Pomegranate ellagitannins inhibit α-glucosidase activity in vitro and reduce starch digestibility under simulated gastro-intestinal conditions.

Abstract Pomegranate extract was tested for its ability to inhibit α-amylase and α-glucosidase activity. Pomegranate extract strongly inhibited rat intestinal α-glucosidase in vitro whereas it was a weak inhibitor of porcine α-amylase. The inhibitory activity was recovered in an ellagitannins-enriched fraction and punicalagin, punicalin, and ellagic acid were identified as α-glucosidase inhibitors (IC50 of 140.2, 191.4, and 380.9 μmol/L, respectively). Kinetic analysis suggested that the pomegranate extract and ellagitannins inhibited α-glucosidase activity in a mixed mode. The inhibitory activity was demonstrated using an in vitro digestion system, mimicking the physiological gastro-intestinal condition, and potatoes as food rich in starch. Pre-incubation between ellagitannins and α-glucosidase increased the inhibitory activity, suggesting that they acted by binding to α-glucosidase. During digestion punicalin and punicalagin concentration decreased. Despite this loss, the pomegranate extract retained high inhibitory activity. This study suggests that pomegranate ellagitannins may inhibit α-glucosidase activity in vitro possibly affecting in vivo starch digestion.


Introduction
High intakes of fruit and vegetables have been associated with a lower incidence of chronic diseases including diabetes, cardiovascular diseases, and cancer (Boeing et al., 2012). It is now widely accepted that the protection supplied by fruit and vegetables against diseases is due to the presence of various bioactive compounds. Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites found in plants. Various in vitro and in vivo evidence show that several (poly)phenol-rich foods are protective against chronic diseases, including cardiovascular disease, neurodegeneration, and cancer (Del Rio et al., 2013).
One of the principal topics concerning the beneficial effects of (poly)phenols is their bio-availability and metabolic fate. Most of the dietary phenolic compounds are subjected to extensive metabolism prior and after the absorption such that, with very few exceptions, only metabolites of the parent compounds enter the circulatory system (Del Rio et al., 2013). As a result, the gastrointestinal tract could be the location for the health benefits derived from a diet rich in (poly)phenols. Phenolic compounds might exert direct protective effects in the gastrointestinal tract, by scavenging reactive oxygen species (Halliwell et al., 2000). The inhibition of intestinal carcinogenesis by red wine (poly)phenols, grape seed extract, and berries has been demonstrated in cell lines, animal model systems, and humans (Adhami et al., 2009;Dolara et al., 2005;Kaur et al., 2006). In addition, (poly)phenols are able to inhibit some intestinal digestive enzymes such as lipase and glucosidases, modulating nutrients bioavailability and resulting in a beneficial effect on obesity and blood glucose control .
The prevalence of type II diabetes is rising exponentially and particular non-insulin-dependent diabetes mellitus is intimately associated to cardiovascular complications as a consequence of post-prandial hyperglycemic condition (Nathanson & Nyström, 2009). Inhibitors of intestinal a-glucosidase enzymes retard the rate of carbohydrate digestion, contributing to reduce postprandial hyperglycemia (Krentz & Bailey, 2005). The use of commercial a-glucosidase inhibitors (acarbose, miglitol, and voglibose) is limited by their gastro-intestinal intolerability and high cost. One intriguing approach to control hyperglycemia could be its prevention by phytochemicals present in the diet. Several reports have been published in recent years showing that berry, red wine, and green tea (poly)phenols are able to inhibit in vitro intestinal glucosidases, potentially suggesting their efficacy in an effective management of diabetes mellitus (Boath et al., 2012a;Kwon et al., 2008).
Pomegranate juice supplementation significantly reduced postprandial blood glucose but not triacylglycerols and cholesterol levels in streptozotocin-induced diabetic mice fed with a high-fat diet (Betanzos Cabrera et al., 2011). Indeed, Punica granatum flower extract was able to reduce post-prandial hyperglycemia in Zucker diabetic fatty rats (Li et al., 2005).
A possible explanation for these observations is that pomegranate juice possesses a-glucosidase or a-amylase inhibitors able to attenuate the post-prandial increase in glycemia. A reduction of a-glucosidase activity was observed in the saliva of healthy humans after the consumption of pomegranate extract during an intervention study (Di Silvestro et al., 2009).
The present study tested a pomegranate (poly)phenol-rich extract for a-glucosidase and a-amylase inhibitory activity to determine its potential mechanism of action as a hypoglycemic agent. Pomegranate extract was subsequently fractionated with the aim to identify compounds that may influence the enzymatic activities. Finally, the inhibitory effect on carbohydrate hydrolysis of pomegranate extract was tested against a real food system (potatoes) using an in vitro digestion model.

Sample preparation and total (poly)phenol determination
Pomegranate juice (poly)phenol-rich extract was obtained using C18 solid-phase extraction (Verzelloni et al., 2007). Columns were pre-conditioned with 60 mL of methanol and subsequently with 40 mL of 0.1% formic acid in water. The pomegranate juice was loaded (20 mL) and 60 mL of 0.1% formic acid was used to elute unbound materials (free sugars, organic acids, and vitamin C). The bound materials, containing pomegranate (poly)phenols, were eluted with 60 mL of methanol. The solvent was removed by a rotary evaporator to near dryness and then freeze-dried. The pomegranate extract was tested for its ability to inhibit the activity of a-glucosidase and a-amylase.
Total (poly)phenols in pomegranate juice and extract were determined using the Folin-Ciocalteau method (Singleton et al., 1999). The total phenolic content was expressed in mmol/L of ellagic acid equivalents, using ellagic acid as a standard at concentrations ranging between 20 and 1000 mmols/L. The choice of the standard was carried out considering that ellagitannins (which are built up with ellagic acid units) are the most predominant phenolic components present in pomegranate juice (Fischer et al., 2011).

Amylase assay
Amylase assay was carried out as reported by  using porcine pancreatic a-amylase and soluble potato starch as a substrate. The reaction was performed in 20 mmol/L sodium phosphate buffer pH 6.9 containing 6.7 mmol/L NaCl. For the reaction, 0.1 mL of 2 U/mL amylase solution (one unit of amylase is defined as the quantity of enzyme that releases 1.0 mg of maltose from starch in 3 min at pH 6.9 at 20 C) was mixed with 0.9 mL of sodium phosphate buffer or different concentrations of pomegranate extract dissolved in the sodium posphate buffer. After 10 min at 37 C, 1 mL of 1% starch solution (dissolved in the sodium posphate buffer) was added and the reaction mixture was incubated at 37 C for 30 min. The reaction was terminated by adding 1 mL of dinitrosalicylic acid solution and boiling for 15 min in a water bath. Enzyme activity was quantified by measuring the mg of maltose released from starch by reading at 540 nm. To calculate the IC 50 value, the enzyme activity was determined in the presence of pomegranate extract with phenol concentrations ranging from 150 to 3000 lmol/L. The IC 50 is defined as the concentration of phenolics required to inhibit 50% of the enzymatic activity.

a-Glucosidase assay
The enzyme a-glucosidase was extracted from rat intestinal acetone powder and assayed according to Oki et al. (1999). The rate of release of p-nitrophenol (PNP) from PNP-gluc was measured at 37 C after incubation for 20 min in the presence of 0.01 U/mL of rat intestinal a-glucosidase (one unit of a-glucosidase is defined as the quantity of enzyme that releases 1.0 lmol of PNP from PNP-gluc per minute at pH 6.8 at 37 C). For the reaction, 0.1 mL of 0.2 U/mL of rat intestinal a-glucosidase was pre-incubated with 0.9 mL of buffer (potassium phosphate buffer 67 mmol/L, pH 6.8) or different concentrations of pomegranate extract dissolved in buffer. After 10 min at 37 C, 1 mL of substrate solution (containing 1 mmol/L PNP-gluc and 0.2 mmol/L of glutathione dissolved in potassium phosphate buffer) was added and the reaction mixture was incubated at 37 C for 20 min. The reaction was terminated by adding 4 mL of 100 mmol/L sodium carbonate solution. Enzyme activity was quantified by measuring the lmol of PNP released from PNP-gluc by reading at 400 nm. To determine the IC 50 value, the enzyme activity was determined in the presence of pomegranate extract with phenols concentrations ranging from 150 to 3000 lmol/L. The IC 50 is defined as the concentration of phenolics required to inhibit 50% of the enzymatic activity.

Pomegranate juice (poly)phenol-rich extract fractionation
To identify compounds responsible for the inhibitory activity, pomegranate extract was fractionated using Sephadex LH-20 with the method adapted from the Tannin Handbook (available at www.users.muohio.edu/hagermae/tannin.pdf).
Sorption to Sephadex LH-20 in aqueous ethanol and selective de-binding with aqueous acetone is an established method for separating tannins from non-tannin phenolics. Briefly, after column preconditioning with 80% ethanol, the pomegranate extract in 80% ethanol was applied to the column. The unbound material (anthocyanins and other monomeric phenolic compounds) was collected after washing with three volumes of 80% ethanol. The bound fraction (ellagitannins) was eluted with three volumes of 50% acetone. Both the fractions were evaporated by a rotary evaporator to near dryness and then freeze-dried. All the fractions were subjected to LC-ESI-MS/MS analysis and tested for their ability to inhibit the hydrolytic enzymes.

LC-ESI-MS/MS analysis
LC-MS/MS analysis was carried out according to Fischer et al. (2011) using an Agilent system 6310A Ion Trap LC-MS n (Agilent, Waldbronn, Germany) equipped with degasser, binary gradient pump, thermo-autosampler, and column oven. The MS/MS system was an ion trap mass spectrometer fitted with an ESI source. Data acquisition and processing were performed using DataAnalysis software (SAS Inc., Cary, NC). Negative ion (ellagitannins) mass spectra of the column eluate were recorded in the range of m/z 50-1300 at a scan speed of 13 000 m/z/s. The mobile phase, solvent A (1% formic acid) and solvent B (acetonitrile), was used under binary linear gradient conditions as follows: 5-15% B (10 min), 15-25% B (20 min), 25-50% B (3 min), 50% B isocratic (4 min); with a flow rate of 1 mL/min.

HPLC-DAD analysis
Individual phenolic compounds were quantified using an HPLC system consisted of a Jasco HPLC system (Orlando, FL) equipped with a diode array detector, a reversed phase column Hamilton HxSil C18 (Hamilton, Reno, Nevada; 250 mm Â 4.6 mm), a volumetric injector Rheodyne (Cotati, CA), and a temperaturecontrolled oven.
For ellagitannins quantification, the monitored wavelength was 360 nm. Identification and quantification of punicalagins A and B, ellagic acid, and punicalin in samples were performed using calibration curves of the respective standards compounds. For this reason, stock solutions of standard compounds were diluted at different concentrations and the solutions were analyzed.
Anthocyanins were quantified at a wavelength of 520 nm as cyanidin-3-glucoside equivalents. The HPLC parameters were the same as reported in the previous section.

Identification of a-glucosidase inhibitors
The ellagitannins recognized by LC-MS/MS were tested for their a-glucosidase inhibitory activity. Ellagic acid and punicalagin (a mixture of A and B isomers) were obtained from Sigma Chemical Co. (Milan, Italy) as pure compounds (95% of purity degree). Punicalin was purified from pomegranate juice following the procedure reported in Aviram et al. (2008). Purified compound was evaporated by a rotary evaporator to near dryness and then freeze-dried. The purified compound was characterized by LC-ESI-MS/MS and the purity assayed with HPLC-DAD (95% of purity degree as deduced from the ratio of the peak area of the isolated compounds and total peak area at 280 nm; see Supplementary Figure). For the calculation of IC 50 values, a-glucosidase assay was carried out in the presence of variable amounts (from 10 to 500 lmol/L) of punicalin, punicalagin, or ellagic acid.

In vitro gastro-intestinal digestion
The gastro-intestinal system was adapted from Tagliazucchi et al. (2012) with some modifications. Potatoes, selected as real starchrich food, were weighed, peeled, and cooked whole in boiling water for 30 min. They were removed and cooled at ambient temperature (21 C) to be handled. Ten grams of cooked potatoes (corresponding to 1.71 g of starch) were homogenized in a laboratory blender for 1 min to simulate mastication in the presence of 5 mL of simulated salivary fluid and 20 mL of different concentrations of pomegranate extract dissolved in a 0.1 M phosphate-buffer (pH 6.9). The artificial saliva consisted of a 0.1 M phosphate-buffer (pH 6.9) containing 1.336 mmol/L CaCl 2 , 0.174 mmol/L MgSO 4 , 12.8 mmol/L KH 2 PO 4 , and 23.8 mmol/L NaHCO 3 , 2 g/L of food casein (known to be a proline-rich protein), and 150 units/L a-amylase.
In the control digestion, the pomegranate extract was omitted and the cooked potatoes (10 g) were homogenized in the presence of 5 mL of simulated salivary fluid and 20 mL of the 0.1 M phosphate-buffer (pH 6.9).
After 10 min of incubation at 37 C in a shaking bath, the pH was adjusted to 2.5 (to simulate gastric pH) with concentrated HCl and after 2 g/L of NaCl and 315 U/mL of pepsin were added. The solution was incubated at 37 C in a shaking bath at 100 rpm for 2 h. At the end of the gastric digestion, the pH was brought to 7.5 with NaHCO 3 (to simulate hepato-pancreatic pH) before adding 0.8 g/L of pancreatin, 5 mg/mL of bile salts, and 2 mL of rat intestinal solution containing 10 U of a-glucosidase. On the basis of the added pancreatin, the amount of digestive enzymes in the intestinal fluid was 80 U/mL of a-amylase, 240 U/mL of proteases, and 384 U/mL of lipase. The solution was then incubated at 37 C in a shaking bath at 100 rpm for a further 2 h.
The amount of glucose released at the end of the digestion was quantified using a hexokinase, glucose-6-phosphate dehydrogenase, phospho-glucose isomerase method (Kunst et al., 1984). The ellagitannins were quantified by HPLC-DAD as reported in the previous section.

Statistical analysis
All data are presented as mean ± SD for three replicates for each prepared sample. Student's t-test and ANOVA with the Tukey post-hoc test was performed using Graph Pad Prism (GraphPad Software, San Diego, CA). The differences were considered significant with p50.05. The IC 50 values were determined using non-linear regression analysis and fitting the data with the log(inhibitor) versus response model (Graph Pad Prism).

Results
The pomegranate juice contained 6.82 ± 0.75 mmol of ellagic acid equivalent (EAE)/L of phenolic compounds. The percentage of the recovery in the C18 bound fraction, corresponding to the pomegranate extract, was 86% of total (poly)phenols (5.87 ± 0.26 mmol of ellagic acid equivalent (EAE)/L).
Pomegranate extract was an effective inhibitor of rat intestinal a-glucosidase with an IC 50 value of 922.8 ± 1.2 lmol of EAE equivalent/L (Figure 1). Acarbose inhibited a-glucosidase in a dose-dependent manner giving an IC 50 value of 69.7 lmol/L. On the contrary, pomegranate extract was a weak inhibitor of a-amylase. At the highest tested concentration, corresponding to a final concentration of pomegranate (poly)phenols in the assay of 3000 lmol of EAE equivalent/L, the a-amylase activity was inhibited by 42%. These results showed that pomegranate extract contained potent inhibitors of rat intestinal a-glucosidase.
Enzymatic analysis showed that only the LH-20 bound fraction caused inhibition of a-glucosidase, whereas the LH-20 unbound fraction did not show any inhibitory activity, even at the highest tested concentrations. It is interesting to note that also the majority of the a-amylase inhibitory activity was recovered in the LH-20 bound fraction, with only a marginal activity found in the LH-20 unbound material.
The retention times, concentration (mmol/L), and mass spectral characteristics of the ellagitannins are specified in Table 1. Punicalin is the major ellagitannins (peak 6; Figure 3A) found in the pomegranate extract; this compound present an [M-H] À ion at m/z 781 and fragments at m/z 601 and 602 for the loss of gallagic acid moiety.
Punicalagin showed an [M-H] À ion at m/z 1083 but it can be also detected as doubly charged ion species at m/z 541. The fragment at m/z 601 in MS/MS experiment showed the loss of a gallagic acid moiety and a fragment with m/z 781 was observed equivalent to the [M-H] À ion of punicalin. The presence of the two isomers A and B (peaks 7 and 8; Figure 3A) was confirmed by the different retention times of the commercial standard isomers.
The compound eluting at 15.0 min exhibited an [M-H] À ion at m/z 783. The loss of water moiety and ellagic acid (m/z 301) in MS/MS experiment produced fragments at m/z 765 and m/z 481, respectively. Based on this fragmentation pathway and a previous study (Okuda et al., 1983), this compound was identified as bis-HHDP-hexoside (pedunculagin A; peak 9; Figure 3A).
The compound which eluted at 19.7 min with fragment at m/z 463 was identified as ellagic acid-hexoside (peak 11; Figure 3A). This compound produced fragments at m/z 300, 301, and 302 in the MS/MS experiment, typical m/z fragments of ellagic acid. Ellagic acid-hexoside has previously reported in pomegranate juice and arils (Fischer et al., 2011).
The last identified compound was ellagic acid (peak 12; Figure 3A). The aglycone moiety (m/z 301) produced characteristic fragments at m/z 229, 201, and 185 in MS/MS experiment. Ellagitannins and ellagic acid were, therefore, identified as the a-glucosidase inhibitors present in the pomegranate extract. The IC 50 values of the individual ellagitannins revealed that punicalagin was the most effective inhibitor of a-glucosidase (IC 50 of 140.2 ± 1.1 lmol/L) followed by punicalin and ellagic acid (IC 50 of 191.4 ± 1.3 lmol/L and 380.9 ± 3.5 lmol/L, respectively).
To gain more information about the role of each identified ellagitannins in the a-glucosidase activity inhibition, their contribution ratio was calculated by dividing the power of inhibitory activity of each identified compound (calculated by dividing the amount of each single compound in the extract in lmol/L by its IC 50 value in lmol/L) with that of the pomegranate extract (calculated by dividing the total (poly)phenolic content of the extract in lmol/L by its IC 50 value in lmol/L) (Toshima et al., 2010). The obtained value was then multiplied by 100 to estimate the contribution ratio as %. For example, the contribution ratio of punicalagin was calculated as follows: (232.2/140.2)*100/(5870/ 922.8) ¼ 26%. The same calculation for punicalin and ellagic acid provides contribution ratio values of 54 and 3%, respectively. The data reported clearly indicated that the a-glucosidase inhibitory activity of pomegranate extract was due to punicalin and punicalagin with a minor contribution of ellagic acid.

Kinetic analysis and mechanism of inhibition
In the original assay, the pomegranate extract was mixed with a-glucosidase and buffer, pre-incubated for 10 min and the reaction started by the addition of the substrate. If the order of addition of components was changed and the reaction started by the addition of the enzyme rather than the substrate, then the pomegranate extract was less effective ( Figure 4A). The same effect was observed when different concentrations of ellagic acid were pre-incubated for 0, 5, 10, 30, or 60 min with a-glucosidase ( Figure 4B). These results suggested that pomegranate ellagitannins interacted directly with the a-glucosidase.
The ellagitannin punicalagin as well as the pomegranate extract was selected as test inhibitors for the kinetic analysis. All the tested samples reduced the V max and increased K M of a-glucosidase (Table 2). These results suggested a mixed-type inhibition with respect to substrate concentration.

Effect of pomegranate extract on potato starch hydrolysis during in vitro gastro-intestinal digestion
The ability of the pomegranate extract to inhibit starch hydrolysis was assessed using a real food during simulated gastro-intestinal conditions. Cooked potatoes were firstly subjected to mastication, in the presence of simulated salivary fluid which contained In the original assay, the pomegranate extract was mixed with the a-glucosidase and buffer, preincubated for 10 min at 37 C and the reaction started by the addition of the substrate. In the revised assay, the pomegranate extract was mixed with the substrate, incubated for 10 min at 37 C and then the reaction was initiated by the addition of the enzyme. The final concentration of pomegranate extract (poly)phenols in the assay was 2 mmol/L. Data are means ± SD (n ¼ 3). (B) Effect of pre-incubation time and ellagic acid concentration on the a-glucosidase inhibitory activity of ellagic acid. Ellagic acid was pre-incubated for 0, 5, 10, 30, and 60 min with a-glucosidase before the addition of the substrate. Tested ellagic acid concentrations were ( ) 75 lmol/L, (oe) 150 lmol/L, ( ) 300 lmol/L, and (g) 600 lmol/L. *p50.05 with respect to the previous time. Data are means ± SD (n ¼ 3). Data are represented as means ± SD (n ¼ 3).
150 units/L of a-amylase. After 10 min, the bolus was subjected to consecutive gastric (2 h) and intestinal (2 h) digestion, in the presence of 80 units/mL of a-amylase and 370 units/L of a-glucosidase.
At the end of the gastro-intestinal digestion, in the absence of the pomegranate extract, the amount of released glucose was 199.5 ± 2.12 mg/g of potato starch. The addition of the pomegranate extract in the digestive system produced a decrease in the amount of released glucose at the end of the gastro-intestinal digestion of 18 and 44% when the digestion was carried out with 2.35 or 4.7 mmol/L of total (poly)phenols, respectively. Control experiments carried out without enzymes showed that there was no hydrolysis of potato starch.
The behavior of the ellagitannins during simulated gastrointestinal digestion of potatoes was followed with HPLC-DAD. The results are detailed in Table 3. The concentration of the ellagitannins punicalin and punicalagin decreased by 22.6 and 30.9% after mastication and by 36.8 and 61.6% after pancreatic digestion, respectively. The amount of ellagic acid increases to 142.8 and 234.2% after mastication and pancreatic digestion, respectively.

Discussion
This is the first report showing that pomegranate juice (poly)phenolic extract is a potent inhibitor of in vitro carbohydrate digestion. Pomegranate extract strongly inhibited the rat intestinal a-glucosidase activity in vitro.
The ability of the pomegranate (poly)phenolic-rich extract to inhibit the starch hydrolysis was also demonstrated using an in vitro digestion system, mimicking the physiological gastrointestinal condition, and potatoes as food rich in starch.
A variety of food (poly)phenolic extracts have been shown to inhibit a-amylase and a-glucosidase activities in vitro. Rat intestinal a-glucosidase inhibitory activity of pomegranate extract (IC 50 value of 278 lg/mL) is lower than that of acarbose (IC 50 of 45 lg/mL), anthocyanins-rich berry extracts (such as blueberry, blackcurrant, rowanberry, and strawberry; IC 50 values from 18 to 42 lg/mL), and black tea (IC 50 of 64 lg/mL) (Koh et al., 2010;. However, the in vitro inhibitory activity of pomegranate extract was similar to that of green tea (IC 50 value of 297 lg/mL) which has been found to be effective in reducing postprandial blood glucose level in vivo (Tang et al., 2013).
Punicalagin, despite its lower IC 50 value against rat intestinal a-glucosidase, was not the most important contributor to the inhibitory activity (26% of contribution). In contrast, punicalin was estimated to be the main contributor to pomegranate extract a-glucosidase inhibition (54% of contribution) owing to its higher content in the extract. The total contribution ratio of all identified ellagitannins in this study was 83%, suggesting that some unidentified compounds with a-glucosidase inhibitory activity can be present in the pomegranate extract or that synergic effects should be considered.
Kinetic analysis suggested that pomegranate extract and ellagitannins inhibited a-glucosidase activity in a mixed mode. The pre-incubation and the order of addition experiments indicate that ellagitannins influence a-glucosidase activity via their ability to bind proteins (Wang et al., 2013). The non-specific binding of ellagitannins with a-glucosidase may alter the structure of the enzyme by reducing the velocity of the catalysis and the accessibility to the active site of the substrate.
Most of the studies previously published on the inhibitory activity of (poly)phenols or (poly)phenols-rich extract against a-amylase and a-glucosidase were carried out using enzymatic assay that did not represent the physiological conditions of the gastro-intestinal tract. One of the most important criticisms in employing the enzymatic assay is the use of starch solution or synthetic substrate solution instead of real food. The importance of utilizing real food lies in the presence of additional molecules (such as proteins, lipids, and fibers), other than starch, that may impede the effect of (poly)phenols on the enzymes. An additional criticism is related to the fact that phenolic compounds are somewhat unstable under real or simulated gastro-intestinal conditions. For example, it has been shown that anthocyanins are degraded in the pancreatic media (Liu et al., 2014) whereas ellagitannins may undergo partial breakdown in the gastrointestinal tract (Larrosa et al., 2010). To overcome this point, we tested the ability of pomegranate (poly)phenols to inhibit the carbohydrate hydrolysis during simulated digestion of potatoes. Results show that the pomegranate extract is able to inhibit in a concentration-dependent manner potato starch digestion under in vitro gastro-intestinal conditions. Despite all the limitations of V max is reported as lmol of p-nitrophenol per min at pH 6.8 at 37 C whereas K M is expressed as mmol/L of p-nitrophenyl a-D-glucoside. Data are represented as means ± SD (n ¼ 3). Values in the same columns with different lowercase letter are significantly different (p50.05). the model system (static model, glucosidase not bound to the enterocyte membrane), our results allow us to infer that pomegranate (poly)phenols may be effective inhibitors of starch digestion also in vivo by inhibiting the activity of a-glucosidase.
Our results show that a portion of pomegranate juice (200 mL) is able to inhibit the starch hydrolysis by about 50% during the digestion of a portion (100 g) of potatoes. As already observed, ellagitannins are not stable under gastro-intestinal condition (Larrosa et al., 2010). We found a decrease in the concentration of the ellagitannins punicalin and punicalagin by 22.6 and 30.9% after mastication, respectively. These decreases may be due to the irreversible binding of ellagitannins to salivary or potato proteins (Wang et al., 2013) or to the hydrolysis of punicalin and punicalagin (Cerdá et al., 2003). In the proposed gastro-intestinal hydrolysis pathway, punicalagin breakdown releases equimolar concentrations of ellagic acid and punicalin which is further hydrolyzed to give equimolar concentrations of gallagic acid and glucose (Cerdá et al., 2003). The loss of punicalagins during the salivary phase of the digestion was not accompanied by the appearance of substantive amounts of ellagic acid; the ellagic acid concentration, in fact, increased after mastication of 31.8 lmol/L, whereas the concentration of punicalagins decreased by about 71.9 lmol/L. This is indicative of the fact that part of the ellagitannins bind potatoes or salivary proteins during the oral phase of the digestion. The concentration of punicalagins remained constant during the gastric phase whereas the intestinal phase caused a further decrease in their concentration. The loss of punicalagins in the intestinal media is totally explained by its hydrolysis to ellagic acid. The punicalagin concentration decreased after intestinal hydrolysis of 71.3 lmol/L which is accompanied by the appearance of 69.3 lmol/L of ellagic acid. It is interesting to note the data of ellagic acid concentration after the gastric phase. The concentration of ellagic acid dropped to 5 lmol/L at the end of the gastric digestion because of its poor solubility in acidic media (Larrosa et al., 2010) and, after the passage in the alkaline intestinal fluid, it returned into the solution. Surprisingly, punicalin concentration did not change further during simulated intestinal digestion with respect to the gastric phase. Punicalin was not stable under intestinal conditions but its loss was compensated by the hydrolysis of punicalagin forming punicalin and ellagic acid.
Thus, the increase of ellagic acid that was observed in the last phase of the intestinal digestion is due mostly to the instability of punicalagin in the intestinal environment, with release of ellagic acid moieties and punicalin.
Despite the binding between ellagitannins and proteins and their hydrolysis in the gastro-intestinal media, the pomegranate extract maintained its ability to inhibit starch digestion. This means that hydrolysis of ellagitannins releases compounds with inhibitory activity. For example, punicalin and ellagic acid that are released from punicalagin are still able to inhibit a-glucosidase and, therefore, starch hydrolysis during the digestion of potatoes.
There is some in vivo and in vitro evidence showing that pomegranate juice may be helpful for type II diabetic subjects. First, there are studies reporting the hypoglycemic activity of pomegranate juice in rats (Betanzos-Cabrera et al., 2011) and in diabetic patients (Rock et al., 2008;Rosenblat et al., 2006). Till now the mechanism has not been elucidated, but our data strongly suggest that the hypoglycemic activity of pomegranate juice is due to the ability of ellagitannins to inhibit starch hydrolysis. Some in vivo studies highlighted the protective effect of pomegranate juice on some oxidative complications in diabetic patients. Rosenblat et al. (2006) demonstrated that the consumption of pomegranate juice by diabetic patients significantly decreased serum oxidative stress and the extent of oxidized LDL uptake by macrophages. This effect was mediated by PPARc activation (Shiner et al., 2007). Moreover, the same research group showed that pomegranate juice consumption by diabetic patients could lead to a delay in the atherosclerosis development by increasing paraoxonase 1 stabilization and association with HDL and stimulating its catalytic activity (Betanzos-Cabrera et al., 2011;Fuhrman et al., 2010). This effect is likely mediated by ellagitannins metabolites such as ellagic acid and urolithins (González-Barrio et al., 2010;Park et al., 2011). Pomegranate ellagitannins, in fact, are not absorbed and bioavailable in the human body but are hydrolyzed during the gastro-intestinal digestion releasing ellagic acid that is afterwards bio-transformed in urolithins by the action of colonic microbiota (González-Barrio et al., 2010). Urolithins are well absorbed in the human colon, mainly urolithin-A or urolithin-B and/or iso-urolithin-A according to urolithin phenotype in each person due to the different microbiota communities (Tomás-Barberán et al., 2014), and although they display low antioxidant activity, they are able in vitro to counteract two key features of diabetic complications, i.e. protein glycation and neurodegeneration (Verzelloni et al., 2011). Thus, pomegranate juice (poly)phenols and metabolites could act at different levels in attenuates type II diabetic complications. They may act at gastro-intestinal level, where the ellagitannins punicalin, punicalagin, and ellagic acid inhibit starch hydrolysis, resulting in a hypoglycaemic effect. At systemic level, the ellagitannins metabolites (ellagic acid, urolithins, and their phase II metabolites) may counteract protein glycation and exert anti-atherosclerotic effects, thus reducing some diabetic complications.

Conclusions
We were able to identify the ellagitannins punicalin and punicalagin as a-glucosidase inhibitors in pomegranate juice. Ellagitannins retained their inhibitory activity in an in vitro model of the digestive system and using cooked potatoes as a source of starch.
In conclusion, our data together with literature data argue with the hypothesis that pomegranate juice can be considered as a rational complementary therapeutic agent to ameliorate postprandial hyperglycaemia linked to type II diabetes and hyperglycaemia-induced vascular complications.