Inhibitory Effects of Pomegranate Extracts on Recombinant Human Maltase–Glucoamylase Kayoko Kawakami, Peng Li, Misugi Uraji, Tadashi Hatanaka, and Hideyuki Ito
α-Glucosidase inhibitors are currently used in the treatment of type 2 diabetes. In this study, we investigated the inhibitory activities of aril and pericarp extracts from pomegranates obtained various regions against recombinant human maltase–glucoamylase (MGAM). The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. The Iranian aril extract was the most effective inhibitor. We investigated the polyphenol content of the pomegranate extracts using the Folin–Ciocalteu method. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content. We further evaluated inhibitory activity against α-glucosidase from the rat small intestine. Pomegranate extract used in this study showed slightly different inhibitory activities according to α-glucosidase origin. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Bioassayguided fractionation of the pomegranate arils led to identification of punicalagin and oenothein B as potent inhibitors of α-glucosidase. Oenothein B showed inhibitory activity with a half-maximal inhibitory concentration (IC50 ) value of 174 μM. Its potency was comparable to that of the α-glucosidase inhibitor acarbose with an IC50 value of 170 μM. Dixon plot kinetic analysis of oenothein B showed a noncompetitive inhibition with a Ki value of 102 μM. These results suggest that pomegranate arils would be useful for suppressing postprandial hyperglycemia.
Abstract:
Keywords: ellagitannin, oenothein B, pomegranate, recombinant human maltase–glucoamylase
Oenothein B found in pomegranate aril show very good inhibition on α-glucosidase. αGlucosidase inhibitors have been used to decrease carbohydrate uptake from foods in diabetic patients. Because pomegranate aril is edible fruits unlike the other plants sources that contain oenothein B, inhibitors from products ingested by consumers are more acceptable as natural ingredients. These results suggest that pomegranate aril is a potent antihyperglycemic food that contains inhibitors of α-glucosidase.
Practical Application:
Introduction
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antioxidant (Kumar and others 2013), anti-inflammatory (Ismail and others 2012), anticancer (Adhami and others 2011), and tyrosinase-inhibitory (Yoshimura and others 2005) effects. The beneficial pomegranate constituents are ellagic acid, ellagitannins, punicic acid, flavonoids, anthocyanidins, anthocyanins, and estrogenic flavonols and flavones (Jurenka 2008). In a previous study, pomegranate flower extract was found to exhibit α-glucosidaseinhibitory activity (Li and others 2005). However, we did not find any studies on α-glucosidase-inhibitory activity in the aril and pericarp parts of the pomegranate. An inhibitory assay using α-glucosidase from small intestine of rats has been investigated (Mai and others 2007). Oku and others (2011) reported an inhibitory assay that used α-glucosidase from the human intestine. However, it is difficult to obtain human intestinal tissue to prepare the enzyme. Rossi and others (2006) reported the production of recombinant human maltaseglucoamylase (MGAM) by using Drosophilia cells. We prepared recombinant human MGAM that was expressed in Pichia pastoris. In this study, we examined the inhibitory activity of aril and pericarp extracts from pomegranate obtained from different regions MS 20140078 Submitted 1/15/2014, Accepted 6/16/2014. Authors against recombinant human MGAM, and tried to identify the Kawakami,Uraji and Hatanaka are with Okayama Prefectural Technology Center active compounds in pomegranate extracts.
Controlling blood glucose levels is an important aspect of the treatment of type 2 diabetes. Dietary carbohydrate is broken down into glucose by α-amylase and small intestinal α-glucosidase, which results in elevated blood glucose concentrations. Therefore, reducing or slowing the digestive availability of carbohydrate-derived glucose is useful in preventing diabetes mellitus. α-Glucosidase inhibitors such as acarbose and miglitol have been used to decrease carbohydrate uptake from foods in diabetic patients (Asano 2003). Because pharmacological drugs may cause adverse effects, much attention has been paid to identifying digestive enzyme-inhibitory compounds from foods that are consumed daily. Green tea (Hara 1997), guava leaves (Deguchi and Miyazaki 2010), tochu tea (Watanabe and others 1997), persimmon leaves (Kawakami and others 2010), and mulberry leaves (Oku and others 2006) are reported to be effective in regulating blood glucose levels. The pomegranate (Punica granatum) is a popular fruit worldwide and has a variety of biological activities, including
for Agriculture, Forestry and Fisheries, Research Inst. for Biological Sciences (RIBS), Okayama 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. Authors Li and Ito are with Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama Univ., 1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan. Authors Ito is with Faculty of Health and Welfare Sciences, Okayama Prefectural Univ., 111 Kuboki, Soja, Okayama, 719-1197, Japan. Direct inquiries to author Ito (E-mail: [email protected]).
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Materials and Methods Materials Pomegranates were collected in August 2012 from the medicinal plant garden of the Faculty of Pharmaceutical Sciences, Okayama R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12568 Further reproduction without permission is prohibited
Pomegranate showed enzyme inhibition . . . MeOH, placed into 2 new 500 mL baffled flasks, and shaken again at 250 rpm for 3 d with MeOH that was added to a final concentration of 3% each day. The cells were then harvested by performing centrifugation at 2000 × g for 30 min. The supernatant was concentrated using ammonium sulfate at 70% saturation and 4 °C. The precipitates, which were collected by centrifugation at 20000 × g for 30 min at 4 °C, were dissolved in distilled water and dialyzed twice against 4.5 L distilled water. Preparation of pomegranate extracts The dialysate was then used as the enzyme preparation. BMGY Pericarps with mesocarps and edible arils of the pomegranates and BMMY were produced according to the manufacturer’s were sampled prior to extraction. The pericarps and arils were instructions (http://tools.invitrogen.com/content/sfs/manuals/ homogenized in 70% aqueous acetone (Ito and others 2014). A easyselect_man.pdf#search=‘Pichia protocol Invitrogen). part (100 mg) of the extracts were prepared using a Mega Bond Elut C18 (1 g, 6 mL; Agilent, Santa Clara, Calif., U.S.A.) cartridge Recombinant human MGAM activity Recombinant human MGAM activity was determined using 2 column developing with H2 O and MeOH (each 20 mL) to remove sugar. The MeOH eluates were used to screen for α-glucosidase mM maltose in 0.1 M phosphate buffer (pH 6.4) at 37 °C. One unit of enzyme activity was defined as the amount of enzyme that inhibitory. required 1 μmol of glucose per min under the assay conditions. Cloning and extracellular production of human MGAM The amount of glucose that was released was determined using a The N-terminal domain of human MGAM is reported to be the Glucose CII Test Wako (Wako Pure Chemical, Osaka, Japan). A catalytic domain of the protein (Rossi and others 2006). Therefore, kinetic study was performed from 0.25 to 2 mM maltose in 0.1 we designed the recombinant protein such that the N-terminal M phosphate buffer (pH 6.4) at 37 °C. The Michaelis constant domain from 86Val to 955Ser was extracellularly expressed by Km of the recombinant human MGAM was determined from using Pichia pastoris. The recombinant protein was also engineered Lineweaver–Burk plots. The Km value for maltose of the resultant such that it possessed c-myc and 6 × His tags at its C-terminus. enzyme was estimated to be 7.2 ± 0.7 mM. This value is close Nested PCR was performed for the human MGAM to 6.4 mM, which has been reported for recombinant human gene. Human adult small intestine cDNA pre- MGAM overexpressed in Pichia pastoris GS155 (Ren and others pared using the PCR Ready First Strand cDNA Kit 2011). (Bio Chain, Hayward, Calif., U.S.A.) was used as a template. We performed primary PCR with 0.2 μM primers (sense and anti- Assay for recombinant human MGAM-inhibitory activity This assay was performed using previous method with some sense: 5 -ACAACTGGTACCACACATGCTAGGACAACG-3 and 5 -AGTGCAGTTTTCGGCAGAAGCACCATTCTC-3 , minor modifications (Rossi and others 2006). Ten microliters of respectively) and DNA polymerase Prime STAR HS, which was samples for extracts or isolated compounds from pomegranate in obtained from Takara Holdings Inc. (Kyoto, Japan), by using dimethyl sulfoxide was added to 50 μL of 4 mM maltose in 0.1 M the following protocol: 30 cycles of 98 °C for 10 s, 60 °C for phosphate buffer (pH 6.4). Then, the mixture was preincubated at 5 s, and 72 °C for 90 s. Next, the MGAM gene was amplified 37 °C for 5 min. The enzymatic reaction was initiated by adding using a secondary PCR similar to the primary PCR, with the 40 μL of the recombinant human MGAM solution. The resulting primary PCR products as a template and with 0.2 μM sense (5 - mixture was incubated at 37 °C for 30 min. After heating at 99 CTGCAGTTTCTGCTGAATGTCCAGTGGTAA-3 ) and an- °C for 5 min, the glucose liberated was measured using a Glucose tisense (5 -CCGCGGACCATTCCACTGTGTATGCTTCTC- CII Test Wako. The half-maximal inhibitory concentration (IC50 ) 3 ) primers (the underlined areas represent the sites for PstI and values were calculated by plotting the logarithm of the concentraSacII, respectively). The 2.2-kbp PCR product was cloned into tion of the sample (μg/mL) compared with the inhibitory activity pCR-Blunt II-TOPO, which was purchased from Invitrogen (%). Each sample was added at final concentrations of 0, 125, 250, Co. (Carlsbad, Calif., U.S.A.), and sequenced. The MGAM gene 500, and 1,000 μg/mL. The inhibition constant (Ki ) and the type was subcloned into pPICZαB (Invitrogen Co.) at its PstI and of inhibition were determined from Lineweaver–Burk and Dixon SacII sites, and the recombinant extracellular expression vector plots. pPICzαB/MGAM was linearized using PmeI. The DNA was purified and transformed to Pichia pastoris KM71H (Invitrogen Assay for rat intestinal α-glucosidase–inhibitory activity Maltase and sucrase in the rat intestine were prepared from Co.). Transformants were screened in the presence of 50 rat intestinal acetone powder (Sigma Aldrich Co., St. Louis, μg/mL Zeocin (Invitrogen Co.). Then, 4 clones were ob- Mo., U.S.A.) by following previous method with modifications tained by performing selection. One clone, A-1, was cho- (Dahlqvist 1964). In brief, rat intestinal acetone powder (100 mg) sen for enzyme production. A colony of A-1 was inocu- was homogenized in 1 mL of phosphate buffer (0.1 M, pH 6.8). lated into a 500 mL baffled flask that contained 25 mL of After centrifugation at 2000 × g for 30 min, the supernatant was buffered glycerol-complex medium (BMGY) with 25 μg/mL used in the assay. The substrates that were used were 4 mM malZeocin and was then shaken at 250 rpm and 30 °C tose for maltase or 8 mM for sucrose for sucrase. The inhibitory for 2 d. This inoculum was used to inoculate eight 500 mL activity was measured using the same method described above. baffled flasks, each of which contained 100 mL of BMGY. After the cells were shaken for 2 d at 250 rpm and 30 °C, they Determination of total polyphenol content were harvested by centrifugation at 2000 × g for 10 min, and The polyphenols were quantified using a colorimetric assay with the supernatant was discarded. The cells were resuspended in 80 absorption at 760 nm according to the Folin–Ciocalteu method mL of buffered methanol-complex medium (BMMY) with 3% (Singleton and Rossi 1965), with the SH-8000Lab microplate Vol. 79, Nr. 9, 2014 r Journal of Food Science H1849
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Univ., Okayama, Japan. Commercial pomegranates from Yunnan and Sichuan were obtained from Mae Chu Co. Ltd. (Nara, Japan). The pomegranates from California were purchased from local markets in Osaka, Japan. The commercial pomegranate aril extract (from Iran) and guava leaf extract were gifts from Morishita Jintan Co. Ltd. (Osaka, Japan) and BHN Co. Ltd. (Tokyo, Japan), respectively.
Pomegranate showed enzyme inhibition . . . Table 1–Recombinant human maltase-glucoamylase and rat intestinal α-glucosidase-inhibitory activities, and polyphenol content of extracts from pomegranate pericarps, arils, and flowers. IC50 (microg/mL) Samples Pericarp California Sichuan Yunnan Okayama Aril California Sichuan Yunnan Okayama Iran Flower Okayama Guava leaf Acarbose
Human MGAM
Rat maltase
Rat sucrase
Polyphenol content (mg/g dry weight)
916 ± 105 995 ± 167 >1000 564 ± 101
333 ± 13 307 ± 15 535 ± 45 412 ± 25
909 ± 203 759 ± 66 >1000 >1000
481 ± 15 512 ± 14 524 ± 9 577 ± 14
462 ± 46 492 ± 42 >1000 793 ± 78 393 ± 5
388 ± 10 >1000 >1000 653 ± 35 527 ± 68
911 ± 92 958 ± 119 >1000 >1000 486 ± 33
464 ± 4 324 ± 6 439 ± 12 389 ± 6 670 ± 4
567 ± 25 >1000 110 ± 6
87 ± 6 514 ± 7 0.43 ± 0.01
324 ± 19 >1000 3.4 ± 0.8
501 ± 10 599 ± 12
Note: The data were expressed as the mean ± SD (n = 3).
reader (Corona Electric Co. Ltd., Ibaraki, Japan). The polyphenol Table 2–Recombinant human maltase-glucoamylase-inhibitory content was calculated in terms of gallic acid equivalents based on activities of eluates of pomegranate aril extracts obtained using a Diaion HP20 column. a gallic acid standard curve. Eluate
HPLC analysis Analytical HPLC was performed using a Shimadzu HPLC system that was equipped with a SPD-6A UV detector and a YMC-Pack SIL A-003 column (250 × 4.6 mm i.d., YMC Co. Ltd., Kyoto, Japan). The separation conditions were as follows: flow rate, 1.5 mL/min; elution solvent, nhexane/MeOH/tetrahydrofuran/formic acid (55:33:11:1) containing oxalic acid (450 mg/L); and UV detection at 280 nm. Pomegranate extract was dissolved in MeOH to a concentration of 1 mg/mL, and 10 μL was injected into the column. Identification of inhibitory compounds The pomegranate arils extract (300 mg) was examined by column chromatography on a Diaion HP-20 column (1.5 × 30 cm; Mitsubishi Chemical Co., Tokyo, Japan) and was developed with water, 10% MeOH, 30% MeOH, 50% MeOH, and 100% MeOH. Each eluate was evaporated to dryness and assayed in terms of the inhibitory activity against recombinant human MGAM. The eluate that exhibited the highest inhibitory activity was further purified using HPLC as follows. The 30% MeOH eluate was analyzed by employing normal phase HPLC to detect punicalagin (8.5 min) and oenothein B (12.5 min), which were identified by comparison with authentic samples (Ito and others 2014). These compounds were then assayed for inhibitory activity.
Results and Discussion
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Screening for α-glucosidase–inhibitory activities of pomegranate extracts Recombinant human MGAM-inhibitory activity was examined using maltose as a substrate. The IC50 value of each pomegranate extract is shown in Table 1. The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. We further evaluated the effects of pomegranate flower, guava leaf extract, and acarbose on the inhibition of α-glucosidases (Table 1). Acarbose, which is a α-glucosidase inhibitor, showed stronger inhibitory activity against α-glucosidases from the rat small intestine than recombinant human MGAM. The aril extract from H1850 Journal of Food Science r Vol. 79, Nr. 9, 2014
0% MeOH 10% MeOH 30% MeOH 50% MeOH 100% MeOH
IC50 (microg/mL) >1000 531 ± 58 362 ± 8 607 ± 52 >1000
Note: The data were expressed as the mean ± SD (n = 3).
Yunnan showed no inhibition of α-glucosidases. Almost all of the pomegranate extracts showed more potent inhibitory activity against rat maltase and recombinant human MGAM than rat sucrase. The aril extracts from Sichuan and Iran inhibited recombinant human MGAM more than the rat maltase and sucrase. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Oku and others (2011) reported that the hydrolyzing activities of disaccharidases are generally similar between humans and rats. However, we found that the inhibitory activity of pomegranate extracts against recombinant human MGAM differs slightly from those against rat maltase and sucrase (Table 1). These results suggest that rat intestinal α-glucosidase might not be suitable as a tool for evaluating the antihyperglycemic activity. α-Glucosidase activities are associated with 2 small intestinal enzymes: MGAM and the sucrase–isomaltase complex in humans (Dahlqvist and Telenius 1969). Each enzyme consists of 2 catalytic subunits: an N-terminal subunit and a C-terminal subunit (Dahlqvist and Telenius 1969). In this study, we examined inhibitory activity against the N-terminal MGAM subunit only. Hence, further studies are required to evaluate the inhibitory effects of pomegranate extract against other subunits. Extensive in vivo studies should be performed on the basis of in vitro studies of such inhibitors.
Total polyphenol content of pomegranate extracts We investigated the polyphenol content of the pomegranate extracts (Table 1). The polyphenol content of the pericarp extracts was higher than that of the aril extracts. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content,
Pomegranate showed enzyme inhibition . . . Table 3–Recombinant human maltase-glucoamylase and rat in- tivity (Mai and others 2007). The aril extract of the Iranian testinal α-glucosidase inhibitory activities of oenothein B and pomegranate was found to have a high polyphenol content and punicalagin. IC50 (μM) Compounds
Human MGAM
Rat maltase
Oenothein B Punicalagin
174 ± 2 305 ± 4
290 ± 13 535 ± 6
Note: The data were expressed as the mean ± SD (n = 3).
strong recombinant human MGAM-inhibitory activity (Table 1). Conversely, the guava leaf and Yunnan pericarp extracts showed Rat sucrase no inhibition of recombinant human MGAM activity, although these extracts contained high amounts of polyphenols. These re213 ± 24 sults imply that recombinant human MGAM-inhibitory activity 369 ± 13 is dependent not only on the amount of polyphenols but also on the diversity of polyphenols and/or synergistic effects of various polyphenols.
which was equivalent to 670.2 mg of gallic acid/g dry weight Identification of recombinant human MGAM inhibitors extract. To identify the inhibitory compounds, we fractionated the In a previous study, plant materials with high polyphenol conaril extract using a Diaion HP-20 column. Among the tent were found to exhibit strong α-glucosidase–inhibitory ac5 eluates, the 30% MeOH eluate showed the highest inhibitory activity against recombinant human MGAM (Table 2). We further analyzed the eluate by utilizing HPLC to identify the active compound. The HPLC profile for the 30% MeOH eluate is shown in Figure 1. A single peak at 8.5 min and another single peak at 12.5 min were identified as punicalagin and oenothein B, respectively, by co-chromatography with authentic specimens (Ito and others 2014). Punicalagin is the main component in pomegranates, whereas, recently presence of oenothein B in pomegranates was first demonstrated (Ito and others 2014). The structures of punicalagin and oenothein B are shown in Figure 2. Oenothein B showed a dose-dependent inhibitory effect on recombinant human MGAM. The IC50 value of oenothein B against recombinant human MGAM was 174 ± 2 μM (Table 3). The inhibition of MGAM activity by oenothein B was comparable to that of acarbose with an IC50 at 170 μM, which was 1.8-fold higher than the IC50 value for punicalagin. This result suggests that oenothein B is a candidate for an efficient inhibitor of recombinant human MGAM. In this study, we first identified punicalagin and oenothein B from the aril extract of Punica granatum as recombinant human Figure 1–HPLC profile of 30% MeOH eluate from pomegranate arils, which MGAM inhibitors. Oenothein B is a macrocyclic ellagitannin
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was separated using a Diaion HP20 column.
Figure 2–Chemical structures of punicalagin and oenothein B. Vol. 79, Nr. 9, 2014 r Journal of Food Science H1851
A
800
1/V(mM/min)
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[I]=500 µM
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Figure 3–(A) Lineweaver–Burk plot of the effect of oenothein B on the rate of maltose digestion by recombinant human MGAM. Maltose was used at final concentrations of 0.25, 0.5, 1, or 2 mM. Oenothein B was added at final concentrations of 0, 62.5, 125, 250, or 500 μM. Symbols: , in the absence of oenothein B; , in the presence of 62.5 μM oenothein B; , in the presence of 125 μM oenothein B; , in the presence of 250 μM oenothein B; ◦, in the presence of 500 μM oenothein B. (B) Dixon plot of the effect of oenothein B on the rate of maltose digestion by MGAM. Maltose was used at final concentrations of 0.25, 0.5, 1, or 2 mM. Oenothein B was added at final concentrations of 0, 62.5, 125, 250, or 500 μM. Symbols: , maltose was used at a final concentration of 0.25 mM; , maltose was used at a final concentration of 0.5 mM; , maltose was used at a final concentration of 1 mM; ◦, and maltose was used at a final concentration of 2 mM. The data were expressed as the mean ± SD (n = 3).
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dimer (Figure 2), and it exhibits various biological activities including antioxidant (Amakura and others 2009), anticancer (Miyamoto and others 1993), anti-inflammatory (Schepetkin and others 2009; Kiss and others 2009), antifungal (Santos and others 2007), and antiviral (Fukuchi and others 1989) activities. Only a few studies have shown that ellagitannins such as oenothein B have α-glucosidase–inhibitory activity (Huang and others 2012). Pomegranate flowers (Li and others 2005) and guava leaf (Wang and others 2010) have previously been reported to exhibit inhibitory activity against α-glucosidase. In this study, the IC50 value of Iranian aril extract was higher than those of the other extracts (Table 1), suggesting that pomegranate aril extract possesses marked antihyperglycemic potential. H1852 Journal of Food Science r Vol. 79, Nr. 9, 2014
Kinetics of oenothein B inhibitory activity against recombinant human MGAM We performed a kinetic study of the inhibitory effect of oenothein B against recombinant human MGAM. The mechanism of inhibition was determined based on the through graphical analysis of primary Lineweaver–Burk and Dixon plots (Figure 3). Our results showed that oenothein B was a noncompetitive inhibitor with a Ki value of 102.3 ± 18.4 μM. In contrast, acarbose, which was used as a positive control, had a Ki of 87.4 ± 14.6 μM against recombinant human MGAM.
Conclusion We examined the inhibitory activities of extracts from pomegtanates obtained from various regions against recombinant human MGAM and rat intestinal α-glucosidase. The aril extract of the Iranian pomegranate was the most effective inhibitor of recombinant human MGAM. We identified a macrocyclic ellagitannin dimer, oenothein B, as an inhibitory constituent of the pomegranate arils. The potency of oenothein B with an IC50 value of 174 μM was comparable to that of acarbose with an IC50 value of 170 μM against recombinant human MGAM. Oenothein B showed noncompetitive inhibition of recombinant human MGAM with a Ki value of 102 μM. Our findings indicate the possibility that pomegranate aril extracts could play important roles in suppression of postprandial hyperglycemia.
Acknowledgments The authors thank Morishita Jintan Co. Ltd., for providing the pomegranate extracts and BHN Co. Ltd., for providing the guava leaf extracts.
Author Contributions K. Kawakami collected test data and drafted the manuscript. P. Li prepared test sample. M. Uraji and T. Hatanaka prepared MGMA. H. Ito designed the study and interpreted the results.
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Li Y, Wen S, Kota BP, Peng G, Li GQ, Yamahara J, Roufogalis BD. 2005. Punica granatum flower extract, a potent alpha-glucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats. J Ethnopharmacol 99:239–44. Mai TT, Thu NN, Tien PG, Van Chuyen N. 2007. Alpha-glucosidase inhibitory and antioxidant activities of Vietnamese edible plants and their relationships with polyphenol contents. J Nutr Sci Vitaminol 53:267–76. Miyamoto K, Nomura M, Sasakura M, Matsui E, Koshiura R, Murayama T, Furukawa T, Hatano T, Yoshida T, Okuda T. 1993. Antitumor activity of oenothein B, a unique macrocyclic ellagitannin. Jpn J Cancer Res 84:99–103. Oku T, Yamada M, Nakamura M, Sadamori N, Nakamura S. 2006. Inhibitory effects of extractives from leaves of Morus alba on human and rat small intestinal disaccharidase activity. Br J Nutr 95:933–8. Oku T, Tanabe K, Ogawa S, Sadamori N, Nakamura S. 2011. Similarity of hydrolyzing activity of human and rat small intestinal disaccharidases. Clin Exp Gastroenterol 4:155–61. Ren L, Qin X, Cao X, Wang L, Bai F, Bai G, Shen Y. 2011. Structural insight into substrate specificity of human intestinal maltase-glucoamylase. Protein Cell 2:827–36. Rossi EJ, Sim L, Kuntz DA, Hahn D, Johnston BD, Ghavami A, Szczepina MG, Kumar NS, Sterchi EE, Nichols BL, Pinto BM, Rose DR. 2006. Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives. FEBS J 273:2673–83.
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α-Glucosidase inhibitors are currently used in the treatment of type 2 diabetes. In this study, we investigated the inhibitory activities of aril and pericarp extracts from pomegranates obtained various regions against recombinant human maltase–glucoamylase (MGAM). The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. The Iranian aril extract was the most effective inhibitor. We investigated the polyphenol content of the pomegranate extracts using the Folin–Ciocalteu method. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content. We further evaluated inhibitory activity against α-glucosidase from the rat small intestine. Pomegranate extract used in this study showed slightly different inhibitory activities according to α-glucosidase origin. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Bioassayguided fractionation of the pomegranate arils led to identification of punicalagin and oenothein B as potent inhibitors of α-glucosidase. Oenothein B showed inhibitory activity with a half-maximal inhibitory concentration (IC50 ) value of 174 μM. Its potency was comparable to that of the α-glucosidase inhibitor acarbose with an IC50 value of 170 μM. Dixon plot kinetic analysis of oenothein B showed a noncompetitive inhibition with a Ki value of 102 μM. These results suggest that pomegranate arils would be useful for suppressing postprandial hyperglycemia.
Abstract:
Keywords: ellagitannin, oenothein B, pomegranate, recombinant human maltase–glucoamylase
Oenothein B found in pomegranate aril show very good inhibition on α-glucosidase. αGlucosidase inhibitors have been used to decrease carbohydrate uptake from foods in diabetic patients. Because pomegranate aril is edible fruits unlike the other plants sources that contain oenothein B, inhibitors from products ingested by consumers are more acceptable as natural ingredients. These results suggest that pomegranate aril is a potent antihyperglycemic food that contains inhibitors of α-glucosidase.
Practical Application:
Introduction
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antioxidant (Kumar and others 2013), anti-inflammatory (Ismail and others 2012), anticancer (Adhami and others 2011), and tyrosinase-inhibitory (Yoshimura and others 2005) effects. The beneficial pomegranate constituents are ellagic acid, ellagitannins, punicic acid, flavonoids, anthocyanidins, anthocyanins, and estrogenic flavonols and flavones (Jurenka 2008). In a previous study, pomegranate flower extract was found to exhibit α-glucosidaseinhibitory activity (Li and others 2005). However, we did not find any studies on α-glucosidase-inhibitory activity in the aril and pericarp parts of the pomegranate. An inhibitory assay using α-glucosidase from small intestine of rats has been investigated (Mai and others 2007). Oku and others (2011) reported an inhibitory assay that used α-glucosidase from the human intestine. However, it is difficult to obtain human intestinal tissue to prepare the enzyme. Rossi and others (2006) reported the production of recombinant human maltaseglucoamylase (MGAM) by using Drosophilia cells. We prepared recombinant human MGAM that was expressed in Pichia pastoris. In this study, we examined the inhibitory activity of aril and pericarp extracts from pomegranate obtained from different regions MS 20140078 Submitted 1/15/2014, Accepted 6/16/2014. Authors against recombinant human MGAM, and tried to identify the Kawakami,Uraji and Hatanaka are with Okayama Prefectural Technology Center active compounds in pomegranate extracts.
Controlling blood glucose levels is an important aspect of the treatment of type 2 diabetes. Dietary carbohydrate is broken down into glucose by α-amylase and small intestinal α-glucosidase, which results in elevated blood glucose concentrations. Therefore, reducing or slowing the digestive availability of carbohydrate-derived glucose is useful in preventing diabetes mellitus. α-Glucosidase inhibitors such as acarbose and miglitol have been used to decrease carbohydrate uptake from foods in diabetic patients (Asano 2003). Because pharmacological drugs may cause adverse effects, much attention has been paid to identifying digestive enzyme-inhibitory compounds from foods that are consumed daily. Green tea (Hara 1997), guava leaves (Deguchi and Miyazaki 2010), tochu tea (Watanabe and others 1997), persimmon leaves (Kawakami and others 2010), and mulberry leaves (Oku and others 2006) are reported to be effective in regulating blood glucose levels. The pomegranate (Punica granatum) is a popular fruit worldwide and has a variety of biological activities, including
for Agriculture, Forestry and Fisheries, Research Inst. for Biological Sciences (RIBS), Okayama 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan. Authors Li and Ito are with Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama Univ., 1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan. Authors Ito is with Faculty of Health and Welfare Sciences, Okayama Prefectural Univ., 111 Kuboki, Soja, Okayama, 719-1197, Japan. Direct inquiries to author Ito (E-mail: [email protected]).
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Materials and Methods Materials Pomegranates were collected in August 2012 from the medicinal plant garden of the Faculty of Pharmaceutical Sciences, Okayama R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12568 Further reproduction without permission is prohibited
Pomegranate showed enzyme inhibition . . . MeOH, placed into 2 new 500 mL baffled flasks, and shaken again at 250 rpm for 3 d with MeOH that was added to a final concentration of 3% each day. The cells were then harvested by performing centrifugation at 2000 × g for 30 min. The supernatant was concentrated using ammonium sulfate at 70% saturation and 4 °C. The precipitates, which were collected by centrifugation at 20000 × g for 30 min at 4 °C, were dissolved in distilled water and dialyzed twice against 4.5 L distilled water. Preparation of pomegranate extracts The dialysate was then used as the enzyme preparation. BMGY Pericarps with mesocarps and edible arils of the pomegranates and BMMY were produced according to the manufacturer’s were sampled prior to extraction. The pericarps and arils were instructions (http://tools.invitrogen.com/content/sfs/manuals/ homogenized in 70% aqueous acetone (Ito and others 2014). A easyselect_man.pdf#search=‘Pichia protocol Invitrogen). part (100 mg) of the extracts were prepared using a Mega Bond Elut C18 (1 g, 6 mL; Agilent, Santa Clara, Calif., U.S.A.) cartridge Recombinant human MGAM activity Recombinant human MGAM activity was determined using 2 column developing with H2 O and MeOH (each 20 mL) to remove sugar. The MeOH eluates were used to screen for α-glucosidase mM maltose in 0.1 M phosphate buffer (pH 6.4) at 37 °C. One unit of enzyme activity was defined as the amount of enzyme that inhibitory. required 1 μmol of glucose per min under the assay conditions. Cloning and extracellular production of human MGAM The amount of glucose that was released was determined using a The N-terminal domain of human MGAM is reported to be the Glucose CII Test Wako (Wako Pure Chemical, Osaka, Japan). A catalytic domain of the protein (Rossi and others 2006). Therefore, kinetic study was performed from 0.25 to 2 mM maltose in 0.1 we designed the recombinant protein such that the N-terminal M phosphate buffer (pH 6.4) at 37 °C. The Michaelis constant domain from 86Val to 955Ser was extracellularly expressed by Km of the recombinant human MGAM was determined from using Pichia pastoris. The recombinant protein was also engineered Lineweaver–Burk plots. The Km value for maltose of the resultant such that it possessed c-myc and 6 × His tags at its C-terminus. enzyme was estimated to be 7.2 ± 0.7 mM. This value is close Nested PCR was performed for the human MGAM to 6.4 mM, which has been reported for recombinant human gene. Human adult small intestine cDNA pre- MGAM overexpressed in Pichia pastoris GS155 (Ren and others pared using the PCR Ready First Strand cDNA Kit 2011). (Bio Chain, Hayward, Calif., U.S.A.) was used as a template. We performed primary PCR with 0.2 μM primers (sense and anti- Assay for recombinant human MGAM-inhibitory activity This assay was performed using previous method with some sense: 5 -ACAACTGGTACCACACATGCTAGGACAACG-3 and 5 -AGTGCAGTTTTCGGCAGAAGCACCATTCTC-3 , minor modifications (Rossi and others 2006). Ten microliters of respectively) and DNA polymerase Prime STAR HS, which was samples for extracts or isolated compounds from pomegranate in obtained from Takara Holdings Inc. (Kyoto, Japan), by using dimethyl sulfoxide was added to 50 μL of 4 mM maltose in 0.1 M the following protocol: 30 cycles of 98 °C for 10 s, 60 °C for phosphate buffer (pH 6.4). Then, the mixture was preincubated at 5 s, and 72 °C for 90 s. Next, the MGAM gene was amplified 37 °C for 5 min. The enzymatic reaction was initiated by adding using a secondary PCR similar to the primary PCR, with the 40 μL of the recombinant human MGAM solution. The resulting primary PCR products as a template and with 0.2 μM sense (5 - mixture was incubated at 37 °C for 30 min. After heating at 99 CTGCAGTTTCTGCTGAATGTCCAGTGGTAA-3 ) and an- °C for 5 min, the glucose liberated was measured using a Glucose tisense (5 -CCGCGGACCATTCCACTGTGTATGCTTCTC- CII Test Wako. The half-maximal inhibitory concentration (IC50 ) 3 ) primers (the underlined areas represent the sites for PstI and values were calculated by plotting the logarithm of the concentraSacII, respectively). The 2.2-kbp PCR product was cloned into tion of the sample (μg/mL) compared with the inhibitory activity pCR-Blunt II-TOPO, which was purchased from Invitrogen (%). Each sample was added at final concentrations of 0, 125, 250, Co. (Carlsbad, Calif., U.S.A.), and sequenced. The MGAM gene 500, and 1,000 μg/mL. The inhibition constant (Ki ) and the type was subcloned into pPICZαB (Invitrogen Co.) at its PstI and of inhibition were determined from Lineweaver–Burk and Dixon SacII sites, and the recombinant extracellular expression vector plots. pPICzαB/MGAM was linearized using PmeI. The DNA was purified and transformed to Pichia pastoris KM71H (Invitrogen Assay for rat intestinal α-glucosidase–inhibitory activity Maltase and sucrase in the rat intestine were prepared from Co.). Transformants were screened in the presence of 50 rat intestinal acetone powder (Sigma Aldrich Co., St. Louis, μg/mL Zeocin (Invitrogen Co.). Then, 4 clones were ob- Mo., U.S.A.) by following previous method with modifications tained by performing selection. One clone, A-1, was cho- (Dahlqvist 1964). In brief, rat intestinal acetone powder (100 mg) sen for enzyme production. A colony of A-1 was inocu- was homogenized in 1 mL of phosphate buffer (0.1 M, pH 6.8). lated into a 500 mL baffled flask that contained 25 mL of After centrifugation at 2000 × g for 30 min, the supernatant was buffered glycerol-complex medium (BMGY) with 25 μg/mL used in the assay. The substrates that were used were 4 mM malZeocin and was then shaken at 250 rpm and 30 °C tose for maltase or 8 mM for sucrose for sucrase. The inhibitory for 2 d. This inoculum was used to inoculate eight 500 mL activity was measured using the same method described above. baffled flasks, each of which contained 100 mL of BMGY. After the cells were shaken for 2 d at 250 rpm and 30 °C, they Determination of total polyphenol content were harvested by centrifugation at 2000 × g for 10 min, and The polyphenols were quantified using a colorimetric assay with the supernatant was discarded. The cells were resuspended in 80 absorption at 760 nm according to the Folin–Ciocalteu method mL of buffered methanol-complex medium (BMMY) with 3% (Singleton and Rossi 1965), with the SH-8000Lab microplate Vol. 79, Nr. 9, 2014 r Journal of Food Science H1849
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Univ., Okayama, Japan. Commercial pomegranates from Yunnan and Sichuan were obtained from Mae Chu Co. Ltd. (Nara, Japan). The pomegranates from California were purchased from local markets in Osaka, Japan. The commercial pomegranate aril extract (from Iran) and guava leaf extract were gifts from Morishita Jintan Co. Ltd. (Osaka, Japan) and BHN Co. Ltd. (Tokyo, Japan), respectively.
Pomegranate showed enzyme inhibition . . . Table 1–Recombinant human maltase-glucoamylase and rat intestinal α-glucosidase-inhibitory activities, and polyphenol content of extracts from pomegranate pericarps, arils, and flowers. IC50 (microg/mL) Samples Pericarp California Sichuan Yunnan Okayama Aril California Sichuan Yunnan Okayama Iran Flower Okayama Guava leaf Acarbose
Human MGAM
Rat maltase
Rat sucrase
Polyphenol content (mg/g dry weight)
916 ± 105 995 ± 167 >1000 564 ± 101
333 ± 13 307 ± 15 535 ± 45 412 ± 25
909 ± 203 759 ± 66 >1000 >1000
481 ± 15 512 ± 14 524 ± 9 577 ± 14
462 ± 46 492 ± 42 >1000 793 ± 78 393 ± 5
388 ± 10 >1000 >1000 653 ± 35 527 ± 68
911 ± 92 958 ± 119 >1000 >1000 486 ± 33
464 ± 4 324 ± 6 439 ± 12 389 ± 6 670 ± 4
567 ± 25 >1000 110 ± 6
87 ± 6 514 ± 7 0.43 ± 0.01
324 ± 19 >1000 3.4 ± 0.8
501 ± 10 599 ± 12
Note: The data were expressed as the mean ± SD (n = 3).
reader (Corona Electric Co. Ltd., Ibaraki, Japan). The polyphenol Table 2–Recombinant human maltase-glucoamylase-inhibitory content was calculated in terms of gallic acid equivalents based on activities of eluates of pomegranate aril extracts obtained using a Diaion HP20 column. a gallic acid standard curve. Eluate
HPLC analysis Analytical HPLC was performed using a Shimadzu HPLC system that was equipped with a SPD-6A UV detector and a YMC-Pack SIL A-003 column (250 × 4.6 mm i.d., YMC Co. Ltd., Kyoto, Japan). The separation conditions were as follows: flow rate, 1.5 mL/min; elution solvent, nhexane/MeOH/tetrahydrofuran/formic acid (55:33:11:1) containing oxalic acid (450 mg/L); and UV detection at 280 nm. Pomegranate extract was dissolved in MeOH to a concentration of 1 mg/mL, and 10 μL was injected into the column. Identification of inhibitory compounds The pomegranate arils extract (300 mg) was examined by column chromatography on a Diaion HP-20 column (1.5 × 30 cm; Mitsubishi Chemical Co., Tokyo, Japan) and was developed with water, 10% MeOH, 30% MeOH, 50% MeOH, and 100% MeOH. Each eluate was evaporated to dryness and assayed in terms of the inhibitory activity against recombinant human MGAM. The eluate that exhibited the highest inhibitory activity was further purified using HPLC as follows. The 30% MeOH eluate was analyzed by employing normal phase HPLC to detect punicalagin (8.5 min) and oenothein B (12.5 min), which were identified by comparison with authentic samples (Ito and others 2014). These compounds were then assayed for inhibitory activity.
Results and Discussion
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Screening for α-glucosidase–inhibitory activities of pomegranate extracts Recombinant human MGAM-inhibitory activity was examined using maltose as a substrate. The IC50 value of each pomegranate extract is shown in Table 1. The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. We further evaluated the effects of pomegranate flower, guava leaf extract, and acarbose on the inhibition of α-glucosidases (Table 1). Acarbose, which is a α-glucosidase inhibitor, showed stronger inhibitory activity against α-glucosidases from the rat small intestine than recombinant human MGAM. The aril extract from H1850 Journal of Food Science r Vol. 79, Nr. 9, 2014
0% MeOH 10% MeOH 30% MeOH 50% MeOH 100% MeOH
IC50 (microg/mL) >1000 531 ± 58 362 ± 8 607 ± 52 >1000
Note: The data were expressed as the mean ± SD (n = 3).
Yunnan showed no inhibition of α-glucosidases. Almost all of the pomegranate extracts showed more potent inhibitory activity against rat maltase and recombinant human MGAM than rat sucrase. The aril extracts from Sichuan and Iran inhibited recombinant human MGAM more than the rat maltase and sucrase. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Oku and others (2011) reported that the hydrolyzing activities of disaccharidases are generally similar between humans and rats. However, we found that the inhibitory activity of pomegranate extracts against recombinant human MGAM differs slightly from those against rat maltase and sucrase (Table 1). These results suggest that rat intestinal α-glucosidase might not be suitable as a tool for evaluating the antihyperglycemic activity. α-Glucosidase activities are associated with 2 small intestinal enzymes: MGAM and the sucrase–isomaltase complex in humans (Dahlqvist and Telenius 1969). Each enzyme consists of 2 catalytic subunits: an N-terminal subunit and a C-terminal subunit (Dahlqvist and Telenius 1969). In this study, we examined inhibitory activity against the N-terminal MGAM subunit only. Hence, further studies are required to evaluate the inhibitory effects of pomegranate extract against other subunits. Extensive in vivo studies should be performed on the basis of in vitro studies of such inhibitors.
Total polyphenol content of pomegranate extracts We investigated the polyphenol content of the pomegranate extracts (Table 1). The polyphenol content of the pericarp extracts was higher than that of the aril extracts. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content,
Pomegranate showed enzyme inhibition . . . Table 3–Recombinant human maltase-glucoamylase and rat in- tivity (Mai and others 2007). The aril extract of the Iranian testinal α-glucosidase inhibitory activities of oenothein B and pomegranate was found to have a high polyphenol content and punicalagin. IC50 (μM) Compounds
Human MGAM
Rat maltase
Oenothein B Punicalagin
174 ± 2 305 ± 4
290 ± 13 535 ± 6
Note: The data were expressed as the mean ± SD (n = 3).
strong recombinant human MGAM-inhibitory activity (Table 1). Conversely, the guava leaf and Yunnan pericarp extracts showed Rat sucrase no inhibition of recombinant human MGAM activity, although these extracts contained high amounts of polyphenols. These re213 ± 24 sults imply that recombinant human MGAM-inhibitory activity 369 ± 13 is dependent not only on the amount of polyphenols but also on the diversity of polyphenols and/or synergistic effects of various polyphenols.
which was equivalent to 670.2 mg of gallic acid/g dry weight Identification of recombinant human MGAM inhibitors extract. To identify the inhibitory compounds, we fractionated the In a previous study, plant materials with high polyphenol conaril extract using a Diaion HP-20 column. Among the tent were found to exhibit strong α-glucosidase–inhibitory ac5 eluates, the 30% MeOH eluate showed the highest inhibitory activity against recombinant human MGAM (Table 2). We further analyzed the eluate by utilizing HPLC to identify the active compound. The HPLC profile for the 30% MeOH eluate is shown in Figure 1. A single peak at 8.5 min and another single peak at 12.5 min were identified as punicalagin and oenothein B, respectively, by co-chromatography with authentic specimens (Ito and others 2014). Punicalagin is the main component in pomegranates, whereas, recently presence of oenothein B in pomegranates was first demonstrated (Ito and others 2014). The structures of punicalagin and oenothein B are shown in Figure 2. Oenothein B showed a dose-dependent inhibitory effect on recombinant human MGAM. The IC50 value of oenothein B against recombinant human MGAM was 174 ± 2 μM (Table 3). The inhibition of MGAM activity by oenothein B was comparable to that of acarbose with an IC50 at 170 μM, which was 1.8-fold higher than the IC50 value for punicalagin. This result suggests that oenothein B is a candidate for an efficient inhibitor of recombinant human MGAM. In this study, we first identified punicalagin and oenothein B from the aril extract of Punica granatum as recombinant human Figure 1–HPLC profile of 30% MeOH eluate from pomegranate arils, which MGAM inhibitors. Oenothein B is a macrocyclic ellagitannin
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was separated using a Diaion HP20 column.
Figure 2–Chemical structures of punicalagin and oenothein B. Vol. 79, Nr. 9, 2014 r Journal of Food Science H1851
A
800
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[I]=500 µM
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Figure 3–(A) Lineweaver–Burk plot of the effect of oenothein B on the rate of maltose digestion by recombinant human MGAM. Maltose was used at final concentrations of 0.25, 0.5, 1, or 2 mM. Oenothein B was added at final concentrations of 0, 62.5, 125, 250, or 500 μM. Symbols: , in the absence of oenothein B; , in the presence of 62.5 μM oenothein B; , in the presence of 125 μM oenothein B; , in the presence of 250 μM oenothein B; ◦, in the presence of 500 μM oenothein B. (B) Dixon plot of the effect of oenothein B on the rate of maltose digestion by MGAM. Maltose was used at final concentrations of 0.25, 0.5, 1, or 2 mM. Oenothein B was added at final concentrations of 0, 62.5, 125, 250, or 500 μM. Symbols: , maltose was used at a final concentration of 0.25 mM; , maltose was used at a final concentration of 0.5 mM; , maltose was used at a final concentration of 1 mM; ◦, and maltose was used at a final concentration of 2 mM. The data were expressed as the mean ± SD (n = 3).
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dimer (Figure 2), and it exhibits various biological activities including antioxidant (Amakura and others 2009), anticancer (Miyamoto and others 1993), anti-inflammatory (Schepetkin and others 2009; Kiss and others 2009), antifungal (Santos and others 2007), and antiviral (Fukuchi and others 1989) activities. Only a few studies have shown that ellagitannins such as oenothein B have α-glucosidase–inhibitory activity (Huang and others 2012). Pomegranate flowers (Li and others 2005) and guava leaf (Wang and others 2010) have previously been reported to exhibit inhibitory activity against α-glucosidase. In this study, the IC50 value of Iranian aril extract was higher than those of the other extracts (Table 1), suggesting that pomegranate aril extract possesses marked antihyperglycemic potential. H1852 Journal of Food Science r Vol. 79, Nr. 9, 2014
Kinetics of oenothein B inhibitory activity against recombinant human MGAM We performed a kinetic study of the inhibitory effect of oenothein B against recombinant human MGAM. The mechanism of inhibition was determined based on the through graphical analysis of primary Lineweaver–Burk and Dixon plots (Figure 3). Our results showed that oenothein B was a noncompetitive inhibitor with a Ki value of 102.3 ± 18.4 μM. In contrast, acarbose, which was used as a positive control, had a Ki of 87.4 ± 14.6 μM against recombinant human MGAM.
Conclusion We examined the inhibitory activities of extracts from pomegtanates obtained from various regions against recombinant human MGAM and rat intestinal α-glucosidase. The aril extract of the Iranian pomegranate was the most effective inhibitor of recombinant human MGAM. We identified a macrocyclic ellagitannin dimer, oenothein B, as an inhibitory constituent of the pomegranate arils. The potency of oenothein B with an IC50 value of 174 μM was comparable to that of acarbose with an IC50 value of 170 μM against recombinant human MGAM. Oenothein B showed noncompetitive inhibition of recombinant human MGAM with a Ki value of 102 μM. Our findings indicate the possibility that pomegranate aril extracts could play important roles in suppression of postprandial hyperglycemia.
Acknowledgments The authors thank Morishita Jintan Co. Ltd., for providing the pomegranate extracts and BHN Co. Ltd., for providing the guava leaf extracts.
Author Contributions K. Kawakami collected test data and drafted the manuscript. P. Li prepared test sample. M. Uraji and T. Hatanaka prepared MGMA. H. Ito designed the study and interpreted the results.
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Vol. 79, Nr. 9, 2014 r Journal of Food Science H1853
