenols (TPLs), mainly including (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC), are wellknown functional nutraceuticals that might have a variety of health benefits such as antidiabetes, anti-inflammation, antioxidative stress, and anticancer effects.18,19 With regard to carbohydrate metabolism, TPLs could ameliorate oxidative stress elicited by postprandial hyperglycemia.20 TPLs have also been proved to be capable of inhibiting the activity of αamylase21 and the absorption of glucose22 in the small intestine, which could lead to a reduction of postprandial blood glucose spike as supported by an in vivo mouse study.23 However, our previous study did not show a significant effect of TPLs on the postprandial glycemic response to both normal corn starch and waxy corn starch in a mouse model study.24 Except for the lower bioavailability of tea catechins,25−27 the instability of tea catechins in the process of digestion might also contribute to the observed effect as shown in the literature that dimers or trimers of catechins or other forms can be formed in simulated digestion process.28,29 Thus, maintenance of the structural
INTRODUCTION Obesity, type 2 diabetes, and cardiovascular diseases have become major public health concerns worldwide. Although the multifactorial etiology of these pandemic diseases and the effect of diet as a contributing factor have been well recognized, glucose homeostasis dysfunction caused by insulin resistance is a common property among these metabolic diseases,1 and glucose release from glycemic carbohydrate digestion is likely the first critical point to control glucose homeostasis. It is known that a large postprandial glycemic fluctuation, which is characteristic of rapidly available carbohydrate or rapidly digestible starch (RDS), can produce excessive free radicals leading to oxidative stress, which is a risk factor for many chronic diseases.2−4 Slowly digestible starch (SDS), as one type of glycemic carbohydrate in diets that could provide a slow and sustained glucose release, is the preferred carbohydrate for glucose homeostasis control and the prevention of oxidative stress.5 However, the fact that there is no commercially available SDS in the market suggests innovative ways from different perspectives are needed to achieve the physiological effects of SDS for glycemic control. Although native cereal starches are inherently SDS materials,6 their direct application in food is strictly limited due to their raw taste and poor functionality, such as a lack of pasting consistency and stability.7,8 Starch modification, as an alternative approach to improve the slow digestion property of starch, has been extensively studied,9−11 and only octenyl succinic anhydride modified starch (OSA-starch) was shown to have a moderately slow digestion property.7,12,13 OSA-starch, which is prepared by a chemical reaction between starch and octenyl succinic © 2015 American Chemical Society
Received: Revised: Accepted: Published: 2820
December 10, 2014 February 26, 2015 February 26, 2015 February 26, 2015 DOI: 10.1021/jf5059705 J. Agric. Food Chem. 2015, 63, 2820−2829
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Journal of Agricultural and Food Chemistry integrity of tea catechins, such as encapsulation with liposome30 or formulation with sucrose or ascorbic acid,31 is necessary for their bioactivities. Literature studies have shown that OSA-starch, as an emulsifier, has been used as a carrier material for the controlled release of bioactive compounds with formed micelles in an aqueous environment.16,17 Therefore, OSA-starch might be prepared as a carrier to protect the structural integrity of tea catechins to improve their bioactivity, and the presence of tea catechins may further improve the slow digestion property of OSA-starch on the basis of a literature report.23 Thus, the in vivo slow digestion property of OSA-starch in the presence of TPLs, as a novel approach to produce functional carbohydrate materials capable of sustaining glucose release and reducing oxidative stress, was studied in the current investigation to improve the knowledge of functional carbohydrates for improved health.
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from the lateral tail vein at 0 (as the control before gavage), 15, 30, 45, 60, 90, and 120 min after gavage. The blood glucose concentration was measured using a glucose analyzer (Johnson & Johnson, China) and expressed as the mean ± standard error (SE). α-Amylase and α-Glucosidase Activity Assays. The activity of small intestine α-amylase was examined using the chromogenic GalG2CNP kit according to the specified instruction (Leadman, Bejing, China). Briefly, the supernatants (40 μL) from the homogenates of ST and LC were combined with 200 μL of GalG2CNP (3 mmol/L) suspended in (2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mmol/L MES in distilled water, pH 6.0), and after incubation at 37 °C for 60 s, the changing rate of OD405 monitored within 2 min at 37 °C was used to calculate the activity of α-amylase. For α-glucosidase, 20 mg/mL maltose (1.0 mL) was hydrolyzed by supernatants (0.1 mL) from homogenates of ST and LC in a sodium phosphate buffer (100 mmol/L, CaCl2 1.0 mmol/L, pH 6.9) at 37 °C for 10 min, and the concentration of glucose was measured using a hexokinase kit (Leadman). The enzyme activities were expressed as percentages compared to the initial values measured before gavaging. An in vitro experiment was also carried out to test the effect of TPLs on the activity of mucosal α-glucosidase of the small intestine. First, the dose−response effect of TPLs (0−10 μg/mL) on the enzyme activity in the supernatant (0.1 mL) of ST homogenate from fasted mice was studied at 37 °C for 10 min in a sodium phosphate buffer (100 mmol/L, CaCl2 1.0 mmol/L, pH 6.9) containing 20 mg/ mL maltose with a final reaction volume of 2.1 mL. Second, the Michaelis−Menten kinetic model was used to evaluate the kinetic parameters in hydrolysis by measuring the amount of liberated glucose under different substrate (maltose) concentrations (0.02−0.50 mg/ mL) in the presence of TPLs (10 μg/mL). A Lineweaver−Burk plot between 1/[substrate] (mg/mL) and 1/[V] (reaction rate, mmol/L/ min) was used to determine the action type of TPLs on α-glucosidase. TPL Concentration Measurement. The content of TPLs in LC and ST was analyzed by HPLC.33 Briefly, the supernatant from LC or ST homogenate was mixed with 2.5 volumes of sodium phosphate buffer (0.4 mol/L, pH 6.8), hydrolyzed by glucuronidase (250 U/ sample) and sulfatase (1 U/sample) at 37 °C for 45 min.23,34,35 After hydrolysis, the sample was extracted twice with ethyl acetate. The organic phase was dried under vacuum, resuspended in 50% methyl alcohol, and analyzed using an Agilent HPLC 1200 instrument coupled with a GraceSmart reverse-phase C18 column (4.6 × 250 mm, particle size = 5 μm) (Deerfield, IL, USA) and a UV detector (280 nm) at a flow rate of 0.8 mL/min at 30 °C. Solvent A composed of acetonitrile, distilled water, and trifluoroacetic acid (TFA) in a ratio of 10:90:0.05 and solvent B in a ratio of 30:70:0.05 were used as the mobile phase. Beginning with 100% solvent A, a linear gradient mobile phase (decreasing polarity) with solvent B was used until 20 min approaching 100% solvent B. After running for 5 min, the mobile phase was returned to 100% solvent A until 30 min to finish the analysis. The measured result represented the content of total catechins, and samples prepared without glucuronidase/sulfatase were used to quantify the unconjugated fractions of catechins. Determination of Malondialdehyde (MDA) and DPPH Scavenging Activities. MDA content in the supernatant of ST homogenate was analyzed using the diagnostic kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instruction of the manufacturer, and the results were expressed as nanomoles per milligram of protein. Total protein contents were determined by Coomassie brilliant blue method using bovine serum albumin as the standard. DPPH free radical scavenging activities of ST homogenate was measured as described by Zhong et al. with slight modification.36,37 DPPH solution in ethanol (0.1 mmol/L) was prepared and used fresh for each test. The DPPH free radical scavenging activities were measured by mixing supernatant from ST homogenate and DPPH solution, and the absorbance at 517 nm was recorded after incubation at room temperature for 30 min. The scavenging rate (%) was calculated with the equation [1 − (A1 − A2)/A0] × 100%, where A0 is the absorbance of the control (2 mL of DPPH solution + 1 mL of NaCl (0.9%)), A1 is the absorbance of the sample (2 mL of DPPH
MATERIALS AND METHODS
Materials. TPLs were obtained from Hetian Biotechnology (Zhejiang, China) with a total tea catechin content of ∼99%, and its composition (EGCG, 66.90%; EGC, 5.88%; ECG, 10.62%; EC, 15.70%) was analyzed using a common HPLC method.32 Waxy maize starch was obtained from National Starch and Chemical Co. (Bridgewater, NJ, USA). The preparation of OSA-starch and measurement of degree of substitution (DS) were based on a literature report,13 and the final DS was estimated to be 0.0194. Octenyl succinic anhydride, glucuronidase (G-7896, EC 3.2.1.31, from Escherichia coli with 9 × 106U/g solid), and sulfatase (S-9754, EC 3.1.6.1, from abalone entrails with 2.3 × 105U/g solid) were purchased from Sigma Chemical (St. Louis, MO, USA). Diphenyl-(2,4,6trinitrophenyl)iminoazanium (DPPH) was purchased from Wolsen Bio. Co. (Shanxi, China). Animals and Treatment. Four-week-old male Kunming mice were purchased from Slac Co. (Shanghai, China) and kept on a 12 h light/dark schedule (lights on from 8:30 a.m. to 8:30 p.m.) in a constant condition (22 ± 1 °C and 60% humidity) with standard chow (Slac Co.) and drinking water provided ad libitum. Experiments were performed 1 week later after overnight fasting. Starch samples (10% w/v in distilled water) with and without TPLs (5%, w/w starch based) were cooked in a boiling water bath for 20 min to gelatinize the starches. After the samples were cooled to room temperature, mice were divided into three groups (20 mice per group) and gavaged with OSA-starch (1000 mg/kg bw, ig) alone or in combination with TPLs (OSAT), and TPLs alone were used as the control (50 mg/kg bw, ig). No chow was provided to mice during the experiment. Mice were sacrificed at time 0 (as the control without feeding) and 30, 60, and 120 min after gavaging. Blood samples were collected into heparin-containing tubes by eyeball extirpating before cervical dislocation and then centrifuged at 3000g for 10 min at 4 °C for plasma insulin analysis. In the meantime, the small intestine tissue (ST) was removed immediately, and luminal contents (LC) was expelled into microcentrifuge tubes with cold saline. ST and LC were snap-frozen and kept at −80 °C for later analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee at Jiangnan University under protocol 20131024-0107. The samples of ST and LC for the measurement of enzyme activity, TPLs’ content, malondialdehyde content, and DPPH scavenging activity were prepared through homogenization of ST and LC by adding ice-cold saline (1:9 (w/v)), which was carried out in a ice bath for a total time period of 30 s (5 s per operation) using a homogenizer (Ultra-Turrax T25, IKA Works Inc., China) at 24000 rpm. The homogenates were centrifuged at 3000g for 10 min at 4 °C, and the supernatant was used as the sample for analysis. Postprandial Glycemic Response Measurement. The postprandial glycemic response was measured by feeding different test diets (TPLs, 50 mg/kg bw; OSA-starch, 1000 mg/kg bw; OSA-starch + 5% TPLs, 1000 mg/kg bw) via gavage. Blood samples were taken 2821
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Journal of Agricultural and Food Chemistry solution + 1 mL of sample supernatant), and A2 is the absorbance of blank (1 mL of tissue supernatant + 2 mL of ethanol). Emulsifying Activity Measurement. The emulsion preparation and emulsifying activity of OSA-starch was measured according to the method proposed by Miao et al. and Meng et al.7,38 with slight modifications. Starch solutions (1.0% w/w) were prepared by dispersing the samples with/without 5% (w/w, based on starch) TPLs in deionized water at room temperature and heating in a boiling water bath with stirring for 20 min to completely dissolve the samples. Oil-in-water (O/W) emulsions with 5% Arawana soybean salad oil (Yihai Kerry, China) were prepared using a high-speed homogenizer (Ultra-Turrax T25, IKA Works Inc., China) at 24000 rpm for 5 min at room temperature to produce a fine emulsion. For the emulsifying activity, 50 μL of each emulsion was pipetted into 5 mL of 0.1% sodium dodecyl sulfate. After emulsion formation, the absorbance (emulsifying activity, EA) was measured at 500 nm. The emulsion stability (ES) of samples was measured after storage at room temperature for 24 h and calculated as follows:
experimental results. Statistical analysis was carried out using the oneway analysis of variance (one-way ANOVA) to determine the statistical significance at p < 0.05 by Tukey’s honest significant difference (HSD) test.
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RESULTS AND DISCUSSION TPLs Modulate the Postprandial Glycemic Response of OSA-Starch. Chemically modified starch has long been used in food applications with different purposes, and nutritionally, most of the chemically modified starches belong to the category of resistant starch. OSA-starch, to our knowledge, is the only one with a substantial fraction of SDS after heat−moisture treatment.13 However, a human study of its effect on postprandial glycemia showed the glycemic profile is still not good enough to provide a sustained glucose release.13 Thus, tea polyphenols, which have been shown to inhibit the activity of α-amylase39,40 and glucose transport of SGLT1,22 were chosen to further improve the slow digestion property of OSA-starch. The dose of TPLs used in the current study was determined to be 5% (w/w, based on dry weight of OSAstarch) because the lowest digestion rate of OSA-starch was observed at this dosage (data not shown). The OSA-starch was produced according to a literature report,13 and the degree of substitution of the final OSA-starch was determined to be 0.0194. The successful esterification of waxy corn starch was proved by FTIR analysis (Figure 1) with two new absorption
ES = (height of remaining emulsion layer/total height) × 100%
Fourier Transform Infrared Spectral (FT-IR) Analysis. OSAstarch (1.0 g) with or without TPLs (5% w/w based on starch) was mixed in 20 mL of distilled water and cooked in a boiling water bath for 20 min with continuous stirring to completely gelatinize the starch. The samples were freeze-dried after the cooked samples were cooled to room temperature. A Nicolet Nexus 470 Fourier transform infrared spectrometer (Thermo Electron Corp.) equipped with an ATR cell and EZ Omnic software (version 7.0) was used to obtain the spectrograms of the OSA-starch. Accurately weighed samples and KBr (100 times the weight of each sample) were fully milled together to get the infrared information from 4000 to 600 cm−1 by accumulating 32 scans per spectra at a resolution of 4 cm−1. Differential Scanning Calorimetry (DSC). DSC measurements were carried out with a Q20 thermal analyzer (TA Instruments, New Castle, DE, USA). OSA-starch (2 mg) and OSA-starch−TPLs mixture (OSA-starch/TPLs = 20:1) were placed in an aluminum pan with water (2:1, v/w) and held for at least 8 h before analysis. The samples were heated from 30 to 120 °C at a heating rate of 10 °C/min in a nitrogen atmosphere. Thermal transitions for gelatinization were characterized by To (onset temperature), Tp (peak temperature), Tc (conclusion temperature), and ΔH (enthalpy of gelatinization). Scanning Electron Microscopy (SEM) Analysis. To analyze the difference of the microstructure of OSA-starch affected by TPLs, freeze-dried OSA-starch with/without 5% TPLs after gelatinization was used as sample for SEM analysis. Scanning electron micrographs were then obtained with a Quanta 200 scanning electron microscope (FEI Co., Eindhoven, The Netherlands) under a vacuum of 13.33 Pa and an operating voltage of 20 kV. Dynamic Laser Scattering (DLS) Analysis. A commercial laser light scattering spectrometer (ALV/DLS/SLS-5022F, ALV Co., Langen, Germany) equipped with an ALV-5000/EPP multi-τ digital time correlator covering 125 ns−37 h in delay time and a He−Ne laser (Uniphase, output power ≈ 20 mW at λ = 632.8 nm) was used to measure the hydrodynamic radius of OSA-starch molecules. An OSAstarch solution (0.1 mg/mL) with or without TPLs (5%, OSA-starch weight base) was mixed and used as the sample for analysis. Each sample solution was first passed through a 0.45 μm Millipore syringe filter into a dust-free cell. The dynamic light scattering measurements were obtained at 90°, and CONTIN FIT (ALV Co.) was performed to obtain the hydrodynamic radius distribution of starch molecules. Viscosity Measurement. OSA-starch (5%) with or without TPLs (5% based on dry weight of starch) was first cocooked at 100 °C for 20 min to completely gelatinize the starch, and then the apparent viscosity (Pa.s) of the samples was measured at 25 °C along the shear rate from 0.1 to 100 s−1 using an AR-G2 rheometer (TA Instruments-Waters LLC, Shanghai, China) with a 40 mm diameter steel plate gapped by 1 mm. Statistical Analysis. Experimental data of in vivo glycemic response, insulin level, and enzyme activity were expressed as means ± SEM, and standard deviation of the mean was used for other
Figure 1. Fourier transform infrared spectrometry of native and OSA starches: (a) waxy starch; (b) OSA-starch. The DS is 0.0194.
bands at 1722 and 1573 cm−1 corresponding to the CO stretching vibration of the ester group and the asymmetric stretching vibration of carboxyl group, respectively.12 The postprandial glycemic response to starch reflects the rate of glucose release from starch digestion and glucose absorption in the small intestine. The triggered secretion of insulin also contributes to a reduced blood glucose level by promoting glucose absorption. The addition of TPLs to OSA-starch significantly improved the postprandial glycemic response using Kunming mouse as the animal model. For OSA-starch, the postprandial blood glucose concentration reached a peak value of 4.9 mmol/L above the baseline after gavaging for 15 min. When OSA-starch was cocooked with TPLs (5%, based on starch dry weight, termed OSAT), a significant decrease of postprandial glycemia (from 4.9 to 3.0 mmol/L at the peak from baseline) with a delayed blood glucose peak (from 15 to 30 min) was observed (Figure 2A), although no significant 2822
DOI: 10.1021/jf5059705 J. Agric. Food Chem. 2015, 63, 2820−2829
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Figure 2. Postprandial glycemic response to OSA-starch affected by the addition of TPLs (A) and insulin levels (B) in mice. Results are shown as means ± SEM (n = 6); (#) p < 0.05 compared to OSA starch; (∗) p < 0.05 compared to TPLs.
Figure 3. Activities of α-glucosidase in the small intestine tissue (A) and luminal contents (B) of mice after feeding different diets. Results are shown as means ± SEM (n = 6); (#) p < 0.05 compared to OSA starch; (∗) p < 0.05 compared to TPLs.
amylase and α-glucosidase in the small intestine were first examined. Because the secretion of digestive enzymes needs the stimuli of food materials, TPLs alone did not show any changes of enzyme activity and can only act as the baseline. Regarding the influence of TPLs on α-amylase activity, it has been well documented in the literature that TPLs mainly inhibit the activity of α-amylase for carbohydrate digestion based on in vitro studies under the optimum condition of enzyme activity.39,40,42−44 Indeed, there was a literature report23 that decreased α-amylase activity was concluded as the reason for decreased postprandial glycemia in a mouse model study using normal corn starch. However, the in vivo activity of α-amylase was not studied, and the conclusion was solely based on an in vitro measurement using commercially sourced α-amylase.23 In the current study, we did not find significant changes of αamylase activities in the small intestine (data not shown). α-Glucosidase inhibitors are commonly prescribed to diabetics to reduce postprandial hyperglycemia induced by the digestion of starchy foods in the small intestine,45 and TPLs are potent α-glucosidase inhibitors from a literature study on Caco-2 cell cultures.46 Thus, the activity of small intestine αglucosidase was examined in both luminal content (LC) and the tissue (ST) of the small intestine. The activities of αglucosidase in LC and ST were significantly decreased in the OSAT-treated mice compared to that in OSA-starch-treated mice (Figure 3). Considering the regulatory effct of starch molecules on the gene expression of starch-hydrolyzing
difference of the area under the glycemic curves (AUC) was found (1140.8 ± 39.2 for OSA-starch, 1173.2 ± 65.9 min· mmoL/L for OSAT). Compared to regular waxy corn starch with a peak glycemia of ∼7.5 mmol/L from baseline in the same mouse model,41 the OSA-starch itself is of slow digestion property, and the addition of TPLs to OSA-starch further significantly decreased its postprandial glycemia. However, the addition of TPLs did not significantly affect the secretion of insulin following oral administration of OSA-starch or OSAT (Figure 2B), indicating insulin is not likely responsible for the decreased glycemia. Additionally, our previous study showed an identical postprandial glycemic response profile to glucose whether in the presence or absence of TPLs, indicating that TPLs have a negligible effect on glucose absorption in the small intestine,22 which is also supported by a study showing that acute administration of EGCG did not inhibit glucose transport across the intestinal wall.23 Therefore, the decreased postprandial glycemia of OSA-starch in the presence of TPLs is likely due to the reduced digestibility of OSA-starch. Effect of TPLs on the Activity of α-Glucosidase. Starch is primarily digested by α-amylase and mucosal α-glucosidase in the small intestine. α-Amylase is responsible for cleaving the large strach molecules into maltooligosaccharides and α-limit dextrins, and α-glucosidase is a group of enzymes anchored on the cell membrane to generate monosaccharides for absorption. As shown above, starch digestion is likely the main reason for decreased postprandial glycemia, so the enzyme activities of α2823
DOI: 10.1021/jf5059705 J. Agric. Food Chem. 2015, 63, 2820−2829
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Figure 4. Effect of TPLs on the activity of small intestine α-glycosidase from mice as expressed by a dose-dependent inhibition pattern (A) and its kinetics (B). Results are shown as means ± SD (n = 3); (∗) p < 0.05; (∗∗) p < 0.01 compared to control.
Figure 5. Concentrations of catechins and EGCG in luminal contents (A, B) and the small intestine tissue (C, D) of mice after oral administration of different diets (TPLs alone and OSAT). Results are shown as means ± SEM (n = 6).
enzymes,47 the decreased activity of α-glucosidase might be due to a decreased gene expression. However, at least 6 h is required for carbohydrate to affect the expression and posttranslation modification of mucosal sucrase-isomaltase (one type of α-glucosidase) in a Caco-2 cell model study.48 Thus, the duration of our in vivo experiment (2 h) is not likely long enough for OSA-starch to affect the expression of small intestine mucosal α-glucosidase, so the decreased activities of αglucosidase in LC and ST were most likely caused by the inhibiton effect of tea catechins in OSAT during digesiton. Indeed, the in vitro study of the effect of TPLs on the activity
α-glucosidase in small intestine homogenate (Figure 4A) showed that its activity was inhibited by TPLs in a dosedependent manner. Kinetic analysis on homogenate αglucosidase showed a decreased reaction velocity (from 1.21 to 0.83 mmol/L/min) without affecting the enzyme’s affinity for substrate, indicating a noncompetitive inhibition function of TPLs (Figure 4B). Therefore, the decreased activity of αglucosidase in the presence of TPLs might be the main reason for the reduced and prolonged postprandial glycemia, which is characteristic of slowly digestible starch.49 A higher enzyme activity of α-glucosidase after feeding OSA-starch alone also 2824
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Figure 6. Differential scanning calorimetry profiles (A) and emulsion property (B) of OSA-starch affected by TPLs.
makes the used method less effective in detecting all of the conjugated catechins, but the actual reason needs further clarification through a systematic study, which is beyond the scope of this paper. Complexation between TPLs and OSA-Starch. Decreased postprandial glycemic response in the presence of TPLs has been shown to be associated with the inhibitory funciton of TPLs on small intestinal α-glucosidase in the current study. However, our previous study of the effect of TPLs on the glycemic responses of normal corn and waxy corn starch did not show significant changes.24 Therefore, it is necessary to understand how TPLs in OSAT sample inhibit α-glucosidase and decrease the postprandial glycemia. Differential scanning calorimetry records the amount of heat involved in the starch gelatinization. TPLs considerably affected the gelatinization properties of OSA-starch (Figure 6A), and there was a clear shift in the endothermic peak toward a lower temperature with significantly decreased enthalpy (from 11.05 ± 0.25 to 9.32 ± 0.12 J/g). The reason for the decreased gelatinization temperature in the presence of TPLs might be due to a rapid hydration of OSA-starch when its hydrophobicity is decreased through a possible hydrophobic interaction with TPLs. Specifically, the interaction between the aromatic rings of TPLs and hydrocarbon chains of OSA groups51 that are mostly present in the amorphous regions of starch granules because the crystalline regions in the granules are difficult for chemical regents to penetrate. In the meantime, the H-bond interactions between the hydrophilic group of TPLs with linear segment33 of amylopectin located in the amorphous region of starch granules might weaken the coupling forces between the crystallites and the amorphous matrix,52 leading to a lower energy required to gelatinize the starch granules. OSA-starch, as an amphiphilic derivative of starch, is an effective stabilizer in water in oil emulsions and some oil-inwater systems.12 As shown in Figure 6B, the emulsifiability of OSA-starch and its stability were decreased significantly after cocooking with TPLs (25.5 and 23.2% declines, respectively), which directly indicates the presence of a hydrophobic interaction between TPLs and OSA-starch leading to a decrease of the hydrophobic property of OSA-starch, and so to a decreased emulsifiability. The above conclusion is supported by a literature report showing that TPLs, especially EGCG, can interact with the lipid layer of emulsions and increase emulsion drop size,53 and this might reflect the same phenomenon of an
supports the inhibition effect of TPLs on the activity of mucosal α-glucosidase. Catechin Release from OSAT Sample. Although TPLs can inhibit the activities of digestive enzymes and alleviate postprandial hyperglycemia, their sensitivity to environmental conditions might affect their bioactivity. In the environment of the small intestine, the alkaline pH makes TPLs more susceptible to degradation, leading to a rapid clearance and a loss of bioactivities.31 The levels of four major catechins in LC after mice were administered OSAT (1000 mg/kg bw, ig) and control of TPLs (50 mg/kg bw, ig) were first analyzed. The results (Figure 5) showed that the concentration of catechins (total and unconjugated) decreased rapidly in LC following administration of the control sample of TPLs and almost undetectable after 120 min, whereas concentration of catechins in the small intestine (ST + LC) of OSAT-treated mice remained relatively stable along the digestion of starch. A lower content of catechins (total and unconjugated forms) was also detected in LC at the early stage of digestion from the OSATtreated mice. All of these results indicate a sustained release of TPLs from OSAT along the digestion process. Conjugated forms of tea catechins are formed during the in vivo elimination process through the action of phase II enzymes. Literature reports showed that glucuronidated EGCG is the major conjugated form when EGCG is orally administered to mice,35 and sulfated and methylated forms are also widely present in the conjugated catechins.27 The biotransformation of TPLs after absorption is intimately associated with the bioactivities of tea cateahins. A literature report showed methylated EGCGs such as 4″-MeEGCG are much less effective than EGCG to induce the apoptosis of prostate cancer cells of LNCaP,50 and some glucuronidated forms of EGCG and EGC are less effective in free radical scavenge activity than their unconjugated forms.51 Thus, we also measured the content of tea catechins in the small intestine tissues, and the results (Figure 5C,D) showed that catechins and EGCG after OSAT treatment were mostly present as the unconjugated forms rather than the conjugated types, indicating the OSAT sample might also have the function to improve the bioactivity of TPLs. However, comparison of the amount of total catechins and unconjugated TPLs showed that the content of total catechins is less than the unconjugated forms, which has also been observed in the literature.23 The reason is likely due to the complex of biotransformation that 2825
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Figure 7. Effect of TPLs on the molecular size distribution (A) and the viscosity of OSA-starch (B).
Figure 8. SEM analysis of freeze-dried OSA-starch (A) and OSAT (B). Scale bar = 100 μm.
intestine is not likely affected even though TPLs do have the capability of inhibiting the activity of α-amylase. However, the complex structure might be a barrier to α-amylase, making it less efficient to hydrolyze starch, which also contributes to a reduction of postprandial glycemia. After TPL-containing (through interaction with OSA group) malto-oligosaccharides or α-limited dextrin (substrate of α-glucosidase) are slowly produced by α-amylase, their accessibility to mucosal αglucosidase might also be affected. A gradual release of TPLs after digestion by α-amylase and α-glucosidase might lead to a high association between TPLs and α-glucosidase due to their closeness in distance, which facilitates the binding of TPLs by α-glucosidase, resulting in activity inhibition. Thus, the OSAT complex causes not only a decreased digestion efficiency by αamylase and a slow production of substrate for mucosal αglucosidase but also an inhibition of α-glucosidase activity by a sustained release of tea catechins from the complex, which all together result in a lower and prolonged postprandial glycemia. Health Implication. Epidemiological studies have associated tea consumption with prevention of chronic and degenerative diseases including cancer,55 cardiovascular disorder,56 obesity, and diabetes.57 However, low bioavailability limits the bioactivities of catechins.31 In our study, a sustained release of unconjugated catechins from OSAT after acute administration was observed (Figure 5). Catechins, mainly EGCG, were present mostly as the more active unconjugated
increase of particle size of OSA-starch molecules (Figure 7A) in which TPLs might bridge more OSA-starch molecules together through hydrophobic interaction. In the meantime, this bridge also changes the conformation or shape of the OSA-starch molecules with a more spherical nature that is less viscous and less shear-thinning (Figure 7B) because shear-thinning behavior is characteristic of linear macromolecules. The influence of TPLs on OSA-starch structure due to hydrophobic interaction was also supported by SEM observation of the freeze-dried samples of OSA-starch and OSAT sample with dramatic difference of macroscopic structures (Figure 8). All of the above experimental results showed the interaciton between OSA-starch and TPLs, and considering the molecular structure of OSA-starch and the TPLs, there might exist a spherical-like OSA-starch−TPL (OSAT) complex in which the hydrophobic interaction between the OSA group of OSA-starch and aromatic hydrocarbons of TPLs is the major force for their complexation.54 When the hydrophobic complexation occurs, they might pull the amylopectin molecules together to shorten the distance between these molecules, facilitating the interaction between linear segments of amylopectin through TPLs’ bridge function as shown in our previous study,33 which further tightens the aggregation of OSA-starch molecules in the OSAT complex. When the complex was digested by α-amylase, the TPLs embedded in the complex might not be accessible to α-amylase, so that the activity of α-amylase in the small 2826
DOI: 10.1021/jf5059705 J. Agric. Food Chem. 2015, 63, 2820−2829
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Journal of Agricultural and Food Chemistry
Figure 9. Level of MDA (A) and DPPH radical scavenging activity (B) in the small intestine tissue of mice. Results are shown as means ± SEM (n = 6); (#) p < 0.05 compared to OSA starch; (∗) p < 0.05 compared to TPLs.
the Ministry of Science and Technology of People’s Republic of China Infrastructure Program (Project 2012BAD33B05).
form in ST rather than the conjugated form. In the meantime, the uptake of unconjugated TPLs might also be increased due to a high concentration of unconjugated TPLs in the luminal side of the small intestine and closeness to the absorptive enterocytes. MDA is a major reactive aldehyde that appears during the peroxidation of fatty acid. Therefore, the content of MDA is widely used as a biomarker of oxidative stress and indicator of tissue damage involving a series of chain reactions.58 We observed a significant increase of MDA level in intestinal tissue of mice due to the fluctuation of plasma glucose caused by OSA starch (Figure 9A). It has been demonstrated that glucose fluctuation is more deleterious to endothelial function through excessive generation of reactive oxygen species (ROS), resulting in oxidative stress.59 Our study showed that the content of MDA decreased significantly in mice dosing OSAT (Figure 9A). Additionally, OSAT feeding also showed stronger DPPH scavenging activity than both OSA-starch and TPLs alone in the ST of mice (Figure 9B; p < 0.05). Thus, the OSAT complex also has an antioxidative bioactivity. In conclusion, experimental results on the physicochemical properties of OSAT sample demonstrate the presence of a hydrophobic interaction-induced complexation between OSAstarch and TPLs. This complex not only changes the digestion property of OSA-starch, leading to a slow elevation of postprandial glycemia and possibly a sustained glucose release, but also has the capability to reduce the oxidative stress, which is likely the first study on functional or bioactive starch material to improve health. Although OSA-starch is not a common food ingredient that is generally consumed in large quantity, the complex formed between OSA-starch and TPLs, as the structural basis for TPLs’ function, indicates some carbohydrate-coupled delivery systems of TPLs might be developed to efficiently decrease the postprandial glycemia for improved health.
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Notes
The authors declare no competing financial interest.
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The current investigation was supported by the National Natural Science Foundation of China (Project 31471585) and 2827
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