Antioxidative and Anti-Inflammatory Activities of Galloyl...

Research ArticleAntioxidative and Anti-Inflammatory Activities of GalloylDerivatives and Antidiabetic Activities of Acer ginnala

Kwan Hee Park Kyu Hyeong Yoon Jun Yin Thi Tam Le Hye Sin AhnSeong Hye Yoon andMinWon Lee

Laboratory of Pharmacognosy and Natural Product Derived Medicine College of Pharmacy Chung-Ang UniversitySeoul 156-756 Republic of Korea

Correspondence should be addressed to Min Won Lee mwleecauackr

Received 11 November 2016 Revised 24 January 2017 Accepted 8 February 2017 Published 2 March 2017

Academic Editor G K Jayaprakasha

Copyright copy 2017 Kwan Hee Park et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chromatographic isolation of the 80MeOH extract ofAcer ginnala (AG) yielded seven galloyl derivatives gallic acid (1) ginnalinB (2) acertannin (3) maplexin D (4) maplexin E (5) quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (6) and kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (7) This is the first study to report the isolation of compounds 4 and 5 from AG Galloylderivatives 3ndash7 exhibited potent radical scavenging activities with 5 and 7 showing particularly strong inhibitory activities againstnitric oxide production in lipopolysaccharides- (LPS-) stimulated RAW2647 cells In addition oral administration of AG extract(500mgkg bw) improved symptoms of hyperglycemia and blunted the increases in serum GOTGPT levels in a rat model ofstreptozotocin-induced diabetes These results suggest that galloyl derivatives (1ndash7) are antioxidant and anti-inflammatory agentsand that AG extract has potential as a functional material or novel herbal medicine for treating diabetes mellitus

1 Introduction

Diabetes one of the deathful diseases is well known world-wide According to the report of WHO the number ofpeople with diabetes in 1980 (108 million) has risen to 422million in 2014 [1] It is reported that diabetes will be the7th leading cause of death in 2030 [2] Between 2 typesof diabetes type 2 diabetes results from ineffective use ofinsulin and the majority of people with diabtes worldwideare this type [3] Oxidative stress is one of the pathways thatcould induce diabetes because the high glucose level couldsimulate free radical production to make imbalance ROS [4]And it is reported that inflammation is associated with thedevelopment of type 2 diabetes such as proinflammatorycytokines interleukin-1 beta and tumor necrosis factor-alpha[5] Thus the antioxidative and anti-inflammatory activitiesare important ways to treat diabetes

The genus Acer (Aceraceae) commonly known as mapleis the largest group of deciduous trees and shrubsThis genuscomprises approximately 130 species occupying a significantpart of the northern hemisphere [6] In Northeast Asia Acerspecies have been used in pharmaceutical preparations such

as decoctions or teas to lower fever improve eyesight andprotect the liver In addition Acer species have been usedby the native population of eastern Canada as traditionalremedies for treating coughs and eye pain [7] In the presentdayAcer species are commonly used in commercial productsin North America

Acer ginnala (AG) otherwise known as ldquoamur maplerdquo isnative to northeastern Asia In Republic of Korea AG hasbeen used as a traditional folk medicine for the treatmentof eye disease wound healing and diarrhea [8] The crys-talline tannin acertannin was first discovered in AG [9] thestructure of this tannin was later fully elucidated as 26-di-O-galloyl-15-anhydro-D-glucitol [10] To date several gal-lotannins flavonoids and terpenoids have been isolated fromAG [11 12] Moreover AG has been reported to demonstrateantioxidative antibacterial and antitumor activities [13 14]

The sap of Acer species that is maple syrup is believedto be a suitable sweetener for the management of diabetesmellitus [15] Recently AG extract was reported to inhibitrat lens aldose reductase and the formation of advanced gly-cation products [16] Furthermore ginnalin A-C from other

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 6945912 8 pageshttpsdoiorg10115520176945912

2 Evidence-Based Complementary and Alternative Medicine

Acer species [17 18] has been reported to exhibit antihyper-glycemic effects in a sucrose-loadedmousemodel of diabetesHowever these previous studies used enzymatic tests or theacute glucose tolerance test which are not sufficient to fullycharacterize the antidiabetic activity of AG [19] The aim ofthis studywas to isolate biological compounds from the leavesof AG and to evaluate the hypoglycemic activity of AG in a ratmodel of streptozotocin- (STZ-) induced diabetes

2 Materials and Methods

21 Plant Material The leaves of AG (1 kg) were collectedfrom the Korea National Arboretum (Pocheon Republic ofKorea) in 2010 A voucher specimen (AG2010) was depositedat the herbarium of the College of Pharmacy Chung-AngUniversity (Seoul Republic of Korea)

22 General Procedures Large-scale chromatographic isola-tionwas performed on a Sephadex LH-20 column (10ndash25120583mGE Healthcare Bio-Sciences AB Uppsala Sweden) Daisogel(40ndash60 120583m Daiso Osaka Japan) was used as the stationaryphase on a middle pressure liquid chromatography (MPLC)system (Gilson Seoul Republic of Korea) The isolationprocess was monitored by thin layer chromatography (TLC)using a precoated silica gel 60 F254 plate (Merck Darm-stadt Germany) Spots were detected under UV radiation(254 nm) and by spraying with FeCl3 and 10 H2SO4 oranisaldehyde-H2SO4 followed by heating The compoundsfrom the leaves of AG were identified by 1D (1H13C) and2D (COSY HSQC and HMBC) nuclear magnetic resonance(NMR) spectroscopy (Varian Palo Alto CA) at Chung-AngUniversity

23 Extraction and Isolation The leaves (1 kg) of AG wereextracted three times with 80 methanol at room temper-ature After removing the methanol under vacuum 3225 gof extract was dissolved in water The resultant aqueoussolutionwas filtered throughCelite 545 (Duksan Pure Chem-icals Ansan Republic of Korea) Next 2275 g of water-soluble concentrated filtrate was applied to a Sephadex LH-20 column (2 kg 10 cm times 80 cm) equilibrated with distilledwater The column was eluted with a water-methanol gra-dient system yielding four fractions Subfraction 1 (64 g)was subjected to Daisogel ODS (300 g 3 times 50 cm) andeluted from the MPLC system using a 20ndash100 methanolgradient (5mLmin 280 nm) and yielded ginnalin B (225mg) Subfraction 2 (795 g) was subjected to DaisogelODS (300 g 3 times 50 cm) and eluted from the MPLC systemusing a 20ndash100methanol gradient (5mLmin 280 nm) andyielded gallic acid (1 25mg) and maplexin E (5 20mg)Subfraction 4 (431 g) was subjected to Daisogel ODS (300 g3 times 50 cm) and eluted from the MPLC system using a 20ndash100 methanol gradient (5mLmin 280 nm) and yieldedgallic maplexin D (4 20mg) quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (6 150mg) and kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (7 850mg) Subfraction 3(1002 g) was recrystallized yielding acertannin (3 80 g)

231 Characterization of Compounds Isolated from AG

Compound 1 [345-Trihydroxybenzoic Acid Gallic Acid](S1 2)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575706 (2H s H-2 6) 13C-NMR (150MHz CD3OD) 120575 10902(C-2 C-6) 120575 12059 (C1) 120575 13815 (C-4) 120575 14495 (C-3 5) 12057516900 (C-7)

Compound 2 [6-Galloyl-15-anhydroglucitol Ginnalin B](S3 4)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575301 (1H t J = 111 Hz H-1b) 120575 311 (2H m H-4 5) 120575 328 (2Hm H-1a 3) 120575 371 (1H m H-2) 120575 413 (1H dd J = 45 117HzH-6b) 120575 439 (1H br d J = 117Hz H-6a) 120575 693 (2H s H-2101584061015840) 13C-NMR (150MHz CD3OD) 120575 6440 (C-6) 120575 6989 (C-1) 120575 6999 (C-4) 120575 7039 (C-2) 120575 7828 (C-3) 120575 7865 (C-5)120575 10898 (C-21015840 C-61015840) 120575 11985 (C11015840) 120575 13866 (C-41015840) 120575 14573(C-31015840 51015840) 120575 16630 (C-71015840)

Compound 3 [26-Digalloyl-15-anhydroglucitol Acertannin](S5 6)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575334 (1H t J = 111 Hz H-1b) 120575 353 (2Hm H-4 5) 120575 372 (1Ht J = 90Hz H-3) 120575 410 (1H dd J = 55 111 Hz H-1a) 120575 438(1H dd J = 45 117Hz H-6b) 120575 454 (1H br d J = 117HzH-6a) 120575 490 (1H m H-2) 120575 709 (2H s H-21015840 61015840) 120575 710 (2Hs H-210158401015840 610158401015840) 13C-NMR (150MHz CD3OD) 120575 6352 (C-6) 1205756653 (C-1) 120575 7049 (C-4) 120575 7177 (C-2) 120575 7550 (C-3) 120575 7866(C-5) 120575 10881 (C-21015840 C-61015840) 120575 10895 (C-210158401015840 610158401015840) 120575 11971 (C11015840)120575 11995 (C-110158401015840) 120575 13845 (C-41015840) 120575 13858 (C-410158401015840) 120575 14502 (C-31015840 51015840) 120575 14507 (C-310158401015840 510158401015840) 120575 16646 (C-71015840) 120575 16701 (C-7101584010158401015840)

Compound 4 [24-Digalloyl-15-anhydroglucitol Maplexin D](S7ndash10)

Gray Amorphous Powder Negative FAB-MS mz 467 [M-H]minus 1H-NMR (600MHz CD3OD) 120575 333 (1H t J = 108HzH-1b) 120575 334 (1H dd J = 6 12Hz H-6b) 120575 338 (1H dd J =24 12HzH-6a) 346 (1H ddd J = 24 54 96HzH-5) 120575 380(1H t J = 90Hz H-3) 120575 398 (1H dd J = 54 108Hz H-1a) 120575480 (2H m H-2 4) 120575 697 (2H s H-21015840 61015840) 120575 697 (2H s H-210158401015840 610158401015840) 13C-NMR (150MHz CD3OD) 120575 6123 (C-6) 120575 6647(C-1) 120575 7200 (C-4) 120575 7230 (C-2) 120575 7295 (C-3) 120575 7994 (C-5) 120575 10923 (C-21015840 C-61015840) 120575 10927 (C-210158401015840 610158401015840) 120575 11951 (C11015840) 12057511973 (C-110158401015840) 120575 13883 (C-41015840) 120575 13893 (C-410158401015840) 120575 14581 (C-3101584051015840) 120575 14583 (C-310158401015840 510158401015840) 120575 16549 (C-71015840) 120575 16577 (C-710158401015840)

Compound 5 [246-Trigalloyl-15-anhydroglucitolMaplexin E] (S11ndash14)

Gray Amorphous Powder Negative FAB-MS mz 619[M-H]minus 1H-NMR (600MHz CD3OD) 120575 345 (1H t J =112Hz H-1b) 120575 385 (1H ddd J = 21 51 96Hz H-5) 120575403 (1H t J = 93Hz H-3) 120575 418 (1H dd J = 54 112HzH-1a) 120575 421 (1H dd J = 51 117Hz H-6b) 120575 442 (1H ddJ = 21 117Hz H-6a) 120575 502 (1H ddd J = 54 96 112Hz

Evidence-Based Complementary and Alternative Medicine 3

H-2) 120575 522 (1H t J = 96Hz H-4) 120575 709ndash711 (2Htimes3 eachs H-21015840 210158401015840 2101584010158401015840 61015840 610158401015840 6101584010158401015840) 13C-NMR (150MHz CD3OD)120575 627 (C-6) 120575 666 (C-1) 120575 711 (C-4) 120575 718 (C-2) 120575 734(C-3) 120575 768 (C-5) 120575 1089ndash1094 (C-21015840 210158401015840 2101584010158401015840 61015840 610158401015840 6101584010158401015840)120575 1196ndash1198 (C-1 11015840 1101584010158401015840) 120575 1385ndash1387 (C-41015840 410158401015840 4101584010158401015840) 1205751450ndash1452 (C-31015840 310158401015840 3101584010158401015840 51015840 510158401015840 5101584010158401015840) 120575 1663ndash1667 (C-71015840710158401015840 7101584010158401015840)

Compound 6 [Quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyra-noside] (S15 16)

Yellow Amorphous Powder 1H-NMR (600MHz CD3OD) 120575103 (3H d J = 54Hz H-610158401015840) 120575 345ndash348 (2H m H-410158401015840 510158401015840)120575 401 (1H dd J = 36 90Hz H-310158401015840) 120575 549 (1H d J = 15HzH-110158401015840) 120575 563 (1H dd J = 15 36Hz H-210158401015840) 120575 619 (1H d J =18Hz H-6) 120575 637 (1H d J = 18Hz H-8) 120575 694 (1H d J =84Hz H-51015840) 120575 717 (2H s H-2101584010158401015840 6101584010158401015840) 120575 733 (1H d J = 84HzH-51015840) 120575 734 (1H dd J = 24 84Hz H-61015840) 120575 736 (1H d J =24Hz H-21015840) 13C-NMR (150MHz CD3OD) 120575 1641 (C-6

10158401015840)120575 6929 (C-310158401015840) 120575 7079 (C-510158401015840) 120575 7210 (C-210158401015840) 120575 7236 (C-410158401015840)120575 9335 (C-8) 120575 9846 (C-6) 120575 9910 (C-110158401015840) 120575 10445 (C-10)120575 10899 (C-2101584010158401015840 6101584010158401015840) 120575 11510 (C-51015840) 120575 11551 (C-21015840) 120575 11981(C-1101584010158401015840) 120575 12141 (C-61015840) 120575 12149 (C-11015840) 120575 13422 (C-3) 120575 13855(C-4101584010158401015840) 120575 14499 (C-5101584010158401015840) 120575 14502 (C-3101584010158401015840) 120575 14842 (C-41015840) 12057515707 (C-9) 120575 15788 (C-2) 120575 16171 (C-5) 120575 16440 (C-7) 12057516606 (C-7101584010158401015840) 120575 17795 (C-4)

Compound 7 [Kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyra-noside] (S17 18)

Yellow Amorphous Powder Negative FAB-MS mz 583 [M-H]minus 1H-NMR (600MHz CD3OD) 120575 102 (3H d J = 60HzH-610158401015840) 120575 344ndash345 (1H m H-510158401015840) 120575 347 (1H t J = 90HzH-410158401015840) 120575 399 (1H dd J = 36 90Hz H-310158401015840) 120575 548 (1H d J= 18Hz H-110158401015840) 120575 562 (1H dd J = 18 36Hz H-210158401015840) 120575 619(1H d J = 18Hz H-6) 120575 637 (1H d J = 18Hz H-8) 120575 696(2H d J = 90Hz H-21015840 61015840) 120575 707 (2H s H-2101584010158401015840 6101584010158401015840) 120575 779(2H d J = 90Hz H-31015840 51015840) 13C-NMR (150MHz CD3OD) 120575164 (C-610158401015840) 120575 693 (C-310158401015840) 120575 708 (C-510158401015840) 120575 721 (C-210158401015840) 120575 723(C-410158401015840) 120575 934 (C-8) 120575 985 (C-6) 120575 991 (C-110158401015840) 120575 1045 (C-10) 120575 1090 (C-2101584010158401015840 6101584010158401015840) 120575 1152 (C-3101584010158401015840 5101584010158401015840) 120575 1198 (C-1101584010158401015840) 1205751211 (C-11015840) 120575 1305 (C-21015840 61015840) 120575 1342 (C-3) 120575 1385 (C-4101584010158401015840) 1205751450 (C-3101584010158401015840 5101584010158401015840) 120575 1571 (C-9) 120575 1578 (C-2) 120575 1602 (C-41015840)120575 1617 (C-5) 120575 1644 (C-7) 120575 1660 (C-7101584010158401015840) 120575 1779 (C-4)

24 Measurement of DPPH Radical Scavenging Activity Eachsample was added to a 01mM DPPH solution in absoluteethanol After mixing gently and incubating for 30minat room temperature the optical density of the solutionwas measured at 540 nm using an ELISA reader (TECANSalzburg Austria) The DPPH radical scavenging activitywas calculated as inhibition rate () = [1 minus (sample opticaldensitycontrol optical density)] times 100 The IC50 value wasdefined as the concentration at which 50 of the DPPH freeradicals were scavenged Ascorbic acid was used as a positivecontrol

25 Measurement of NBTSuperoxide Scavenging ActivityEach sample was added to a substrate mixture containing

EDTA (005mM) hypoxanthine (02mM) and nitrobluetetrazolium (NBT 1mM) in 50mM phosphate buffer (pH75) The reaction was then initiated by the addition ofxanthine oxidase (12 U120583L Sigma) The NBTsuperoxideradical scavenging activity was calculated as inhibition rate() = [1 minus (sample optical densitycontrol optical density)]times 100 The IC50 value was defined as the concentration atwhich 50 of the NBTsuperoxide radicals were scavengedAllopurinol (Sigma) was used as a positive control

26 Measurement of Inhibition of NO Production RAW2647mouse macrophage cells were seeded onto 96-well plates (3 times104 cellswell) and incubated for 2 h at 37∘C in a humidifiedatmosphere (5 CO2) The cells were then incubated inmediumcontaining 1120583gmL lipopolysaccharide (LPS Sigma)and the sample of interest After incubating for an additional24 h Griess reagent (01 naphthylethylenediamine and 1sulfanilamide in 5 H3PO4 solution Sigma) was mixed withthe culture supernatant The absorbance of the samples wasthen measured at 540 nm using a microplate reader andthe amount of nitrite in each sample was calculated froma sodium nitrite standard curve The inhibition rate of NOproduction was calculated as inhibition rate () = [1 minus(sample optical density minus blank optical density)(controloptical density minus blank optical density)] times 100The IC50 valuewas defined as the concentration at which the nitrite radicalswere reduced by 50 L-NMMA was used as a positivecontrol

27 Induction of Diabetes and Experimental Design MaleSprague-Dawley rats (130ndash150 g each) were housed in thedepartmental animal facility for two weeks before the exper-iment Environmental conditions included a temperature of22ndash24∘C a 12 h day12 h night cycle and relative air humidityof 40ndash60The animals were provided with standard rodentdiet and water ad libitum and were divided into 4 groups(119899 = 6 per group) For the induction of experimentaldiabetes 45mgkg bw (01M citrate buffer of pH 45 ina volume of 1mLkg) of STZ (Sigma USA) was injectedintraperitoneally Mice with a blood glucose level greaterthan 300mgdL were considered to be diabetic and wereincluded in the experiment The 24 rats were divided intothe following 4 groups Group I normal control Group IIdiabetic control Group III diabetic rats treated daily withAGextract (500mgkg bw) Group IV diabetic rats treated dailywith 3 (AT 100mgkg bw) respectively

28 Biochemical Analysis Rats were fasted overnight afterwhich blood samples were collected from the inferior venacava Blood glucose levels were detected using a portableblood glucose meter (Roche Diagnostics GmbH Germany)GOT and GPT levels were determined using commercial kits(Asan Pharmaceutical Republic of Korea)

29 Statistical Analysis The DPPH NBTsuperoxide andnitrite radical scavenging activities of each compound wereanalyzed by one-way analysis of variance (ANOVA) followedby the Student-Newman-Keuls (SNK) post hoc testThe body


HO

HO

HO OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

O

O

OO

OO

O

OR1O

R3O OR2

G = galloyl

R

R

H

R1

GGHG

R2

HGGG

R3

HHGG

1

2345

67

Figure 1 Structures of compounds 1ndash7 from AG

weight blood glucose and GOTGPT levels of the rats wereanalyzed by the MannndashWhitney 119880 test

3 Results and Discussion

31 Isolation and Structural Identification The repeatedchromatographic isolation of the 80 MeOH extract ofAG yielded seven galloyl derivatives gallic acid (1) [8]ginnalin B (2) [8 18] acertannin (3) [8 17] maplexin D(4) [20] maplexin E (5) [20] quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (6) [21] and kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (7) [21] (Figure 1) All iso-lated compounds were identified by comparing the spectraldata with values reported in the literature This study is thefirst to report the isolation of compounds 4 and 5 from AG

32 Evaluation of Antioxidative and Anti-Inflammatory Activ-ities To assess the antioxidative activities of the compoundsisolated from AG the DPPH radical scavenging ability ofeach compound was measured All compounds exhibitedpotent antioxidative activities as shown in Table 1 Ofparticular note compounds 3ndash5 exhibited significantly morepotent scavenging activity than the positive control (ascorbic

acid) (119901 lt 005) These gallotannins (3ndash5) all have morethan two galloyl groups which are known to eliminate freeradicalsMoreover the extent of theDPPHradical scavengingactivity depended on the number of galloyl groups (1 2 lt 34 5) Flavonoids are also well-known natural antioxidantsIn particular flavonoids with a keto-enol moiety at positionsC-4 and C-5 and phenolic hydroxyl groups in the B-ringare known to act as crucial electron acceptors Moreovercompounds 6 and 7 possessed an additional galloyl moietyat the O-2 position of the rhamnopyranoside ring We foundthat the DPPH radical scavenging activity of compound 6which possessed a 3101584041015840-dihydroxyphenyl B-ring was morepotent than that of compound 7 which possessed a 41015840-hydroxyphenyl B-ring Moreover this effect was significantlygreater than that of ascorbic acid (119901 lt 005) (Table 1)

The enzyme xanthine oxidase catalyzes the oxidation ofhypoxanthine and xanthine to uric acid producing a super-oxide radical and hydrogen peroxide Thus the superoxideradical scavenging activities of the compounds isolated fromAGwere evaluated using a colorimetric NBT reduction assaySimilar to the DPPH radical scavenging results compounds3ndash5 scavenged superoxide radicals more effectively thancompounds 1 and 2 indicating that the number of galloyl


Table 1 IC50 values of compounds 1ndash7 for DPPH scavenging NBTsuperoxide radical scavenging and inhibition of nitric oxide productionby LPS-stimulated RAW2647 cells

CompoundIC50 (120583M)

a

DPPH radical scavengingactivity

NBTsuperoxidescavenging activity

Inhibition of NOproduction

1 1044 plusmn 077bc 1708 plusmn 133e 100lt2 1214 plusmn 003c 2080 plusmn 084e 100lt3 687 plusmn 105a 296 plusmn 014a 100lt4 692 plusmn 052a 301 plusmn 012a 100lt5 572 plusmn 030a 283 plusmn 009a 3608 plusmn 212b

6 1244 plusmn 030c 520 plusmn 057b 7646 plusmn 468c

7 1892 plusmn 033d 1244 plusmn 154cd 3562 plusmn 125b

Vitamin C 1311 plusmn 012c mdash mdashAllopurinol mdash 975 plusmn 122c mdashL-NMMA mdash mdash 1723 plusmn 125aaValues are presented as mean plusmn SD (119899 = 3)Different superscript letters indicate a significant difference (119901 value lt 005)

groups is crucial for the elimination of superoxide radicals(119901 lt 005) (Table 1) Flavonoids are also well-knownnatural xanthine oxidase inhibitors and superoxide radicalscavengers The hydroxyl groups at positions C-5C-7 andthe double bond between positions C-2 and C-3 have bothbeen shown to be essential for effective inhibition of xanthineoxidase while the hydroxyl group at position C-31015840 hasbeen reported to influence superoxide radical scavenging[22] With respect to these structure-activity relationshipscompounds 6 and 7 (flavonols) showed either the sameor significantly more potent scavenging activity than thepositive control allopurinol (119901 lt 005) (Table 1) Thus theseresults indicate that other bioavailable galloyl derivatives suchas oligo-galloyl-substituted acer-tannins or galloyl flavonoidsfrom AG contribute more to the elimination of intracellularoxidative stress compared to compound 2

Next we confirmed the inhibitory effects of the com-pounds isolated from AG on nitric oxide production in LPS-stimulated RAW2647 cells Macrophages produce variousproinflammatory mediators including the short-lived freeradical NO Moreover lipopolysaccharide (LPS) a compo-nent of the cell wall of Gram-negative bacteria is one ofthe most powerful activators of macrophages and stimu-lates NO production Of the gallotannins only compound5 (a trigalloyl-substituted tannin) inhibited nitric oxideproduction in contrast treatment with compounds 1ndash4did not have any significant effect on NO production Wehypothesize that compounds 1ndash4 were too hydrophilic toreduce intracellular NO production (Table 1) The 119909 log119875scores of compounds 1ndash4 as provided by PubChem wereall negative Consistent with this tendency the inhibitoryactivity of compound 7 which possessed a 41015840-hydroxyphenylB-ring was more potent than that of compound 6 whichpossessed a 3101584041015840-dihydroxyphenyl B-ring (119901 lt 005) (Table 1)[19] Of particular note trigalloyl-substituted tannins andkaempferol-derived flavonoids have been reported to reduceiNOS expression via NF-kB inactivation [23 24]

33 Evaluation of Antidiabetic Activity Streptozotocin iswidely applied to induce experimental diabetes due to its abil-ity to selectively target and destroy insulin-producing pan-creatic islet beta cells [25] In the present study we inducedexperimental diabetes through IP injection of STZ and eval-uated the antidiabetic effect of AG via oral administration ofAG extract (500mgkg bw) Alternatively we administeredthe major compound of AG ginnalin A (3) (100mgkg bw)The STZ IP injection group exhibited significantly elevatedfasting blood glucose and serum GOTGPT levels comparedto the normal control group Oral administration of AGextract significantly reduced these increases in fasting bloodglucose and serumGOTGPT levels compared to the diabeticcontrol group However treatment group of 3 did not exhibitany significant changes even though the median values weredecreased In addition STZ injection resulted in a significantdecrease inwhole bodyweight whichwas restored by the oraladministration of AG Similar to above this rescue was notobserved in treatment group of 3 (Figure 2)

Diabetes is a pathological process related to an unbal-anced production of ROS such as hydroxyl radicals (∙OH)superoxide anions (O2) and H2O2 and RNS such as nitricoxide (NO) and peroxynitrite (ONOOminus) [26] Excessiveoxidative stress elevates aldose reductase activity and stimu-lates the hexosamine biosynthetic pathway as well as increas-ing formation of advanced glycation end-products [27] Inaddition lipid peroxide production and abnormal immuneresponses result in various complications such as diabeticretinopathy nephropathy neuropathy and atherosclerosis[28] Thus the application of natural antioxidants that offerresistance against oxidative stress to diabetes has been a topicof interest

STZ has been reported to generate robust production ofnitric oxide and hydroxyl radicals or ROS [29] leading toinflammation and apoptosis of 120573-cells in the pancreas andthereby mimicking autoimmune diabetes Moreover at highconcentrations acute toxicity in extrapancreatic organs such


lowastlowast

lowastlowast

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lowast

ATAGDCNC ATAGDCNC

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ght (

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lood

glu

cose

(mg

dL)

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GO

T (K

amen

mL)

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12000

GPT

(Kam

enm

L)

Figure 2 Effects of AG and AT (acertannin) on whole body weight fasting blood glucose and GOT and GPT levels in STZ-induced diabeticrats The experimental diabetic rats were orally given AG (500mgkg bw) or AT (100mgkg bw) for 2 weeks (119899 = 6 per group) Results areexpressed as median and 75th25th percentiles (lowast119901 lt 005 and lowastlowast119901 lt 001 versus diabetic control group 119901 lt 001 versus normal controlgroup)

as the liver has been reported [30] Oral administration ofAG to diabetic rats effectively recovered fasting blood glucoseand serum GOTGPT levels These results suggest that AGhas a protective effect against oxidative stress in STZ-induceddiabetic rats

In contrast acertannin (3) the main crystalline tannin ofAG did not show significant antidiabetic activity Consistentwith this result 3 and the monogalloyl- and digalloyl-substituted tannins (1 2 and 4) did not have any effect onNO production in LPS-stimulated RAW2647 cells120572-Glucosidase secreted from intestinal chorionic epithe-

lium is a carbohydrate hydrolase 120572-Glucosidase (EC 32120)inhibitors as a new antidiabetic drug from 1980s slow downthe process of digestion and absorption of carbohydrates bycompetitively blocking the glucosidase activity A previousreport [31] found that galloyl substitution (more than threesubstitutions at the 15-anhydroglucitol core) resulted inpotent inhibition of 120572-glucosidase with a galloyl group atC-6 being essential for this activity In particular the 120572-glucosidase inhibitory activity of 3 (IC50 = 8842 plusmn 694 120583M)

was reported to be 10 times less than that of 5 (IC50 =826 plusmn 037 120583M) [31] Moreover flavonoid hydroxylation andgalloylation have been reported to improve the inhibitoryactivity of120572-glucosidase [32]This finding suggests that oligo-galloyl-substituted acer-tannins or galloyl flavonoids fromAGalso directly influence glucosemetabolism Future studieswill aim to elucidate the active constituents of AG and toelucidate the mechanism of action of this extract

4 Conclusion

Chromatographic isolation of the 80 MeOH extract ofAcer ginnala (AG) yielded seven galloyl derivatives gal-lic acid (1) ginnalin B (2) acertannin (3) maplexinD (4) maplexin E (5) quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (6) and kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (7) Compounds 3ndash7 exhibited par-ticularly potent radical scavenging activities and 5 and 7strongly inhibited nitric oxide production in LPS-stimulatedRAW2647 cells In addition oral administration of AG


extract (500mgkg bw) for 2 weeks resulted in significantlylower fasting blood glucose and serum GOTGPT levelsin STZ-induced experimental diabetic rats These resultssuggest that the leaves of AG could be useful in the treatmentof diabetes mellitus However identification of the activeconstituents of AG responsible for its antidiabetic activity willrequire further in vivo studies Such studies could be designedbased on reports that acertannin exhibits antidiabetic activityin vitro and in the acute glucose tolerance test in vivo

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

KwanHee Park and Kyu Hyeong Yoon contributed equally tothis work

Acknowledgments

This study was supported by the Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF-2016R1D1A1B03930307) which is funded by the Min-istry of Education Science and Technology

References

[1] World Health Organization Global Report on Diabetes WHOGeneva Switzerland 2016

[2] C D Mathers and D Loncar ldquoProjections of global mortalityand burden of disease from 2002 to 2030rdquo PLoS Medicine vol3 no 11 article e442 2006

[3] WHO ldquoDefinition diagnosis and classification of diabetes mel-litus and its complications Part 1 diagnosis and classificationof diabetes mellitusrdquo Tech Rep WHONCDNCS992 WorldHealth Organization Geneva Switzerland 1999

[4] T Inoguchi P Li F Umeda et al ldquoHigh glucose level and freefatty acid stimulate reactive oxygen species production throughprotein kinase Cmdashdependent activation of NAD(P)H oxidasein cultured vascular cellsrdquoDiabetes vol 49 no 11 pp 1939ndash19452000

[5] M Crook ldquoType 2 diabetes mellitus a disease of the innateimmune system An updaterdquo Diabetic Medicine vol 21 no 3pp 203ndash207 2004

[6] D M Van Gelderen P C De Jong and H J OterdomMaplesof the World Timber Press Portland Ore USA 1994

[7] T Arnason R J Hebda and T Johns ldquoUse of plants for foodand medicine by Native Peoples of eastern Canadardquo CanadianJournal of Botany vol 59 no 11 pp 2189ndash2325 1981

[8] S E Choi K H Park M H Oh et al ldquoAntioxidative activitiesand quantitative determination of gallotannins from barks ofAcer ginnalaMaximrdquoKorean Journal of Pharmacognosy vol 41no 3 pp 174ndash179 2010

[9] A G Perkin and Y Uyeda ldquoXIIImdashOccurrence of a crystallinetannin in the leaves of the Acer ginnalardquo Journal of the ChemicalSociety Transactions vol 121 pp 66ndash76 1922

[10] K Bock N Faurschou laCour S R Jensen and B J NielsenldquoThe structure of acertanninrdquo Phytochemistry vol 19 no 9 p2033 1980

[11] Y K Son and Y N Han ldquoIsolation of triterpenoid saponinsfrom the stems of Acer ginnala Maximrdquo Korean Journal ofPharmacognosy vol 33 no 4 pp 301ndash304 2002

[12] R-L Lu F-L Hu and T Xia ldquoActivity-guided isolation andidentification of radical scavenging components in Gao-Chateardquo Journal of Food Science vol 75 no 8 pp H239ndashH243 2010

[13] S S Han S C Lo Y W Choi J H Kim and S H BaekldquoAntioxidant activity of crude extract and pure compounds ofAcer ginnala Maxrdquo Bulletin of the Korean Chemical Society vol25 no 3 pp 389ndash391 2004

[14] H C Yong S H Sung O L Hyun andH B Seung ldquoBiologicalactivity of bioactive components from Acer ginnala MaxrdquoBulletin of the Korean Chemical Society vol 26 no 9 pp 1450ndash1452 2005

[15] N Nagai Y Ito and A Taga ldquoComparison of the enhancementof plasma glucose levels in type 2 diabetes Otsuka Long-EvansTokushima Fatty rats by oral administration of sucrose ormaplesyruprdquo Journal of Oleo Science vol 62 no 9 pp 737ndash743 2013

[16] Y M Lee Y S Kim J H Kim and J S Kim ldquoScreening ofKorean herbal medicines with inhibitory activity on advancedglycation end products formation (IX)rdquo Korean Journal ofPharmacognosy vol 44 no 3 pp 298ndash304 2013

[17] A Honma T Koyama and K Yazawa ldquoAnti-hyperglycemiceffects of sugar maple Acer saccharum and its constituentacertanninrdquo Food Chemistry vol 123 no 2 pp 390ndash394 2010

[18] A Honma T Koyama and K Yazawa ldquoAnti-hyperglycaemiceffects of the Japanese red maple Acer pycnanthum and its con-stituents the ginnalins B and Crdquo Journal of Enzyme Inhibitionand Medicinal Chemistry vol 26 no 2 pp 176ndash180 2011

[19] W Bi Y Gao J Shen et al ldquoTraditional uses phytochemistryand pharmacology of the genus Acer(maple) a reviewrdquo Journalof Ethnopharmacology vol 189 pp 31ndash60 2016

[20] C Wan T Yuan L Li et al ldquoMaplexins new 120572-glucosidaseinhibitors from redmaple (Acer rubrum) stemsrdquo Bioorganic andMedicinal Chemistry Letters vol 22 no 1 pp 597ndash600 2012

[21] Y Kim D-S Jang S-H Park et al ldquoFlavonol glycoside gallateand ferulate esters from Persicaria lapathifolia as inhibitorsof superoxide production in human monocytes stimulated byunopsonized zymosanrdquo PlantaMedica vol 66 pp 72ndash74 2000

[22] P Cos L Ying M Calomme et al ldquoStructure-activity relation-ship and classification of flavonoids as inhibitors of xanthineoxidase and superoxide scavengersrdquo Journal of Natural Prod-ucts vol 61 no 1 pp 71ndash76 1998

[23] V Garcıa-Mediavilla I Crespo P S Collado et al ldquoTheanti-inflammatory flavones quercetin and kaempferol causeinhibition of inducible nitric oxide synthase cyclooxygenase-2 and reactive C-protein and down-regulation of the nuclearfactor kappaB pathway in Chang Liver cellsrdquo European Journalof Pharmacology vol 557 no 2-3 pp 221ndash229 2007

[24] M-S Kim S-B Park K Suk et al ldquoGallotannin isolated fromeuphorbia species 126-Tri-O-galloyl-120573-D-allose decreasesnitric oxide production through inhibition of nuclear factor-120581 gtB and downstream inducible nitric oxide synthase expres-sion in macrophagesrdquo Biological and Pharmaceutical Bulletinvol 32 no 6 pp 1053ndash1056 2009

[25] E B Johansson and H Tjalve ldquoStudies on the tissue-depositionand fate of [14C] streptozotocin with special reference to thepancreatic isletsrdquo Acta Endocrinologica vol 89 pp 339ndash3511978


[26] P Subash-Babu A A Alshatwi and S Ignacimuthu ldquoBeneficialantioxidative and antiperoxidative effect of cinnamaldehydeprotect streptozotocin-induced pancreatic 120573-cells damage inwistar ratsrdquo Biomolecules andTherapeutics vol 22 no 1 pp 47ndash54 2014

[27] M C Eppens M E Craig J Cusumano et al ldquoPrevalence ofdiabetes complications in adolescents with type 2 comparedwith type 1 diabetesrdquoDiabetes Care vol 29 no 6 pp 1300ndash13062006

[28] V Granberg N Ejskjaer M Peakman and G SundkvistldquoAutoantibodies to autonomic nerves associated with cardiacand peripheral autonomic neuropathyrdquo Diabetes Care vol 28no 8 pp 1959ndash1964 2005

[29] N S Kwon S H Lee C S Choi T Kho and H S Lee ldquoNitricoxide generation from streptozotocinrdquo FASEB Journal vol 8no 8 pp 529ndash533 1994

[30] R H Bell Jr and R J Hye ldquoAnimal models of diabetes mellitusphysiology and pathologyrdquo Journal of Surgical Research vol 35no 5 pp 433ndash460 1983

[31] Z Yin W Zhang F Feng Y Zhang and W Kang ldquo120572-Glucosidase inhibitors isolated from medicinal plantsrdquo FoodScience and Human Wellness vol 3 no 3-4 pp 136ndash174 2014

[32] J Xiao G Kai K Yamamoto and X Chen ldquoAdvance in dietarypolyphenols as 120572-glucosidases inhibitors a review on structure-activity relationship aspectrdquo Critical Reviews in Food Scienceand Nutrition vol 53 no 8 pp 818ndash836 2013

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Acer species [17 18] has been reported to exhibit antihyper-glycemic effects in a sucrose-loadedmousemodel of diabetesHowever these previous studies used enzymatic tests or theacute glucose tolerance test which are not sufficient to fullycharacterize the antidiabetic activity of AG [19] The aim ofthis studywas to isolate biological compounds from the leavesof AG and to evaluate the hypoglycemic activity of AG in a ratmodel of streptozotocin- (STZ-) induced diabetes

2 Materials and Methods

21 Plant Material The leaves of AG (1 kg) were collectedfrom the Korea National Arboretum (Pocheon Republic ofKorea) in 2010 A voucher specimen (AG2010) was depositedat the herbarium of the College of Pharmacy Chung-AngUniversity (Seoul Republic of Korea)

22 General Procedures Large-scale chromatographic isola-tionwas performed on a Sephadex LH-20 column (10ndash25120583mGE Healthcare Bio-Sciences AB Uppsala Sweden) Daisogel(40ndash60 120583m Daiso Osaka Japan) was used as the stationaryphase on a middle pressure liquid chromatography (MPLC)system (Gilson Seoul Republic of Korea) The isolationprocess was monitored by thin layer chromatography (TLC)using a precoated silica gel 60 F254 plate (Merck Darm-stadt Germany) Spots were detected under UV radiation(254 nm) and by spraying with FeCl3 and 10 H2SO4 oranisaldehyde-H2SO4 followed by heating The compoundsfrom the leaves of AG were identified by 1D (1H13C) and2D (COSY HSQC and HMBC) nuclear magnetic resonance(NMR) spectroscopy (Varian Palo Alto CA) at Chung-AngUniversity

23 Extraction and Isolation The leaves (1 kg) of AG wereextracted three times with 80 methanol at room temper-ature After removing the methanol under vacuum 3225 gof extract was dissolved in water The resultant aqueoussolutionwas filtered throughCelite 545 (Duksan Pure Chem-icals Ansan Republic of Korea) Next 2275 g of water-soluble concentrated filtrate was applied to a Sephadex LH-20 column (2 kg 10 cm times 80 cm) equilibrated with distilledwater The column was eluted with a water-methanol gra-dient system yielding four fractions Subfraction 1 (64 g)was subjected to Daisogel ODS (300 g 3 times 50 cm) andeluted from the MPLC system using a 20ndash100 methanolgradient (5mLmin 280 nm) and yielded ginnalin B (225mg) Subfraction 2 (795 g) was subjected to DaisogelODS (300 g 3 times 50 cm) and eluted from the MPLC systemusing a 20ndash100methanol gradient (5mLmin 280 nm) andyielded gallic acid (1 25mg) and maplexin E (5 20mg)Subfraction 4 (431 g) was subjected to Daisogel ODS (300 g3 times 50 cm) and eluted from the MPLC system using a 20ndash100 methanol gradient (5mLmin 280 nm) and yieldedgallic maplexin D (4 20mg) quercetin-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (6 150mg) and kaempferol-3-O-(210158401015840-galloyl)-120572-L-rhamnopyranoside (7 850mg) Subfraction 3(1002 g) was recrystallized yielding acertannin (3 80 g)

231 Characterization of Compounds Isolated from AG

Compound 1 [345-Trihydroxybenzoic Acid Gallic Acid](S1 2)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575706 (2H s H-2 6) 13C-NMR (150MHz CD3OD) 120575 10902(C-2 C-6) 120575 12059 (C1) 120575 13815 (C-4) 120575 14495 (C-3 5) 12057516900 (C-7)

Compound 2 [6-Galloyl-15-anhydroglucitol Ginnalin B](S3 4)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575301 (1H t J = 111 Hz H-1b) 120575 311 (2H m H-4 5) 120575 328 (2Hm H-1a 3) 120575 371 (1H m H-2) 120575 413 (1H dd J = 45 117HzH-6b) 120575 439 (1H br d J = 117Hz H-6a) 120575 693 (2H s H-2101584061015840) 13C-NMR (150MHz CD3OD) 120575 6440 (C-6) 120575 6989 (C-1) 120575 6999 (C-4) 120575 7039 (C-2) 120575 7828 (C-3) 120575 7865 (C-5)120575 10898 (C-21015840 C-61015840) 120575 11985 (C11015840) 120575 13866 (C-41015840) 120575 14573(C-31015840 51015840) 120575 16630 (C-71015840)

Compound 3 [26-Digalloyl-15-anhydroglucitol Acertannin](S5 6)

Gray Amorphous Powder 1H-NMR (600MHz CD3OD) 120575334 (1H t J = 111 Hz H-1b) 120575 353 (2Hm H-4 5) 120575 372 (1Ht J = 90Hz H-3) 120575 410 (1H dd J = 55 111 Hz H-1a) 120575 438(1H dd J = 45 117Hz H-6b) 120575 454 (1H br d J = 117HzH-6a) 120575 490 (1H m H-2) 120575 709 (2H s H-21015840 61015840) 120575 710 (2Hs H-210158401015840 610158401015840) 13C-NMR (150MHz CD3OD) 120575 6352 (C-6) 1205756653 (C-1) 120575 7049 (C-4) 120575 7177 (C-2) 120575 7550 (C-3) 120575 7866(C-5) 120575 10881 (C-21015840 C-61015840) 120575 10895 (C-210158401015840 610158401015840) 120575 11971 (C11015840)120575 11995 (C-110158401015840) 120575 13845 (C-41015840) 120575 13858 (C-410158401015840) 120575 14502 (C-31015840 51015840) 120575 14507 (C-310158401015840 510158401015840) 120575 16646 (C-71015840) 120575 16701 (C-7101584010158401015840)

Compound 4 [24-Digalloyl-15-anhydroglucitol Maplexin D](S7ndash10)

Gray Amorphous Powder Negative FAB-MS mz 467 [M-H]minus 1H-NMR (600MHz CD3OD) 120575 333 (1H t J = 108HzH-1b) 120575 334 (1H dd J = 6 12Hz H-6b) 120575 338 (1H dd J =24 12HzH-6a) 346 (1H ddd J = 24 54 96HzH-5) 120575 380(1H t J = 90Hz H-3) 120575 398 (1H dd J = 54 108Hz H-1a) 120575480 (2H m H-2 4) 120575 697 (2H s H-21015840 61015840) 120575 697 (2H s H-210158401015840 610158401015840) 13C-NMR (150MHz CD3OD) 120575 6123 (C-6) 120575 6647(C-1) 120575 7200 (C-4) 120575 7230 (C-2) 120575 7295 (C-3) 120575 7994 (C-5) 120575 10923 (C-21015840 C-61015840) 120575 10927 (C-210158401015840 610158401015840) 120575 11951 (C11015840) 12057511973 (C-110158401015840) 120575 13883 (C-41015840) 120575 13893 (C-410158401015840) 120575 14581 (C-3101584051015840) 120575 14583 (C-310158401015840 510158401015840) 120575 16549 (C-71015840) 120575 16577 (C-710158401015840)

Compound 5 [246-Trigalloyl-15-anhydroglucitolMaplexin E] (S11ndash14)

Gray Amorphous Powder Negative FAB-MS mz 619[M-H]minus 1H-NMR (600MHz CD3OD) 120575 345 (1H t J =112Hz H-1b) 120575 385 (1H ddd J = 21 51 96Hz H-5) 120575403 (1H t J = 93Hz H-3) 120575 418 (1H dd J = 54 112HzH-1a) 120575 421 (1H dd J = 51 117Hz H-6b) 120575 442 (1H ddJ = 21 117Hz H-6a) 120575 502 (1H ddd J = 54 96 112Hz
















HO

HO

HO OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

O

O

OO

OO

O

OR1O

R3O OR2

G = galloyl

R

R

H

R1

GGHG

R2

HGGG

R3

HHGG

1

2345

67











a














lowastlowast

lowastlowast

lowastlowast

lowast

ATAGDCNC ATAGDCNC

ATAGDCNC ATAGDCNC

00

10000

20000

30000

40000

Who

le b

ody

wei

ght (

g)

00

10000

20000

30000

40000

50000

60000

Fasti

ng b

lood

glu

cose

(mg

dL)

00

2000

4000

6000

8000

10000

GO

T (K

amen

mL)

00

2000

6000

4000

8000

10000

12000

GPT

(Kam

enm

L)






4 Conclusion




Competing Interests




Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































HO

HO

HO OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

O

O

OO

OO

O

OR1O

R3O OR2

G = galloyl

R

R

H

R1

GGHG

R2

HGGG

R3

HHGG

1

2345

67











a














lowastlowast

lowastlowast

lowastlowast

lowast

ATAGDCNC ATAGDCNC

ATAGDCNC ATAGDCNC

00

10000

20000

30000

40000

Who

le b

ody

wei

ght (

g)

00

10000

20000

30000

40000

50000

60000

Fasti

ng b

lood

glu

cose

(mg

dL)

00

2000

4000

6000

8000

10000

GO

T (K

amen

mL)

00

2000

6000

4000

8000

10000

12000

GPT

(Kam

enm

L)






4 Conclusion




Competing Interests




Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















a














lowastlowast

lowastlowast

lowastlowast

lowast

ATAGDCNC ATAGDCNC

ATAGDCNC ATAGDCNC

00

10000

20000

30000

40000

Who

le b

ody

wei

ght (

g)

00

10000

20000

30000

40000

50000

60000

Fasti

ng b

lood

glu

cose

(mg

dL)

00

2000

4000

6000

8000

10000

GO

T (K

amen

mL)

00

2000

6000

4000

8000

10000

12000

GPT

(Kam

enm

L)






4 Conclusion




Competing Interests




Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowast

lowastlowast

lowast

ATAGDCNC ATAGDCNC

ATAGDCNC ATAGDCNC

00

10000

20000

30000

40000

Who

le b

ody

wei

ght (

g)

00

10000

20000

30000

40000

50000

60000

Fasti

ng b

lood

glu

cose

(mg

dL)

00

2000

4000

6000

8000

10000

GO

T (K

amen

mL)

00

2000

6000

4000

8000

10000

12000

GPT

(Kam

enm

L)






4 Conclusion




Competing Interests




Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests




Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Antioxidative and Anti-Inflammatory Activities of Galloyl...

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