research article polyploidy analysis and attenuation of...

Research ArticlePolyploidy Analysis and Attenuation of OxidativeStress in Hepatic Tissue of STZ-Induced Diabetic RatsTreated with an Aqueous Extract of Vochysia rufa

Izabela Barbosa Moraes12 Camilla Manzan-Martins1

Neire Moura de Gouveia1 Luciana Karen Calaacutebria13 Karen Renata Nakamura Hiraki4

Alberto da Silva Moraes4 and Foued Salmen Espindola1

1 Instituto de Genetica e Bioquımica Universidade Federal de Uberlandia Avenida Para 1720 38400-902 Uberlandia MG Brazil2Faculdade Sao Francisco de Barreiras BR 135 Km 01 2341 47800-970 Barreiras BA Brazil3Faculdade de Ciencias Integradas do Pontal Universidade Federal de Uberlandia Rua 20 1600 38304-402 Ituiutaba MG Brazil4Instituto de Ciencias Biomedicas Universidade Federal de Uberlandia Avenida Para 1720 38400-902 Uberlandia MG Brazil

Correspondence should be addressed to Foued Salmen Espindola fouedespindolagmailcom

Received 15 August 2014 Revised 23 December 2014 Accepted 24 December 2014

Academic Editor Wenchuan Lin

Copyright copy 2015 Izabela Barbosa Moraes et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Diabetes mellitus (DM) is characterized by hyperglycemia and alterations in the metabolism of lipids carbohydrates and proteinsDue to its hypoglycemic effect Vochysia rufa is frequently used in Uberlandia Brazil to treat DM Despite its popularity there islittle information about its effect on hepatic tissue Therefore we evaluated the histoarchitecture oxidative stress parameters andpolyploidy of liver tissue from streptozotocin- (STZ-) induced diabetic rats treated with aqueous extract of Vochysia rufa (AEV)Histology was determined by fixing the livers processing and staining with HE Oxidative stress was determined by evaluatingCAT GPx and SOD activity in liver homogenates and hepatic mitochondria fraction and by measuring GST GSH levels and lipidperoxidation (MDA) Polyploidy was determined by subjecting isolated hepatocyte nuclei to flow cytometry In the diabetic groupGST activity and GSH rates decreased whereas liver homogenate analysis showed that GPx SOD activity and MDA increasedAEV treatment restored all parameters to normal levels The oxidative stress analysis of hepatic mitochondria fraction showedsimilar results Lower polyploid cell populations were found in the diabetic rat livers even after glibenclamide treatmentThus AEVtreatment efficiently reduced hepatic oxidative stress caused by STZ-induced diabetes and produced no morphological changes inthe histological analysis

1 Introduction

Diabetes mellitus is a metabolic disorder characterized byhyperglycemia resulting from insufficient secretion of orreceptor insensitivity to endogenous insulin [1] More-over DM causes alterations in carbohydrate protein andlipid metabolism [2] Diabetic complications are linkedto hyperglycemia-induced oxidative stress which eventu-ally overcomes the endogenous antioxidant defense systemthrough glucose autoxidation induction of nonenzymaticglycosylation of various macromolecules and generation of

reactive oxygen species (ROS) [3]The human body possessesseveral enzymes associated with antioxidant defense andrepair mechanisms against oxidative stress such as catalase(CAT) superoxide dismutase (SOD) glutathione peroxi-dase (GPx) reduced glutathione (GSH) and glutathione S-Transferase (GST) [4]

The liver is the main detoxifying organ in the bodybut also plays a central role in metabolic homeostasis [5]Alterations in hepatic glucosemetabolism are associatedwithdiabetes and changes to many hepatic enzymes occur indiabetic individuals [6]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 316017 8 pageshttpdxdoiorg1011552015316017

2 Evidence-Based Complementary and Alternative Medicine

For years various people around the world have usedmedicinal plants to manage diabetes [7ndash12] Studies haveshown that plants can have beneficial effects on diabeticcomplications [13 14] especially on hepatic oxidative stress[14ndash16] V rufa Mart popularly known as ldquosweet barkrdquo hasbeen used in folk medicine to treat diabetes mellitus type1 and type 2 in Uberlandia Brazil Several species of thegenus Vochysia have important therapeutic and medicinalproperties Phytochemical characterization of work carriedout with the genus led to the isolation of polyphenols andtriterpenes [17] Unlike our study the main compoundsfound by Silva [18] present in themethanol extract ofVochysiadrums were phenolic compounds coumarins saponins andtriterpenoids However there is not any report about thesugars hitherto let alone its antidiabetic activity in experi-mental model of the diabetes Therefore the present studyinvestigates the effect preliminary of an aqueous extract of Vrufa (AEV) on the hepatic tissue and hepatic mitochondriafraction of diabetic rats by examining GPx GST SOD CATactivity lipid peroxidation GSH levels histoarchitecture andpolyploidy

2 Materials and Methods

21 Plant Material and the Aqueous Extract Stem bark ofV rufa Mart was collected from the Cerrado biome inthe outskirts of Abadia dos DouradosMG Brazil (latitude18∘27101584050510158401015840 and longitude 47∘23101584037210158401015840) from October 2010to February 2011 The plant was identified and a voucherspecimen deposited (number 58888) at theHerbariumUber-landensis of the Universidade Federal de Uberlandia Thebark was dried at 40∘C and ground to a powderThe aqueousextract was obtained using a commonprocedure that involvesthe maceration of 200 g of bark in 1 L of distillated water for24 h (1 5 wv) at room temperature The resulting extractwas then filtered and centrifuged at 2000timesg at 4∘C for15min Finally the supernatant was collected frozen andlyophilized

22 Quantification of Reducing Sugars The presence ofreducing sugars was determined by the Lane-Eynon methodin which cupric salts in alkaline tartrate solution can bereduced by heating aldoses and ketoses turning into redcuprous salts [19] In this procedure 5mL of solution A and5mL of Fehling solution B were transferred to a 250mLErlenmeyer flask with the aid of a pipette and then 50mLof distilled water was added for heating until boiling Thenthe test sample was transferred to a 25mL burette and addeddropwise over Fehlingrsquos solution boiling with continuousstirring until the solution changed from blue to colorlessA reddish residue was formed in the flask bottom and 2-3drops of methylene blue were added to it and the titrationwas completed according to the change of colorThe Fehlingrsquossolution was previously calibrated with a 05 glucose solu-tion (vv in water) with three replications Calculation 100times119860 times 119886119875 times 119881 = Reducing carbohydrates in glucose (ww)The mL of the sample solution = spend the titration 119886 = titleFehling (number of grams of glucose corresponding to 10mL

of Fehling solution) 119875 = 119892 of sample (or initial volume ofthe sample) 119881 = volume of the sample spent titration Thesimplified formula can be expressed as

Gear carbohydrates into glucose () = 119886 times 100119881 (1)

A solution of the aqueous extract of Vochysia 05 was pre-pared and carried out the same procedures to glucose

23 Animals Adult Wistar male rats (200ndash250 g) wereobtained from ANILAB (Animais de Laboratorio Criacaoe Comercio Ltda-EPP) Paulınia Brazil The animals weremaintained under standard conditions (22 plusmn 1∘C 60 plusmn 5humidity and a 12 h light 12 h dark cycle) The animalswere fed a commercial pellet diet (6582 carbohydrate536 fiber 210 protein and 496 fat) (BioBase SCBrazil) and received water ad libitum All procedures for thehandling use and euthanasia of the animals were approvedby the Ethics Committee for Animal Research at the FederalUniversity of Uberlandia Brazil (CEUAUFU 06010)

24 Induction of Diabetes The rats were intraperitoneallyanesthetized with xylazineketamine (1 1 vv) Diabetes wasinduced in overnight-fasted animals by an intraperitonealinjection (40mgkg) of STZ (Sigma-Aldrich St Louis MOUSA) freshly dissolved in 001M citrate buffer (pH 45)Citrate buffer was also administered to the animals in thenondiabetic group The animals were fasted for another90min after the injection Ten days after the STZ injectiona portable glucometer was used to measure fasting bloodglucose in blood collected from the tail veins of all animalsAnimals with blood glucose levels gt250mgdL were consid-ered diabetic

25 Experimental Procedure The animals were divided intosix groups of 10 animals a nondiabetic control (ND) adiabetic control (DB) nondiabetics treated with 500mgkgof aqueous extract ofV rufa (ND-AEV) nondiabetics treatedwith 6mgkg of glibenclamide (ND-GB) diabetics treatedwith 500mgkg of aqueous extract of V rufa (DB-AEV)and diabetics treated with 6mgkg of glibenclamide (DB-GB) Body weight was measured at the beginning and endof the study and the animals had free access to food andwater On day 43 of the treatment the rats were anesthetizedwith xylazineketamine (1 1 vv) Livers were then removedrinsed in saline solution to remove blood weighed andimmediately frozen and stored at minus80∘C for assays (119899 = 6)Livers were also fixed in buffered formaldehyde (10) forhistological analysis (119899 = 4)

26 Liver Homogenization for Evaluation of Oxidative StressLiver samples (1 g) were homogenized on ice in four volumesof homogenization buffer (20mM Tris-HCl pH 74 2mMdithiothreitol 1mM benzamidine 05mM phenylmethane-sulfonyl fluoride 05mM aprotinin and 01mM pefabloc)Next the homogenate was centrifuged at 800timesg for 10minat 4∘C and protein concentrations were thenmeasured by theBradford method [20]

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Effects of aqueous extract of Vochysia rufa and glibenclamide on body and liver weight in STZ-induced diabetic and nondiabeticrats

CAT120583gseg

GPxmmolminmL

GSHmM

GST120583molsdotminminus1sdotgminus1

SODUmg protein

MDAnmolmgprotein

ND 3345 plusmn 231 22 plusmn 02 37 plusmn 03 1212 plusmn 69 06 plusmn 01 15 plusmn 01

ND-AEV 4396 plusmn 378lowasta

43 plusmn 05lowasta

48 plusmn 03lowasta

1112 plusmn 45 28 plusmn 07lowasta

14 plusmn 01

ND-GB 3889 plusmn 287 24 plusmn 02 26 plusmn 03lowasta

1053 plusmn 08 06 plusmn 01 15 plusmn 02

DB 3482 plusmn 233 38 plusmn 03lowasta

27 plusmn 03lowasta

848 plusmn 51a

13 plusmn 02lowasta

19 plusmn 01lowasta

DB-AEV 3121 plusmn 226 23 plusmn 03lowastb

37 plusmn 02lowastb

1217 plusmn 157lowastb

12 plusmn 01 13 plusmn 01lowastb

DB-GB 3209 plusmn 200 24 plusmn 02lowastb

32 plusmn 02 936 plusmn 74 06 plusmn 01b

16 plusmn 02

Data are shown as means plusmn SEM 119899 = 6 lowast119875 lt 005 119875 lt 0005aCompared with a nondiabetic control groupbDiabetic treated group compared with a diabetic controlND nondiabetic DB STZ-induced diabetic AEV treated with aqueous extract of Vochysia rufa GB treated with glibenclamide

27 Mitochondrial Fraction A 1 g liver sample was homog-enized in 4mL of homogenization buffer (25mM sucrose20mM HEPES 1mM EDTA pH 74 2mM dithiothreitol1mMbenzamidine 05mMphenylmethanesulfonyl fluoride05mM aprotinin and 01mM Pefabloc) The homogenatewas then filtered and centrifuged at 800timesg at 4∘C for 10minThe supernatant was centrifuged again at 10000timesg at 4∘Cfor 10min to obtain the mitochondrial fraction Finally themitochondrial pellet was suspended by the same buffer andstored at minus80∘C for analysis

28 Oxidative Stress Analysis Lipid peroxidation was bio-chemically assessed by determining malondialdehyde levels(MDA) [21] Catalase (CAT) activity wasmeasured accordingto the method of Aebi [22] Superoxide dismutase (SOD) wasmeasured as described by Boveris [23] Glutathione peroxi-dase (GPx) activity was assayed by the method described byFlohe and Gunzler [24] and glutathione S-transferase (GST)activity wasmeasured according to Habig et al [25] Reducedglutathione (GSH) was measured with 2-nitrobenzoic acid(DTNB) reagent as described by Beutler et al [26] Totalprotein in the tissue homogenate and mitochondrial fractionwas estimated by the method of Bradford [20]

29 Histological Analysis Histology was determined by fix-ing the livers in 10 phosphate-buffered formalin process-ing embedding in paraffin and then staining with HampEHepatic polyploidy was assessed cytophotometrically bysubjecting liver tissue to a Feulgen reaction with room-temperature hydrolysis (4N HCl) Stained slides were cap-tured on a LEICA DM500 microscope equipped with aLEICA Application Suite (Version 181) camera Digitalizedimages were analyzed using Image J open source softwareand the integrated optical density parameter which shows theaverage DNA quantity in the nuclei of each population

210 Flow Cytometry Hepatocyte nuclei were isolatedaccording to the method described by Blobel and Potter[27] Immediately after tissue homogenization using a Potterhomogenizer in 025M sucrose in TKM (Tris 002M KCl

0025M and MgCl20005M) buffer the homogenate was

filtered mixed with 23M sucrose in TKM and applied to23M sucrose in TKM solution This process produced agradient that was centrifuged at 124000timesg at 4∘C for 30minPelleted nuclei were suspended in TKM buffer and stainedwith propidium iodide for analysis by flow cytometry (BDAccuri C6 Flow Cytometer) Analysis was then performedusing FlowJo software

211 Statistical Analysis Oxidative stress data were analyzedby the Student 119905-test (SigmaStat 35 software Systat SoftwareInc IL USA) and expressed as means plusmn SEM Medians ofthe flow cytometry data were analyzed in Minitab softwareusing Moodrsquos Median Test A 119875 value lt 005 was consideredsignificant for all tests

3 Results

31 Quantification of Reducing Sugars Thepresence of reduc-ing sugars was determined by the Lane-Eynon methodwherein the Vochysia extract contained 70 reducing car-bohydrates Furthermore we observed that there is noabsorption spectrum in HPLC as analyzed in HPLC profilewhere there is no separation of the compounds howeverthese molecules have not yet been isolated and characterized

32 Effects of AEV on Body Weight Blood Glucose Level andLiver Weight in STZ-Induced Diabetic Rats After 43 days ofAEV treatment blood glucose levels of the diabetic groupdecreased (3146 plusmn 873mgdL) although it is not sig-nificantly compared to the diabetic control group (535 plusmn43mgdL) However glucose levels of glibenclamide treateddiabetics were significantly lower than diabetic controls (97plusmn19mgdL)

The mean body weight of diabetic rats was significantlylower than that of nondiabetic controls (Table 1) NeitherAEV nor glibenclamide treatment changed this parameter inthe nondiabetic and diabetic groups Liver weight decreasedafter diabetes but tended to revert to levels observed in


Table 2 Measurements of oxidative stress parameters in liverhomogenates of STZ-induced diabetic and nondiabetic rats treatedor not with AEV and glibenclamide

Initial BW(g)

Final BW(g) LW (g) Relative LW

()

ND 2646 plusmn 08 3569 plusmn 105 117 plusmn 05 33 plusmn 01

ND-AEV 2687 plusmn 385 3505 plusmn 94 99 plusmn 03a 29 plusmn 01a

ND-GB 2693 plusmn 38 3555 plusmn 89 101 plusmn 03a28 plusmn 01

a

DB 2370 plusmn 81 2259 plusmn 84lowasta90 plusmn 02

lowasta40 plusmn 01

lowasta

DB-AEV 2248 plusmn 109 2202 plusmn 100 96 plusmn 02 43 plusmn 02

DB-GB 2317 plusmn 99 2490 plusmn 146 123 plusmn 07lowastb49 plusmn 04

Data are shown as means plusmn SEM 119899 = 10 lowast119875 lt 0001 119875 lt 005aCompared with a nondiabetic control groupbDiabetic treated group compared with a diabetic controlND nondiabetic DBSTZ-induced diabetic AEV treated with aqueousextract of Vochysia rufa GB treated with glibenclamide BW body weightLW liver weight

Table 3 Evaluation of CAT GPx and SOD activity in the hepaticmitochondria of STZ-induced diabetic and nondiabetic rats treatedwith aqueous extract of Vochysia rufa and glibenclamide

CAT120583gseg

GPxmmolminmL

SODUmgprotein

ND 4842 plusmn 412 24 plusmn 03 17 plusmn 017

ND-AEV 7593 plusmn 887lowasta 566 plusmn 04Ca

18 plusmn 032

ND-GB 6893 plusmn 807lowasta


DB 3529 plusmn 141lowasta

51 plusmn 03a

08 plusmn 015lowasta

DB-AEV 4654 plusmn 241lowastb

39 plusmn 03Cb

128 plusmn 008b

DB-GB 4863 plusmn 311lowastb

16 plusmn 01Cb

09 plusmn 009

Data are shown as means plusmn SEM 119899 = 6 lowast119875 lt 005 119875 lt 0005 C119875 lt0001aCompared with a nondiabetic control groupbDiabetic treated group compared with diabetic controlND nondiabetic DB STZ-induced diabetic AEV treated with aqueousextract of Vochysia rufa GB treated with glibenclamide

nondiabetic animals after AEV treatment and reverted com-pletely after glibenclamide treatment Liverweightwas higherin diabetic animals These results indicate that diabetesdecreased body mass more drastically than liver mass andthat while AEV and glibenclamide were unable to restorebody weight to normal levels these treatments were able toincrease liver weight back to control values

33 Oxidative Stress Analysis MDA SOD and GPx activityincreased and GST and GSH activity decreased after diabetes(Table 2) AEV treatment reverted all these changes exceptSOD In diabetic rats glibenclamide treatment restored GPxand SOD to normal and tended to restore GSH GST andMDA In some cases (CAT GPx and SOD) AEV treatmentproduced opposite results in nondiabetic and diabetic rats

CAT and SOD activities were significantly lower in thehepatic mitochondrial tissue (Table 3) of the diabetic controlgroup than in the nondiabetic control Both CAT and SOD

Table 4 Streptozotocin-induced diabetes mellitus alters ploidylevels in rats

Group ND ND-AEV ND-GB DB DB-AEV DB-GB2n4n 063a 053ac 079

bc086

bc074

abc181

d

2n4n medians of the ratio between diploid and tetraploid cells 119899 = 6different letters in the same line indicate statistical significance (119875 lt 005)ND nondiabetic DB STZ-induced diabetic AEV treated with aqueousextract of Vochysia rufa GB treated with glibenclamide

activity increased in the diabetic group treated with AEV butonly CAT activity increased in the diabetic group treatedwithglibenclamide Finally GPx activity was higher in the livermitochondria of the diabetic group but lower in both treateddiabetic groups

34 Histological Analysis Analysis of HE-stained liver slices(Figure 1) showed preservation of liver histoarchitecturehepatocytes with a normal cytoplasmic eosinophilic aspectand one or two nuclei with loose chromatin and evidentnucleolus No evidence of inflammation necrosis fibrosisproliferation destruction or parenchyma regeneration andtherefore no visual signs of hepatotoxicity were observed

35 Polyploidy Hepatic polyploidy was estimated by ana-lyzing isolated hepatocyte nuclei via flow cytometry Theresults showed that streptozotocin induced diabetes reducedthe ratio of tetraploid nuclei to diploid nuclei (Table 4)In other words diabetes reduced polyploidy relative to thenondiabetic control group Polyploidy was not statisticallyhigher in the diabetic group treated with EAV than in thecontrol however the data did show a tendency for polyploidyto increase towards normal rates after this treatment Onthe other hand glibenclamide treatment of both diabeticand nondiabetic rats significantly decreased numbers oftetraploid nuclei relative to diploid nuclei Images of Feulgen-stained liver slices confirmed these results (data not shown)

4 Discussion

STZ-induced diabetes is one of the most commonly usedexperimental models of human diabetes used in research[28] STZ induces hyperglycemia by destroying pancreatic 120573-cells and consequently reducing insulin production Hyper-glycemia is associated with metabolic change in the liverespecially loss of body weight and antioxidant imbalance [2930] which occurred in the present study Medicinal plantscan reduce the effects of diabetes mainly because of theirhypoglycemic properties and capacity to counteract oxidativestress [15 16 31]

Despite its popularity information is lacking on theefficiency of V rufa as a treatment for diabetes Neverthe-less several species of the Vochysia genus have importanttherapeutic andmedicinal properties Phytochemical charac-terization of species of this genus has isolated polyphenolsand triterpenes [17] However this study demonstrated thepresence of sugars in the aqueous extract ofV rufa Similar toour results [32] demonstrated that Coptis chinensis contains


ND

CVCon

trol

(a)

DB

CV

(b)

CV

AEV

(c)

CV

(d)

CV

GB

(e)

CV

(f)

Figure 1 Photomicrographs of liver sections from nondiabetic control rats (ND) (a) diabetic control (DB) (b) nondiabetic treated withaqueous extract of V rufa (AEV) (c) diabetic treated with AEV (d) nondiabetic treated with glibenclamide (GB) (e) and diabetic treatedwith (GB) (f) HampE (Bar = 25120583m) CV central vein Kupffer cells with a normal aspect are indicated by arrows

963 carbohydrate 48 uronic acid and 061 protein andis mainly composed of glucose arabinose xylose galactoseand galacturonic acid In recent years the antidiabetic effectof polysaccharide has widely been reported [33ndash35] Zhanget al [36] demonstrated that the orally administered Tcuspidata polysaccharides had an effective hypoglycemiceffect and could effectively alleviate the impaired oxidativestress in the kidney and liver of the diabetic rats induced

by STZ Jiang et al [32] demonstrated that Coptis chinen-sis polysaccharides could produce a potent and efficaciousantidiabetic effect and antioxidant activity in diabetic mice

Increased lipid peroxidation as observed in the currentstudy is associated with increased oxidative stress especiallyin the STZ-induced diabetic model [31 37 38] Underhyperglycemic conditions glucose autooxidizes and pro-duces superoxide leading to the production of free radicals


that in turn cause lipid peroxidation in lipoproteins [15] Inthe present study MDA formed in the diabetic group Incontrast MDA formation was lower in the diabetic grouptreated with AEV Similar results were found for otherresearchers [14] CAT is produced by peroxisomes and playsa role in cell defense by breaking down hydrogen peroxide(H2O2) into H

2O andO

2 No differences in liver homogenate

analysis were observed however CAT activity was lowerin the mitochondria fraction of the diabetic control groupLow MDA content combined with significant increases incatalase activity was observed in diabetic rats treated with theextract confirming the antioxidant effect of this treatmentThe extract may therefore lead to significant elimination offree radicals caused by diabetes

The SOD oxide-reductase group is responsible for super-oxide anion (O

2

minus) dismutation into molecular oxygen andwater In the present study SOD activity was higher in theliver homogenates of STZ-induced diabetic rats in responseto greater hepatic oxidative stress [16 29] However SODactivity was lower in the hepatic mitochondria fraction ofthe diabetic group Kapoor and Kakkar [39] showed thatin cultured hepatocytes MnSOD (mitochondrial SOD) andCuSOD (cytosolic SOD) are differentially expressed whenexposed to high glucose concentrations (40mM) Althoughhigh-glucose induces low MnSOD expression it also leadsto high CuSOD expression These differences could beexplained by potential differences in SOD concentrationbetween liver homogenates and hepatic mitochondria Treat-ment with V rufa extract increases SOD activity in livermitochondria fraction which may protect against oxidativeinjury

GPx activity has been shown to be higher in liverhomogenates than in hepatic mitochondria fraction [12 40]whereas GSH levels have been shown to be lower [15 38]The glutathione system is the main intracellular antioxidantmechanism It is dependent on GSH concentration and theGSHGSSG ratio which is regulated by de novo synthe-sis of glutathione in a redox cycle mediated by reactionscontrolled by GPx and GR Additionally lower GSH levelshave been considered as the hallmark of oxidative stress[12] Furthermore high GPx activity in diabetic animals isrelated to higher oxidative stress in the liver The decreasesin GST activity for the diabetic group in the present studycorroborate the findings of other studies [38] GST is afamily of isoenzymes that participate in conjugating toxicelectrophiles to GSH Therefore decreased GST activity indiabetic rats may be related to lower GSH availability

Abundant clinical evidence demonstrated that the dia-betes correlated closely with oxidative stress resulting in anincreased ROS production or a reduction in the antioxidantdefense system [41] Oxidative stress decreased after AEVtreatment as evidenced by the reduction of lipid peroxidationand GPx activity to normal levels GST activity increasedprotecting cells against the toxic effects of harmful com-pounds and preventing oxidative damage [42] Higher SODand CAT activity in the mitochondria fraction of diabetictreated rats is related to nondiabetic control levels and highGSH levels Many reports on medicinal plant treatments fordiabetes show reduced oxidative stress in the liver [15 28 43]

These results indicated an obvious antioxidant effect ofV rufaon STZ-induced diabetic rats in hepatic tissue due to presenceof polysaccharides Polysaccharide mechanism involved inantidiabetic activity may include increased levels of seruminsulin reduction of blood glucose level and improvedtolerance of glucose [44] This is done by our group a studyto evaluate the effect of V rufa extract in pancreas induceddiabetic rats In this study we evaluated the ability of theextract to increase insulin secretion

Polyploid cells form is present in several tissue types[45] including liver tissue Gentric et al [46] describedthree principal effects of polyploidy on liver tissue Firstpolyploidy could protect hepatocytes from genotoxic damageby increasing the number of copies of functional genesThis function may be especially important for an organwhose primary role is to metabolize and eliminate toxiccompounds Second polyploidy could be an economicalsolution to growth problems that occur when an organ workswithin its capabilities avoiding the great energy demands ofcell division Finally polyploidy could alter the expression ofspecific genes Our study showed reduced levels of polyploidyin STZ-induced diabetic rats If this phenomenon benefitsthe tissue diabetes could leave the hepatic tissue unprotectedCelton-Morizur et al [45] showed that tetraploid hepatocytenumbers decreased in rats treated with streptozotocin Thisstudy also demonstrated that insulin regulates the genesisof binucleated tetraploid liver cells Because STZ destroysbeta-cells insulin production decreases to the point that liverdevelopment is impaired

Increased polyploidy in the liver has been associatedwith several health conditions including aging Ghiraldiniet al [47] demonstrated that NOD mice with severe hyper-glycemia have greater ploidy levels than nondiabetic miceOur contradictory results might suggest that the ploidyparameter is determined purely by STZ administration andnot by hyperglycemia Therefore the toxic effects of STZmay directly affect hepatic metabolism and thus impair thecapacity of hepatocytes to increase their degree of ploidy viaendoreplication

STZ is a glucosamine-nitrosourea compound whichenters cells through the GLUT2 transporter and interactswith DNA causing severe mutations Thus STZ can beused to kill cancerous beta-pancreatic cells when surgeryis inviable However GLUT2 is also the primary glucosetransporter in liver cells allowing it to enter hepatocytes[48] Approximately 15ndash67 of patients treated with strepto-zotocin chemotherapy develop some kind of hepatocellulardamage [49] Although no morphological evidence of hepa-totoxicity was observed in the present study STZ treatmentmay still have directly altered the liver parameters investi-gated However given that AEV treatment reverted manyalterations it indicates that STZ-induced hyperglycemia mayalso have influenced hepatic change

5 Conclusion

We showed that STZ-induced diabetes increased oxidativestress led to higher relative liver weight and reduced liverpolyploidy suggesting that these changes may have been


caused by the pathology of diabetes in the liver NeverthelessSTZ could also be directly responsible for these alterationsgiven that the drug itself is hepatotoxic The action of thesugars presents in AEV extract improved the antioxidantenzymes activity and the elevated lipid peroxidation in STZ-induced diabetic rats implied its free radical scavengingpotential and hence it has the ability to prevent diabetic asso-ciated complication However since AEV treatment reducedthe deleterious effects on liver tissue it seems that both STZ(principally) and diabetes (secondarily) are responsible

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

Foued Salmen Espindola received a fellowship for ResearchProductivity from CNPQ and grant for the program ofMineirorsquos Researcher from FAPEMIG (CBB-PPM-00270-12)This work was supported by the FAPEMIG Brazil (Grant noAPQ-01271-10 to FSE) This work also received support byfellowship toNMG (PNPDCAPES) and IBM (CNPq) and byRede Fitocerrado which is a nongovernmental organizationbased in Uberlandia MG Brazil Thanks are due to DrPaulo S Pereira (Federal Institute of Education Science andTechnology Rio Verde Goias) and Dra Suzelei C Franca(Department of Biotechnology Ribeirao Preto UniversityUNAERP Ribeirao Preto Sao Paulo Brazil) for their provi-sion of the quantification of reducing sugars

References

[1] A P Rolo and C M Palmeira ldquoDiabetes and mitochondrialfunction role of hyperglycemia and oxidative stressrdquoToxicologyand Applied Pharmacology vol 212 no 2 pp 167ndash178 2006

[2] B Halliwell and J Gutterridge Free Radical in Biology andMedicine Oxford Science New York NY USA 2003

[3] A O Ademiluyi and G Oboh ldquoAttenuation of oxidativestress and hepatic damage by some fermented tropical legumecondiment diets in streptozotocin-induced diabetes in ratsrdquoAsian Pacific Journal of Tropical Medicine vol 5 no 9 pp 692ndash697 2012

[4] M Z Gul F Ahmad A K Kondapi I A Qureshi and I AGhazi ldquoAntioxidant and antiproliferative activities ofAbrus pre-catorius leaf extractsmdashan in vitro studyrdquo BMC Complementaryand Alternative Medicine vol 13 article 53 2013

[5] L P Bechmann R A Hannivoort G Gerken G S Hotamis-ligil M Trauner and A Canbay ldquoThe interaction of hepaticlipid and glucose metabolism in liver diseasesrdquo Journal ofHepatology vol 56 no 4 pp 952ndash964 2012

[6] L Rossetti A Giaccari N Barzilai K Howard G Sebel andM Hu ldquoMechanism by which hyperglycemia inhibits hepaticglucose production in conscious rats Implications for thepathophysiology of fasting hyperglycemia in diabetesrdquo TheJournal of Clinical Investigation vol 92 no 3 pp 1126ndash11341993

[7] S Celikler S Tas O Vatan S Ziyanok-Ayvalik G Yildiz andR Bilaloglu ldquoAnti-hyperglycemic and antigenotoxic potentialof Ulva rigida ethanolic extract in the experimental diabetes

mellitusrdquo Food and Chemical Toxicology vol 47 no 8 pp 1837ndash1840 2009

[8] S Gupta S B Sharma S K Bansal and K M Prabhu ldquoAnti-hyperglycemic and hypolipidemic activity of aqueous extract ofCassia auriculata L leaves in experimental diabetesrdquo Journal ofEthnopharmacology vol 123 no 3 pp 499ndash503 2009

[9] Y-J Hsu T-H Lee C L-T Chang Y-T Huang and W-CYang ldquoAnti-hyperglycemic effects and mechanism of Bidenspilosawater extractrdquo Journal of Ethnopharmacology vol 122 no2 pp 379ndash383 2009

[10] N Hamza B Berke C Cheze et al ldquoPrevention of type 2diabetes induced by high fat diet in the C57BL6J mouse by twomedicinal plants used in traditional treatment of diabetes in theeast of Algeriardquo Journal of Ethnopharmacology vol 128 no 2pp 513ndash518 2010

[11] M S Islam ldquoEffects of the aqueous extract of white tea(Camellia sinensis) in a streptozotocin-induced diabetes modelof ratsrdquo Phytomedicine vol 19 no 1 pp 25ndash31 2011

[12] M Dıaz-Flores S Angeles-Mejia L A Baiza-Gutman et alldquoEffect of an aqueous extract of Cucurbita ficifolia Bouche onthe glutathione redox cycle inmice with STZ-induced diabetesrdquoJournal of Ethnopharmacology vol 144 no 1 pp 101ndash108 2012

[13] I Ahmed E Adeghate E Cummings A K Sharma andJ Singh ldquoBeneficial effects and mechanism of action ofMomordica charantia juice in the treatment of streptozotocin-induced diabetes mellitus in ratrdquo Molecular and Cellular Bio-chemistry vol 261 no 1 pp 63ndash70 2004

[14] R Babujanarthanam P Kavitha U SMahadeva Rao andM RPandian ldquoQuercitrin a bioflavonoid improves the antioxidantstatus in streptozotocin Induced diabetic rat tissuesrdquoMolecularand Cellular Biochemistry vol 358 no 1-2 pp 121ndash129 2011

[15] A A Abolfathi D Mohajeri A Rezaie and M NazerildquoProtective effects of green tea extract against hepatic tissueinjury in streptozotocin-induced diabetic ratsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID740671 10 pages 2012

[16] G R Rodrigues F C di Naso M Porawski et al ldquoTreatmentwith aqueous extract from Croton cajucara Benth reduceshepatic oxidative stress in Streptozotocin-diabetic ratsrdquo Journalof Biomedicine and Biotechnology vol 2012 Article ID 9023517 pages 2012

[17] Y L Zucaro Z R S Compagnone S C Hess and F DelleMonache ldquoHydroxymaslinic acid a triterpene from Vochysiaferrugineardquo Journal of the Brazilian Chemical Society vol 11 no3 pp 241ndash244 2000

[18] M A B Silva Levantamento etnobotanico e triagem hipoli-pidemica de Plantas Medicinais do Cerrado Mato-grossenseAvaliacao da Atividade hipolipidemica da Simaba ferruginea StHil e Vochysia rufaMart [Dissertacao] Universidade Federal deMato GrossomdashFaculdade de Ciencias Medicas Cuiaba Brazil2009

[19] Instituto Adolfo Lutz Metodos fısico-quımicos para analise dealimentos IAL Sao Paulo Brazil 4th edition 2004

[20] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[21] M Hermes-Lima W G Willmore and K B Storey ldquoQuan-tification of lipid peroxidation in tissue extracts based onFe(III)xylenol orange complex formationrdquo Free Radical Biologyamp Medicine vol 19 no 3 pp 271ndash280 1995


[22] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[23] A Boveris ldquoDetermination of the production of superoxideradicals and hydrogen peroxide in mitochondriardquo in Methodsin Enzymology pp 429ndash435 Academic Press New York NYUSA 1984

[24] L Flohe and W A Gunzler ldquoAssays of glutathione peroxidaserdquoMethods in Enzymology vol 105 pp 114ndash121 1984

[25] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[26] E Beutler O Duron and BM Kelly ldquoImprovedmethod for thedetermination of blood glutathionerdquoThe Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[27] G Blobel and V R Potter ldquoNuclei from rat liver isolationmethod that combines purity with high yieldrdquo Science vol 154no 757 pp 1662ndash1665 1966

[28] P Sarkhail S Rahmanipour S Fadyevatan et al ldquoAntidiabeticeffect of Phlomis anisodonta effects on hepatic cells lipid per-oxidation and antioxidant enzymes in experimental diabetesrdquoPharmacological Research vol 56 no 3 pp 261ndash266 2007

[29] T Matsunami Y Sato T Sato S Ariga T Shimomura and MYukawa ldquoOxidative stress and gene expression of antioxidantenzymes in the streptozotocin-induced diabetic rats underhyperbaric oxygen exposurerdquo International Journal of Clinicaland Experimental Pathology vol 3 no 2 pp 177ndash188 2010

[30] R Pazdro and J R Burgess ldquoThe role of vitamin E and oxidativestress in diabetes complicationsrdquo Mechanisms of Ageing andDevelopment vol 131 no 4 pp 276ndash286 2010

[31] A V Da Costa L K Calabria F B Furtado et al ldquoNeuropro-tective effects of Pouteria ramiflora (Mart) Radlk (Sapotaceae)extract on the brains of rats with streptozotocin-induced dia-betesrdquoMetabolic Brain Disease vol 28 no 3 pp 411ndash419 2013

[32] S Jiang P Du L An G Yuan and Z Sun ldquoAnti-diabeticeffect of Coptis Chinensis polysaccharide in high-fat diet withSTZ-induced diabetic micerdquo International Journal of BiologicalMacromolecules vol 55 pp 118ndash122 2013

[33] R-J Li S-D Qiu H-X Chen H Tian and H-X Wang ldquoTheimmunotherapeutic effects ofAstragalus polysaccharide in type1 diabetic micerdquo Biological and Pharmaceutical Bulletin vol 30no 3 pp 470ndash476 2007

[34] C Sunil V Duraipandiyan P Agastian and S IgnacimuthuldquoAntidiabetic effect of plumbagin isolated from Plumbagozeylanica L root and its effect on GLUT4 translocation instreptozotocin-induced diabetic ratsrdquo Food and Chemical Toxi-cology vol 50 no 12 pp 4356ndash4363 2012

[35] X-Q Mao Y Wu K Wu et al ldquoAstragalus polysaccharidereduces hepatic endoplasmic reticulum stress and restoresglucose homeostasis in a diabetic KKAy mouse modelrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1947ndash1956 2007

[36] D Zhang H Meng and H-S Yang ldquoAntidiabetic activity ofTaxus cuspidata polysaccharides in streptozotocin-induced dia-betic micerdquo International Journal of Biological Macromoleculesvol 50 no 3 pp 720ndash724 2012

[37] S R Deconte R J da Silva Oliveira L K Calabria et al ldquoAlter-ations of antioxidant biomarkers and type I collagen depositionin the parotid gland of streptozotocin-induced diabetic ratsrdquoArchives of Oral Biology vol 56 no 8 pp 744ndash751 2011

[38] D Prabakaran and N Ashokkumar ldquoProtective effect ofesculetin on hyperglycemia-mediated oxidative damage in

the hepatic and renal tissues of experimental diabetic ratsrdquoBiochimie vol 95 no 2 pp 366ndash373 2013

[39] R Kapoor and P Kakkar ldquoProtective role of Morin a flavonoidagainst high glucose induced oxidative stress mediated apopto-sis in primary rat hepatocytesrdquo PLoS ONE vol 7 no 8 ArticleID e41663 2012

[40] H R Yilmaz E Uz N Yucel I Altuntas and N OzcelikldquoProtective effect of caffeic acid phenethyl ester (CAPE) onlipid peroxidation and antioxidant enzymes in diabetic rat liverrdquoJournal of Biochemical and Molecular Toxicology vol 18 no 4pp 234ndash238 2004

[41] K Susztak A C RaffM Schiffer and E P Bottinger ldquoGlucose-induced reactive oxygen species cause apoptosis of podocytesand podocyte depletion at the onset of diabetic nephropathyrdquoDiabetes vol 55 no 1 pp 225ndash233 2006

[42] M A Amer M H Ghattas D M Abo-Elmatty and S HAbou-El-Ela ldquoInfluence of glutathione S-transferase polymor-phisms on type-2 diabetesmellitus riskrdquoGenetics andMolecularResearch vol 10 no 4 pp 3722ndash3730 2011

[43] J F da Costa Guerra C L de Brito Magalhaes D C Costa ME Silva and M L Pedrosa ldquoDietary acai modulates ROS pro-duction by neutrophils and gene expression of liver antioxidantenzymes in ratsrdquo Journal of Clinical Biochemistry and Nutritionvol 49 no 3 pp 188ndash194 2011

[44] S B Mishra C V Rao S K Ojha M Vijakumar and AVerma ldquoAn analytical review of plants for anti diabetic activitywith their phytoconstituents and mechanism of actionrdquo Inter-national Journal Pharmacological Sciences and Research vol 1pp 29ndash38 2010

[45] S Celton-Morizur G Merlen D Couton and C DesdouetsldquoPolyploidy and liver proliferation Central role of insulinsignalingrdquo Cell Cycle vol 9 no 3 pp 460ndash466 2010

[46] G Gentric C Desdouets and S Celton-Morizur ldquoHepatocytespolyploidization and cell cycle control in liver physiopathologyrdquoInternational Journal ofHepatology vol 2012 Article ID 2824308 pages 2012

[47] F G Ghiraldini I S Silva and M L S Mello ldquoPolyploidy andchromatin remodeling in hepatocytes from insulin-dependentdiabetic and normoglycemic aged micerdquo Cytometry Part A vol81 no 9 pp 755ndash764 2012

[48] W J Schnedl S Ferber J H Johnson and C B Newgard ldquoSTZtransport and cytotoxicity specific enhancement in GLUT2-expressing cellsrdquo Diabetes vol 43 no 11 pp 1326ndash1333 1994

[49] P S Schein M J OrsquoConnell J Blom et al ldquoClinical antitumoractivity and toxicity of streptozotocin (NSC-85998)rdquo Cancervol 34 no 4 pp 993ndash1000 1974

Submit your manuscripts athttpwwwhindawicom

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


For years various people around the world have usedmedicinal plants to manage diabetes [7ndash12] Studies haveshown that plants can have beneficial effects on diabeticcomplications [13 14] especially on hepatic oxidative stress[14ndash16] V rufa Mart popularly known as ldquosweet barkrdquo hasbeen used in folk medicine to treat diabetes mellitus type1 and type 2 in Uberlandia Brazil Several species of thegenus Vochysia have important therapeutic and medicinalproperties Phytochemical characterization of work carriedout with the genus led to the isolation of polyphenols andtriterpenes [17] Unlike our study the main compoundsfound by Silva [18] present in themethanol extract ofVochysiadrums were phenolic compounds coumarins saponins andtriterpenoids However there is not any report about thesugars hitherto let alone its antidiabetic activity in experi-mental model of the diabetes Therefore the present studyinvestigates the effect preliminary of an aqueous extract of Vrufa (AEV) on the hepatic tissue and hepatic mitochondriafraction of diabetic rats by examining GPx GST SOD CATactivity lipid peroxidation GSH levels histoarchitecture andpolyploidy

2 Materials and Methods

21 Plant Material and the Aqueous Extract Stem bark ofV rufa Mart was collected from the Cerrado biome inthe outskirts of Abadia dos DouradosMG Brazil (latitude18∘27101584050510158401015840 and longitude 47∘23101584037210158401015840) from October 2010to February 2011 The plant was identified and a voucherspecimen deposited (number 58888) at theHerbariumUber-landensis of the Universidade Federal de Uberlandia Thebark was dried at 40∘C and ground to a powderThe aqueousextract was obtained using a commonprocedure that involvesthe maceration of 200 g of bark in 1 L of distillated water for24 h (1 5 wv) at room temperature The resulting extractwas then filtered and centrifuged at 2000timesg at 4∘C for15min Finally the supernatant was collected frozen andlyophilized

22 Quantification of Reducing Sugars The presence ofreducing sugars was determined by the Lane-Eynon methodin which cupric salts in alkaline tartrate solution can bereduced by heating aldoses and ketoses turning into redcuprous salts [19] In this procedure 5mL of solution A and5mL of Fehling solution B were transferred to a 250mLErlenmeyer flask with the aid of a pipette and then 50mLof distilled water was added for heating until boiling Thenthe test sample was transferred to a 25mL burette and addeddropwise over Fehlingrsquos solution boiling with continuousstirring until the solution changed from blue to colorlessA reddish residue was formed in the flask bottom and 2-3drops of methylene blue were added to it and the titrationwas completed according to the change of colorThe Fehlingrsquossolution was previously calibrated with a 05 glucose solu-tion (vv in water) with three replications Calculation 100times119860 times 119886119875 times 119881 = Reducing carbohydrates in glucose (ww)The mL of the sample solution = spend the titration 119886 = titleFehling (number of grams of glucose corresponding to 10mL

of Fehling solution) 119875 = 119892 of sample (or initial volume ofthe sample) 119881 = volume of the sample spent titration Thesimplified formula can be expressed as

Gear carbohydrates into glucose () = 119886 times 100119881 (1)

A solution of the aqueous extract of Vochysia 05 was pre-pared and carried out the same procedures to glucose

23 Animals Adult Wistar male rats (200ndash250 g) wereobtained from ANILAB (Animais de Laboratorio Criacaoe Comercio Ltda-EPP) Paulınia Brazil The animals weremaintained under standard conditions (22 plusmn 1∘C 60 plusmn 5humidity and a 12 h light 12 h dark cycle) The animalswere fed a commercial pellet diet (6582 carbohydrate536 fiber 210 protein and 496 fat) (BioBase SCBrazil) and received water ad libitum All procedures for thehandling use and euthanasia of the animals were approvedby the Ethics Committee for Animal Research at the FederalUniversity of Uberlandia Brazil (CEUAUFU 06010)

24 Induction of Diabetes The rats were intraperitoneallyanesthetized with xylazineketamine (1 1 vv) Diabetes wasinduced in overnight-fasted animals by an intraperitonealinjection (40mgkg) of STZ (Sigma-Aldrich St Louis MOUSA) freshly dissolved in 001M citrate buffer (pH 45)Citrate buffer was also administered to the animals in thenondiabetic group The animals were fasted for another90min after the injection Ten days after the STZ injectiona portable glucometer was used to measure fasting bloodglucose in blood collected from the tail veins of all animalsAnimals with blood glucose levels gt250mgdL were consid-ered diabetic

25 Experimental Procedure The animals were divided intosix groups of 10 animals a nondiabetic control (ND) adiabetic control (DB) nondiabetics treated with 500mgkgof aqueous extract ofV rufa (ND-AEV) nondiabetics treatedwith 6mgkg of glibenclamide (ND-GB) diabetics treatedwith 500mgkg of aqueous extract of V rufa (DB-AEV)and diabetics treated with 6mgkg of glibenclamide (DB-GB) Body weight was measured at the beginning and endof the study and the animals had free access to food andwater On day 43 of the treatment the rats were anesthetizedwith xylazineketamine (1 1 vv) Livers were then removedrinsed in saline solution to remove blood weighed andimmediately frozen and stored at minus80∘C for assays (119899 = 6)Livers were also fixed in buffered formaldehyde (10) forhistological analysis (119899 = 4)

26 Liver Homogenization for Evaluation of Oxidative StressLiver samples (1 g) were homogenized on ice in four volumesof homogenization buffer (20mM Tris-HCl pH 74 2mMdithiothreitol 1mM benzamidine 05mM phenylmethane-sulfonyl fluoride 05mM aprotinin and 01mM pefabloc)Next the homogenate was centrifuged at 800timesg for 10minat 4∘C and protein concentrations were thenmeasured by theBradford method [20]



CAT120583gseg

GPxmmolminmL

GSHmM


SODUmg protein

MDAnmolmgprotein



43 plusmn 05lowasta

48 plusmn 03lowasta


14 plusmn 01




27 plusmn 03lowasta

848 plusmn 51a

13 plusmn 02lowasta

19 plusmn 01lowasta


37 plusmn 02lowastb





16 plusmn 02









3 Results






Initial BW(g)


()




a


lowasta40 plusmn 01

lowasta





CAT120583gseg

GPxmmolminmL

SODUmgprotein



18 plusmn 032




51 plusmn 03a



39 plusmn 03Cb

128 plusmn 008b


16 plusmn 01Cb

09 plusmn 009







bc086

bc074

abc181

d





4 Discussion




ND

CVCon

trol

(a)

DB

CV

(b)

CV

AEV

(c)

CV

(d)

CV

GB

(e)

CV

(f)







2O andO




2








5 Conclusion






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CAT120583gseg

GPxmmolminmL

GSHmM


SODUmg protein

MDAnmolmgprotein



43 plusmn 05lowasta

48 plusmn 03lowasta


14 plusmn 01




27 plusmn 03lowasta

848 plusmn 51a

13 plusmn 02lowasta

19 plusmn 01lowasta


37 plusmn 02lowastb





16 plusmn 02









3 Results






Initial BW(g)


()




a


lowasta40 plusmn 01

lowasta





CAT120583gseg

GPxmmolminmL

SODUmgprotein



18 plusmn 032




51 plusmn 03a



39 plusmn 03Cb

128 plusmn 008b


16 plusmn 01Cb

09 plusmn 009







bc086

bc074

abc181

d





4 Discussion




ND

CVCon

trol

(a)

DB

CV

(b)

CV

AEV

(c)

CV

(d)

CV

GB

(e)

CV

(f)







2O andO




2








5 Conclusion






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Initial BW(g)


()




a


lowasta40 plusmn 01

lowasta





CAT120583gseg

GPxmmolminmL

SODUmgprotein



18 plusmn 032




51 plusmn 03a



39 plusmn 03Cb

128 plusmn 008b


16 plusmn 01Cb

09 plusmn 009







bc086

bc074

abc181

d





4 Discussion




ND

CVCon

trol

(a)

DB

CV

(b)

CV

AEV

(c)

CV

(d)

CV

GB

(e)

CV

(f)







2O andO




2








5 Conclusion






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ND

CVCon

trol

(a)

DB

CV

(b)

CV

AEV

(c)

CV

(d)

CV

GB

(e)

CV

(f)







2O andO




2








5 Conclusion






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2O andO




2








5 Conclusion






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article polyploidy analysis and attenuation of...

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