Anti-Glycation Effects of Pomegranate (Punica granatum L.) Fruit Extract and Its

Transcription

1 Anti-Glyction Effects of Pomegrnte (Punic grntum L.) Fruit Extrct nd Its Components In Vivo nd In Vitro Yuy Kumgi, 1 Schie Nktni, 1 Hideki Onoder, 1 Akifumi Ngtomo, 2 Norihis Nishid, 2 Yoichi Mtsuur, 2 Kenji Kot 1 nd Mshiro Wd 1,* 1 Deprtment of Phrmceutil nd Helth Sciences, Fculty of Phrmceuticl Sciences, Josi University, Sitm, Jpn. 2 Morishit Jintn Co., Ltd., Osk, Jpn. Corresponding Author * Mshiro Wd. 1-1 Keykidi, Skdo, Sitm , Jpn. Tel: Fx: E-mil: mwd@josi.c.jp. 1

2 ABSTRACT Accumultion of dvnced glyction end products (AGEs) leds to vrious diseses such s dietic complictions nd rteriosclerosis. In this study, we exmined the effect of pomegrnte fruit extrct (PFE) nd its constituent polyphenols on AGEs formtion in vivo nd in vitro. PFE, fed with high-ft nd high-sucrose (HFS) diet to KK-A y mice, significntly reduced glyction products such s glycolumin (22.0 ± 2.4%), hemogloin A1c (5.84 ± 0.23%), nd serum AGEs (8.22 ± 0.17 µg/ml), s compred to control HFS group (30.6 ± 2.6%, 7.45 ± 0.12% nd 9.55 ± 0.17 µg/ml, respectively, P < 0.05). In nti-glyction ssys, PFE, puniclin, puniclgin, ellgic cid, nd gllic cid suppressed the formtion of AGEs from ovine serum lumin nd sugrs. In this study, we discuss the mechnism of the nti-glyction effects of PFE nd its components in vivo nd in vitro. Key words: dvnced glyction end products (AGEs), pomegrnte, ellgic cid, puniclgin, dietes 2

3 INTRODUCTION Glyction is non-enzymtic multistge rection strting with the inding of protein nd reducing sugr such s glucose nd fructose. 1 3 In the first stge of the rection, Schiff se is generted etween n mino group of the protein nd the cronyl group of the sugr, producing n Amdori compound such s glycolumin nd hemogloin A1c (HA1c). In the ltter stges, the Amdori compound is oxidized, dehydrted, nd condensed to finlly generte dvnced glyction end products (AGEs). 1,2 Some AGEs formed from cronyl compound such s glycerldehyde nd methylglyoxl re not generted through Amdori compounds. 4 AGEs re thought to e induced y ging nd hyperglycemi. The resulting AGEs ccumultion itself ccelertes ging nd cuses dietic complictions in which protein denturtion, inflmmtion, nd oxidtive stress occur. 5 There re three wys to suppress the dverse effect of AGEs: (1) inhiition of AGEs genertion, (2) degrdtion of the generted AGEs, nd (3) inhiition of inding of AGEs to their receptors. 1,6,7 Severl studies hve reported tht polyphenolic compounds inhiit the formtion of 3

4 AGEs, y inhiiting glyction rections. 8,9 Pomegrnte (Punic grntum L.) is deciduous shru of the Lythrcee fmily tht contins vrious polyphenols such s ellgic cid (EA), puniclin (PC), nd puniclgin (PG) within its fruit (Figure 1). 10 Some in vitro studies showed tht pomegrnte extrct nd EA inhiited AGEs formtion in glyction ssy using ovine serum lumin (BSA) nd glucose However, there re no in vivo studies designed to clrify whether the intke of pomegrnte inhiits AGEs formtion. In this study, we exmined the effect of pomegrnte fruit extrct (PFE) intke on AGEs formtion in the lood of type 2 dietic model mice. Moreover, we compred the inhiitory effects of pomegrnte-derived polyphenols on AGEs formtion in vitro. MATERIALS AND METHODS Chemicls PFE ws prepred y drying the polyphenol frction of Indin pomegrnte fruits. Briefly, the juice from the fruits ws pssed through synthetic resin to dsor polyphenols. Then, 4

5 the resin ws extrcted y hydrous ethnol nd the extrct ws dried to fford the PFE powder. The polyphenol contents of the PFE were determined y n HPLC method ccording to Zhng, Y. et l. 14 The contents of gllic cid (GA), EA, PC nd PG were 1.0, 0.63, 2.7 nd 9.1% (w/w), respectively. PC nd PG were isolted from the PFE. Glucose, fructose, EA, nd minogunidine (AG) were purchsed from Wko Pure Chemicl Ind., Ltd. (Osk, Jpn). Glycerldehyde nd GA were purchsed from Sigm-Aldrich Chemicl Co. (St. Louis, MO, USA). BSA ws purchsed from Jckson ImmunoReserch Inc. (West Grove, PA, USA). In vivo experiments Six-week-old mle type 2 dietic mice (KK-Ay/TAJc1) were purchsed from CLEA Jpn, Inc. (Tokyo, Jpn). A Norml group (n = 12) ws fed norml diet (AIN-93G) throughout the experimentl period. The other mice (n = 39) were fed high-ft (28%) nd high-sucrose (30%) diet modified from AIN-93G (HFS) for 2 weeks to induce hyperglycemi. The hyperglycemic mice were further red with HFS (control, n = 13), HFS contining 1.5% PFE 5

6 (n = 13), nd HFS contining 0.3% AG (positive control, n = 13). The PFE concentrtion ws set to the mximum concentrtion tht hd not ffected to the food intke in preliminry experiment. In the experimentl diet, PFE nd AG were replced with the cornstrch of HFS. The mice were housed individully t 21 ± 1 C, 40 50% humidity, nd 12:12-h light-drk cycle (lights on t 07:00) with free ccess to wter nd limited ccess to the experiment feed only for 16 h (18:00 10:00). Mouse weight ws mesured once per week. The til vin lood fter 10 hours of strvtion ws collected to mesure glucose levels y the mutrotse nd glucose oxidse ssy 15 (Glucose C II-Test Wko, Wko Pure Chemicl Industries, Ltd.) nd glycolumin concentrtion using the enzymtic ssy 16 (Lucic GA-L, Ashi Ksei Phrm Co., Tokyo, Jpn) once every two weeks. At the end of the feeding period, whole lood ws collected from the postcvl vein to mesure HA1c using the enzymtic ssy 17 (Norudi-HA1c kit, Sekisui Medicl Co., Ltd., Tokyo, Jpn), serum AGEs using the ELISA ssy (Rt/mouse Glycerldehyde-derived AGEs ELISA Kit, Up Well Co. Ltd., Fukuok, Jpn) nd lipid peroxide using the thiorituric cid rective sustnces ssy 18 (Oxiselect 6

7 TBARS Assy Kit, Cell Biols, Inc., Sn Diego, USA) ccording to the mnufcturer s instructions. The present study ws conducted in ccordnce with the Animl Experiment Guidelines of Josi University. In vitro experiments BSA (50 mg/ml) nd sugr (100 mm glucose, 100 mm fructose nd 10 mm glycerldehyde) were dissolved in 0.2 M phosphte uffer (ph 7.4). Ech PFE-relted smple ws dded to this solution to finl concentrtions of 10, 100, nd 1000 µg/ml for PFE, nd 10, 100, nd 1000 µm for GA, EA, PC, nd PG. The non-supplemented BSA + sugr solution ws used s control. The mixture ws incuted t 37 C in totl drkness: 15 dys for the glucose-contining mixture, 5 dys for the fructose-contining mixture, nd 1 dy for the glycerldehyde-contining mixture. After incution, the mixture ws trnsferred to 96-well lck plte (Greiner Bio-One, Tokyo, Jpn) to mesure the fluorescence intensity (excittion = 370 nm, emission = 440 nm) using Spectr Mx M2 (Moleculr Devices, Sunnyvle, CA, 7

8 USA). The rte of AGES formtion ws clculted s follows: AAAAAA ffffffffffffffffff rrrrrrrr (%) = ffffffffffffffffffffffff iiiiiiiiiiiiiiiiii oooo ssssssssssss ( iiiiiiiiiiiiiiiiiiii iiiiiiiiiiiiiiiiiiii) ffffffffffffffcceeeeeeee iiiiiiiiiiiiiiiiii oooo cccccccccccccc ( iiiiiiiiiiiiiiiiiiii iiiiiiiiiiiiiiiiiiii) 100 Sttisticl nlysis Sttisticl nlysis ws performed with SttMte III softwre (ATMS Co., Ltd., Tokyo, Jpn). The dt were nlyzed y Dunnett's test nd Tukey's test. Differences were considered significnt t p < The dt re expressed s mens ± SE. 8

9 RESULTS Food intke nd ody weight To evlute the nti-glyction effect of PFE in vivo, glyction mrkers such s glycolumin, HA1c, nd serum AGEs of KK-A y mice fed the smple (PFE with HFS) diet for 8 weeks fter induction of hyperglycemi were nlyzed. Tle 1 shows totl cloric intke nd totl food intke during the experimentl diet, nd ody weight t the end of the experiment. Although the mount of food intke during the smple feeding ws significntly lower in HFS-sed groups (HFS, PFE, nd AG) thn the Norml group, the totl clorie consumption of ll groups ws virtully identicl. The net intke of PFE ws clculted s 1.5 mg/ g/ dy. Finl ody weight ws significntly higher in the HFS sed-groups thn in the Norml group. There were no differences in cloric intke nd weight gin etween the HFS groups. Blood glucose, glyction products, nd lipid peroxide levels The fsting lood glucose concentrtion ws consistently higher in the HFS-sed groups 9

10 thn in the Norml group (Figure 2). Although the glycolumin levels of the HFS group tended to e higher thn those of Norml group, feeding of PFE nd AG with HFS significntly decresed those levels fter 4 nd 6 weeks, respectively (Figure 3A). PFE lso significntly reduced HA1c levels t 8 weeks s well s AG (Figure 3B). Serum AGEs concentrtion showed trend similr to tht of glycolumin nd HA1c (Figure 3C). Among significnt increses in serum lipid peroxide in ll HFS-sed groups, PFE slightly suppressed the level s compred to HFS (p = 0.059) (Figure 4). In vitro experiments The AGEs formtion derived from BSA with glucose, fructose, nd glycerldehyde in vitro ws concentrtion-dependently suppressed y ddition of PFE nd its components (Figure 5A C). An even lower concentrtion of PFE (10 µg/ml) significntly suppressed the AGEs formtion from fructose nd glycerldehyde (Figure 5A). The nti-ages-formtion potency of the PFE components ws similr in the ssys using different sugrs. The potency of EA 10

11 nd GA ws comprle to tht of AG. PC nd PG showed higher potency thn EA nd GA. The lowest concentrtion of PC nd PG (10 µm) remrkly inhiited the AGEs formtion, especilly with fructose (Figure 5B). DISCUSSION Most reports concerning the nti-glyction effect of PFE hve een performed in vitro, in which the suppression of AGEs formtion ws vi inhiition of the inding of proteins with sugrs However, it hs een uncler precisely wht components of pomegrnte contriute to this suppression. Only one in vivo study reported tht PFE inhiited collgen-derived AGEs in humns. 19 However, there hs een no investigtion out the nti-glyction effects of pomegrnte in vivo. Therefore, we hve evluted the nti-glyction effects of pomegrnte in type 2 dietes model mice, in which it is reltively esy to induce conditions tht promote glyction. Additionlly, the effect of the individul components of PFE ws investigted y n nti-glyction in vitro ssy. The results indicte tht 11

12 pomegrnte contins severl components tht hve the ility to suppress AGEs formtion in type 2 dietic mice. Orl dministrtion of PFE might not ffect glucose sorption from mels in the intestine ecuse there were no significnt differences in cloric intke nd ody weight gin etween HFS nd HFS plus PFE. Higher levels of the fsting lood glucose concentrtion in the HFS plus PFE group indicte tht PFE could not completely reverse the hyperglycemi. Therefore, the PFE-medited reduction in HA1c nd glycolumin might e cused y inhiition of the rection etween the protein nd glucose, tht is, glyction. The ccumultion of glycted lumin lredy strted to decrese t 4 weeks fter orl dministrtion of PFE, lso suggesting tht PFE inhiited the erly stges of glyction. The suppression of HA1c y PFE lso supports our hypothesis. The decresed ccumultion of these glyction products proly resulted in the lower concentrtion of AGEs in PFE-fed mice. The dditionl enefit of PFE could e ntioxidnt effects tht were oserved s slight decline in lood lipid peroxide levels. PFE nd its constituent polyphenols hve shown in vitro nti-glyction effects s evluted y the formtion of glucose- nd fructose-derived 12

13 AGEs. 20,21 In this study, we reveled tht PFE nd its component polyphenols suppressed the genertion of glycerldehyde-derived AGEs in vitro, especilly the ellgitnnins PC nd PG comprle to the nti-glyction therpeutic minogunidine. Glycerldehyde more esily rects with protein thn fructose nd glucose, 2,22 nd the glycerldehyde-derived AGEs re thought to e highly toxic. 4 The nti-ages-formtion potency seemed to depend on the numer of phenolic groups, suggesting tht these polyphenols cted s n ntioxidnt in the ltter stges of glyction in vitro. However, orlly dministered ellgitnnins hrdly trnsfer to the lood ecuse of their reltively high moleculr weight Ellgitnnins like PC nd PG re digested y gstric cid to EA some of which will pper in the lood. The rest of EA is metolized y intestinl cteri into urolithin which lso will pper in the lood. 23,24,26,27 Liu, W. et l. Reported tht urolithin possessed nti-glyction effect similr to minogunidhine. 13 Therefore, the nti-glyction effect of PFE shown in this in vivo study might e induced y these metolites tht hd een degrded from ellgitnnins in the 13

14 digestive trct. Additionlly, ellgitnnins might contriute to the inhiition of exogenous AGEs formtion in the gstrointestinl trct. Further study is needed to clrify the mechnism of how the metolites incorported into the lood inhiit erly stges of glyction. 14

15 Acknowledgements This work ws supported y the susidized projects support y Ministry of Agriculture, Forestry nd Fisheries Green nd Wter Environment Technology nd Innovtion Project, The 6 th Industry Genertion for Agriculture, Forestry nd Fisheries Community Progrm

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20 Figure Cptions Figure 1. Structures of pomegrnte polyphenols. Figure 2. Fsting serum glucose levels in dietic model mice fed with Norml (n=11-12), HFS (n=12-13), HFS+PFE (n=12-13), nd HFS+AG (n=11-13) diets. Dt re shown s the mens ± SE. (n = 10 13), Tukey s test p < Figure 3. Serum glyction mrkers of (A) glycolumin levels (n=11-13) t 4, 6 nd 8 weeks, (B) ha1c levels (n=11-13) t 8 weeks, nd (C) serum AGEs (n=4) t 8 weeks in dietic model mice fed norml, HFS, HFS+PFE, nd HFS+AG diets. Dt re shown s the mens ± S.E., Tukey s test p < Figure 4. Serum lipid peroxide in dietic model mice fed norml, HFS, HFS+PFE, nd HFS+AG diets. Dt re shown s the mens ± SE. (n = 10), Tukey s test p <

21 Figure 5. Anti-glyction effects of PFE nd its polyphenols on (A) glucose-derived, (B) fructose-derived, nd (C) glycerldehyde-derived AGEs formtion. Dt re shown s the mens ± SE. (n = 3), Dunnett s test, *p < 0.05, p <

22 Tle 1. Totl food intke, totl cloric intke nd finl ody weight of dietic model mice fed with norml (n=13), HFS (n=13), HFS+PFE (n=13), nd HFS+AG (n=13) diets. Dt re shown s the mens ± SE. Tukey s test p < Prmeter Norml HFS HFS+PFE HFS+AG Totl clorie intkes (kcl) 1262 ± ± ± ± 40 Totl food intkes (g) 318 ± ± ± ± 7.7 Body weight (g) 44.9 ± ± ± ± 1.4 1

23 Figure 1. ellgic cid puniclin puniclgin 2

24 Figure Norml HFS HFS+PFE HFS+AG Serum glucose (mg/dl) w 2 w 4 w 6 w 8 w 3

25 Figure 3. A) Glycolumin level Norml HFS HFS+PFE HFS+AG Glycolumin / totl lumin (%) c 4w 6w 8w c B) HA1c level 10 HA1c / H (%) c 0 Norml HFS HFS+PFE HFS+AG C) Serum AGEs level Serum AGEs concentrtion (µg/ml) Norml HFS HFS+PFE HFS+AG c 4

26 Figure 4. Serum lipid peroxide (mm) Norml HFS HFS+PFE HFS+AG 5

27 Figure 5. A) Glucose-derived AGEs AGEs Formtion rte (%) PFE (µg/ml) EA (µm) GA (µm) PC (µm) PG (µm) AG (µm) B) Fructose-derived AGEs 140 AGEs Formtion rte (%) * * PFE (µg/ml) EA (µm) GA (µm) PC (µm) PG (µm) AG (µm) C) Glycerldehyde-derived AGEs 140 AGEs Formtion rte (%) * * PFE (µg/ml) EA (µm) GA (µm) PC (µm) PG (µm) AG (µm) 6

28 Grphic for tle of contents 7