researcharticle withania coagulans fruit extract reduces

Research ArticleWithania coagulans Fruit Extract ReducesOxidative Stress and Inflammation in Kidneys ofStreptozotocin-Induced Diabetic Rats

Shreesh Ojha1 Juma Alkaabi2 Naheed Amir1 Azimullah Sheikh1 Ahmad Agil3

Mohamed Abdelmonem Fahim4 and Abdu Adem1

1 Department of Pharmacology andTherapeutics College of Medicine and Health Sciences United Arab Emirates UniversityPO Box 17666 Al Ain UAE

2Department of Internal Medicine College of Medicine and Health Sciences United Arab Emirates UniversityPO Box 17666 Al Ain UAE

3Department of Pharmacology and Neurosciences Institute School of Medicine University of Granada 18012 Granada Spain4Department of Physiology College of Medicine and Health Sciences United Arab Emirates University PO Box 17666 Al Ain UAE

Correspondence should be addressed to Abdu Adem abduademuaeuacae

Received 4 April 2014 Revised 18 July 2014 Accepted 20 July 2014 Published 14 September 2014

Academic Editor Kota V Ramana

Copyright copy 2014 Shreesh Ojha et al This 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

The present studywas carried out to investigate the changes in oxidative and inflammatory status in streptozotocin-induced diabeticratrsquos kidneys and serum following treatment with Withania coagulans a popular herb of ethnomedicinal significance The keymarkers of oxidative stress and inflammation such as inflammatory cytokines (IL-1120573 IL-6 and TNF-120572) and immunoregulatorycytokines (IL-4 and IFN-120574) were increased in kidneys along with significant hyperglycemia However treatment of four-monthdiabetic rats with Withania coagulans (10mgkg) for 3 weeks significantly attenuated hyperglycemia and reduced the levels ofproinflammatory cytokines in kidneys In additionWithania coagulans treatment restored the glutathione levels and inhibited lipidperoxidation alongwithmarked reduction in kidney hypertrophyThe present study demonstrates thatWithania coagulans correctshyperglycemia and maintained antioxidant status and reduced the proinflammatory markers in kidneys which may subsequentlyreduce the development and progression of renal injury in diabetesThe results of the present study are encouraging for its potentialuse to delay the onset and progression of diabetic renal complications However the translation of therapeutic efficacy in humansrequires further studies

1 Introduction

Diabetes a rising epidemic throughout the world has nosigns of abatement and remains one of the most challeng-ing health problems People with diabetes suffer from thedetrimental vascular which accounts for high morbidityand mortality [1] Among several vascular complicationschronic renal failure and end stage renal diseases appearfirst and often associated with metabolic and hemodynamicalternations The development and progression of diabetesand associated vascular complications are largely precipitatedby chronic hyperglycemia-induced oxidative stress [2] Inaddition to oxidative stress immune-mediated low grade

chronic inflammatory mechanisms have been demonstratedto play a significant role in pathogenesis of renal injuryin long term diabetes [3] Convincing number of studiesdemonstrates that oxidative stress and immune inflammatoryprocesses intimately linked together causing renal damagethrough multiple mechanisms [3ndash6]

Themanagement of diabetic renal complications is basedon the approaches to delay the development and progres-sion by keeping strict control of blood pressure or plasmaglucose [7] However controlling the blood pressure andplasma glucose levels to prevent the renal complicationsis far from satisfactory [7] This imperfection points tothe need for newer therapeutic agents that have potential

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 201436 9 pageshttpdxdoiorg1011552014201436

2 Oxidative Medicine and Cellular Longevity

to target these intimately linked cascade oxidative stress-inflammatory cytokine signaling and delay the progressionand development of renal complications in diabetes [7]Therefore in search of newer therapeutic agents medicinalplants considered as a major source of drug discovery fromnatural origin have been extensively explored [1]

Subsequently many plant-derived natural products havethe potential to be effective in diabetic renal complicationsby attenuating oxidative stress and proinflammatory andimmunoregulatory cytokines [8ndash10] The challenge is toidentify the most promising compounds and evaluate theirprotective mechanism The fruits of Withania coagulansbelonging tofamily Solanaceae have received considerableattention for their benefits in chronic degenerative diseasesincluding diabetesTheplantWithania coagulans commonlyknown as Indian Rennet vegetable rennet (English) Panirdodi (Hindi) and Ning gu shui qie (Chinese) has beenreported to possess a variety of ethnomedicinal uses [11]The extract has shown potential activities namely anticancer[12] woundhealing [13] immunomodulating [14] antihyper-glycemic [15] and hypolipidemic [16] activities

Despite several reports of its benefits in diabetes [1115 17ndash19] and considering its potential to target the com-plex interplay of oxidative stress and inflammatory andimmunoregulatory cytokines in diabetic renal complicationit is worthwhile to study the effect of Withania coagulans inkidneys of streptozotocin- (STZ-) induced diabetes In orderto understand the mechanism the present study examinedthe effect ofWithania coagulans on antioxidant defense lipidperoxidation and immunoregulatory and proinflammatorycytokines

2 Material and Methods

21 Chemicals STZ sodium citrate citric acid bovineserum albumin 5-sulfosalicylic acid (SSA) naphthalenediamine dihydrochloride sulphanilamide phosphoric acidHEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) sucrose 14-dithiothreitol (DTT) CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonatesodium chloride protease inhibitors phenylmethylsulfonylfluoride (PMSF) tween 20 sodium nitrate 33551015840-Te-tramethylbenzidine (TMB) glutathione (GSH) assay kitand all other required chemicals if not specified werepurchased from Sigma-Aldrich (Sigma Chemical Co StLouis MO USA) All chemicals used in the present studywere of analytical grade Malondialdehyde (MDA) assaykit was purchased from Northwest Life Science Specialties(WA USA) Cytokines duo set ELISA kits were purchasedfrom RampD Systems (Minneapolis MN USA) EnzChekmyeloperoxidase (MPO) activity assay kit was purchasedfrom Life Technologies (NY USA)

22 Animals andDiet MaleWistar rats (230 to 250 g) bred inthe animal research facility of College ofMedicine andHealthSciencesUnitedArabEmiratesUniversity AlAinUAEwereused The animals were housed under standard laboratoryconditions (22 plusmn 2∘C and 65 plusmn 5 humidity) and maintained

on a 12-hour lightdark cycle The animals had free accessto food and water and were fed commercially availablestandard rat diet A maximum of four rats were housed percage and acclimatized to the laboratory conditions prior tothe commencement of the experiment The experimentalprotocols were approved by the Institutional Animal EthicsCommittee of College of Medicine and Health Sciences(IAEC CMHS) United Arab Emirates University Al AinUAE and conducted according to the criteria outlined inthe guide for the care and use of laboratory animals by theNational Academy of Sciences

23 Preparation of the Withania coagulans Aqueous FruitExtract A standard protocol was followed for the extractionof Withania coagulans The fruits of Withania coagulans(028 g100mL) were soaked in distilled water overnightfollowed by a mechanical dispersion using a sterile cottonwood (Hardwood Products Company Guilford CT USA)and filtration through cheese clothThe dose of 10mgkg wasselected based on a dose response pilot study in our labora-tory A total of five doses (0 10 125 625 and 1250mgkg)were screened to find out the optimal dose following a doseresponse curve in a postprandial glucose test based doseresponse study Five groups of six STZ diabetic rats eachwere fasted overnight and used in the experiment GroupI served as diabetic control and received vehicle (distilledwater only) Rats of groups II III IV and V received dosesof 10 125 625 and 1250mgkg respectively of aqueous fruitextract suspended in distilled water The level of baselineblood glucose was measured at 0 hr followed by an oraladministration of either distilled water (diabetic controlgroup) or Withania coagulans extract The rats were allowedto have free access to food and waterThe blood samples werecollected from tail vein at 1 2 3 and 4 hrs after giving theextract using anACCU-CHEKperforma glucometer Amongthe doses studied the dose of 10mgkg was found mostpotent in exhibiting the antihyperglycemic activity (resultsnot shown) For further experiments the dose of 10mgkgwas chosen and a detailed study was performed

24 Induction of Experimental Diabetes in Rats A single doseof 60mgkg STZ was dissolved in freshly prepared citratebuffer (pH 45 01M) and injected intraperitoneally to inducediabetes The age matched control rats received an equalamount of citrate buffer andwere used alongwith the diabetescontrol group Diabetes was confirmed by using Accucheckperforma glucometer (Roche Diagnostics NSW Australia)after 48 hours of STZ injection The rats having plasmaglucose levels ofgt350mgdLwere considered as diabetics andwere used in the present study The rats injected with STZprovide a relatively inexpensive and easily accessible rodentmodel that is not extremely obese and simulates the naturalhistory andmetabolic characteristics of patients with diabetesmellitus [20]

25 Experimental Design The rats were divided into threeexperimental groups each consisting of six rats Group 1served as nondiabetic controls group The group 2 and 3

Oxidative Medicine and Cellular Longevity 3

Male Wistar rats

STZ(+)

STZ(+) Treatment for 21 days

- Sacrifice- Organ

collection- Analysis

no ip injection

STZ(minus)

Diabetic controlvehicle oral gavage

daily (n = 6)

Diabetic treated

aqueous extract oral gavage daily (n = 6)

Withania coagulans

Nondiabetic group

60mgkg ip

60mgkg ip

no treatment (n = 6)

Figure 1 Schematic diagram of the experimental groups and treatment protocol

rats were four-month diabetic at the start of the experi-ment Group 2 served as STZ-induced diabetic group group3 served as diabetic group treated orally with Withaniacoagulans (10mgkgday bw for 3 weeks) The schematicrepresentation of the experimental groups and treatmentprocedure are presented in Figure 1 During the experimentalperiod the body weight and blood glucose were determinedat regular intervals The blood glucose level was measuredbefore treatment and after the 3-week treatment over a periodof 4 h At the end of the experimental period rats were euth-anized and the kidneys were removed and processed for theestimation of reduced glutathione (GSH) malondialdehyde(MDA) nitric oxide (NO) and cytokines (IL-1120573 IL-4 IL-6TNF-120572 and IFN-120574) using the specific kits

26 Preparation of Kidney Tissue Homogenate The kidneyswere removed weighed washed in ice-cold PBS and mincedinto 2ndash5mm fragments followed by homogenization using apolytron homogenizer (IKA Laboratory Germany) with 5volumes of ice-cold buffer containing 100mM HEPES pH75 10 sucrose 10mM DTT 01 CHAPS 150mM NaClprotease inhibitors tablet and 1mM PMSF The sampleswere centrifuged at 10000timesg for 10min and the obtainedsupernatant was removed and stored at minus80∘C until theassessment of MPO activity and cytokines using ELISA kits

27 Determination of Oxidative Stress Markers The levelsof GSH and MDA were determined using commerciallyavailable kits in serum and kidney The level of NO wasmeasured only in kidney tissues

28 Estimation of Reduced Glutathione (GSH) The GSHcontent in serum and kidney homogenate was estimatedfollowing manufacturer protocol of the assay kit Brieflythe measurement of GSH uses a kinetic assay in whichcatalytic amounts (nmoles) of GSH cause a continuousreduction of 55-dithiobis (2-nitrobenzoic acid) to nitroben-zoic acid (TNB) and the glutathione disulfide (GSSG)formed was recycled by glutathione reductase and NADPHThe yellow color product 5-thio-2-TNB was measuredspectrophotometrically at 412 within 5min of 55-dithio-bis(2-nitrobenzoic acid) addition against a blank with nohomogenate GSH concentration was expressed as 120583M ofGSH per milligram of tissue or per 001mL of serum

29 Estimation of Malondialdehyde (MDA) The lipid perox-idation product MDA in the kidney homogenate from eachgroup was measured using the MDA assay kit Briefly theassay is based on the reaction of MDA with thiobarbituricacid (TBA) to form aMDA-TBA adduct that absorbs stronglyat 532 nm Briefly the deproteinated tissue sample wasadded to 1M phosphoric acid and butylated hydroxytoluenein ethanol and then the mixture was heated at 60∘C for60minThe suspension was cooled to room temperature andcentrifuged at 10000timesg for 2-3min and the pink coloredsupernatant was taken for spectroscopic measurements at532 nm for the assay of MDA The concentration of MDAwas expressed as 120583M per 10 milligram of tissue or per 01mLserum

210 Assay of Myeloperoxidase (MPO) Activity The chlorina-tion assay forMPO activity in serum and kidney homogenate(ngmg tissuewet weight) was performed in amicrotiter plateusing the EnzChekMPOactivity assay kit Briefly 50120583Lof 2times31015840-(p-aminophenyl) fluorescein working solution was addedto 50120583L of sample The reaction mixture was then incubatedin the dark at 37∘C for 20min The fluorescence intensity ofeach sample was recorded at 485 nm excitation and 530 nmemission on a Perkin Elmer luminescence spectrofluorome-ter

211 Estimation of Nitric Oxide (NO) Accumulation ofnitric oxide was used to determine the production of NOaccording to the Griess reagent (02 naphthylene diaminedihydrochloride and 2 sulphanilamide in 5 phosphoricacid) method Briefly 100 120583L of sample was mixed withan equal volume of Griess reagent and incubated at roomtemperature for 10ndash15min The absorbance at 492 nm wasmeasured in an automated microplate reader (Tecan GroupLimited Mannedorf Switzerland) The nitrite concentrationwas quantitated using NaNO

2as standard and was expressed

as micromolar concentrations of NO per mg tissue

212 Determination of Proinflammatory Cytokines in KidneyEnzyme immunoassay of IL-1120573 IL-4 IL-6 TNF-120572 and IFN-120574 in kidney homogenate was performed by using commer-cial sandwich RampD duoset ELISA kit (Minneapolis USA)Briefly the wells of a 96-well microtiter plate were coatedwith respective primary antibody in phosphate buffer saline


Table 1 Effect ofWithania coagulans on weight changes of body and kidney to body weight ratio Twenty-one-day treatment withWithaniacoagulans extract caused a significant improvement in the body weight and kidney to body weight ratio compared to diabetic controls

Groups Body weight (gms) Kidney weight body weightBefore treatment During treatment

Nondiabetic controls 368166 plusmn 1720 41933 plusmn 22lowastlowastlowast 00029 plusmn 000012Diabetic controls 2664 plusmn 561 2592 plusmn 539lowastlowastlowast 000469 plusmn 000017lowastlowastlowast

W coagulans treated 26957 plusmn 709 292 plusmn 1249lowastlowast 000408 plusmn 0000084lowastlowastlowast

Results are means plusmn SEM 119899 = 6 rats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 from nondiabetic controls 119875 005 from diabetic controls

(PBS) (100 120583Lwell) overnight at room temperature washedwith phosphate-buffered saline containing 005 Tween-20(PBST) and then blocked with 1 bovine serum albuminin PBS for one hour After washing plates were incubatedwith serum kidney homogenates and respective standardsfor 2 hours After washing with PBST a detection antibodywas added for 2 hours and 100 120583L of HRP was added forhalf an hour after the washing The TMB-ELISA substratewas added and the color intensity read at 450 nm with amicroplate reader (Tecan Group Ltd Mannedorf Switzer-land) Cytokines levels were expressed as pg per milligram oftissue wet weight and per mL of serum

213 Statistical Analysis Data was analyzed statistically usingSPSS 190 software The means of the data are presented withthe standard error mean (SEM) The results were analyzedusing one-way ANOVA to determine the significance of themean between the groupsValues of119875 lt 005were consideredsignificant

3 Results

31 Effect of Withania coagulans on Body Weight and Kidneyto Body Weight Ratio Table 1 shows the changes in bodyweight and the ratio of kidneybody weight in differentexperimental groups There was a significant (119875 lt 0001)decrease in the body weight of rats administered STZ incomparison with rats of nondiabetic control group Diabeticrats treated with Withania coagulans show a significant(119875 lt 005) improvement in body weight when comparedto diabetic control rats Ratio of kidneybody weight is anindex of renal hypertrophy and a significant (119875 lt 0001)increase in kidneybody weight indicates renal injury inSTZ administered rats However treatment with Withaniacoagulans to the diabetic rats has significantly (119875 lt 005)reduced renal hypertrophy as evidenced by reduction ofkidneybody weight when compared to the diabetic control

32 Effect of Withania coagulans on Blood Glucose BUN andCreatinine The changes in the level of blood glucose andserum insulin in the rats of different experimental groupsare represented in Figure 2 A significant (119875 lt 0001)and persistent rise in plasma glucose level was observed inSTZ administered rats as compared with nondiabetic controlgroup However a significant (119875 lt 0001) reduction wasobserved in the plasma glucose level of diabetic rats treatedwithWithania coagulanswhen compared to diabetic controls

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

Before treatmentAfter 3-week treatment

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowast

treatedW coagulans

leve

l (m

gdL

)Ra

ndom

blo

od g

luco

se

Figure 2 Effect of Withania coagulans on blood glucose level Thediabetic treated rats showed significant decrease in blood glucoselevels compared to diabetic controls Results aremeansplusmn SEM 119899 = 6rats lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 fromnondiabetic controls119875 lt 001 from diabetic controls

The BUN and creatinine levels were not different between thedifferent groups (results not shown)

33 Effect of Withania coagulans on Glutathione Animalsadministered STZ showed a significant (119875 lt 005) decreasein the serum GSH level when compared to the nondiabeticcontrol group (Figure 3(a)) However no significant changein kidney GSH level was observed in diabetic rats whencompared to the nondiabetic control group Treatment withWithania coagulans extract significantly (119875 lt 005) inducedthe level of GSH both in serum and in kidney of diabetic ratswhen compared to diabetic control group (Figure 3(a))

34 Effect of Withania coagulans Lipid Peroxidation The ratsadministered STZ showed a significant increase in the MDAlevels of serum (119875 lt 005) and kidney (119875 lt 0001) ascompared to the nondiabetic control group (Figure 3(b))However treatment withWithania coagulans has not reducedthe level ofMDA in serum and showed a slight nonsignificantdecrease in the kidney compared to diabetic control group(Figure 3(b))

35 Effect of Withania coagulans on MPO Activity Amodestbut insignificant increase in MPO levels in kidney of thediabetic control group was observed when compared to non-diabetic control group (Figure 3(c))However treatmentwith


0

50

100

150

200

250

300

350

Serum Kidney

Nondiabetic controlDiabetic control

lowast

lowastlowastlowast

W coagulans treated

GSH

(120583M

)

(a)

00

10

20

30

40

50

60

70

Serum Kidney

lowastlowastlowast

lowastlowastlowastlowast

lowast


W coagulans treated

Mal

ondi

alde

hyde

(120583M

)

(b)

0

10

20

30

40

50

60

Nondiabeticcontrol

Diabeticcontrol

Kidn

ey M

PO (n

gm

g)

W coagulans treated

(c)

Figure 3 Effect ofWithania coagulans on serum and kidney levels of (a) GSH (b) MDA and (c) MPO Results are means plusmn SEM 119899 = 6 ratslowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 from nondiabetic controls 119875 005 from diabetic controls

0

5

10

15

20

25

30

35

40

45

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

NO

(120583M

)

Figure 4 Effect of Withania coagulans on levels of NO in kidneyResults are means plusmn SEM 119899 = 6 rats 119875 005 from diabetic controls

Withania coagulanswas found todecrease MPO levels in kid-ney as compared to the diabetic control group (Figure 3(c))The decrease in MPO levels was not significant in any group

36 Effect of Withania coagulans on Nitric Oxide A modestnonsignificant decrease in NO levels in kidney of the diabeticcontrol group was observed when compared to nondiabeticcontrol group (Figure 4) However treatment withWithaniacoagulans has significantly (119875 lt 005) increased theNO levelsin kidney as compared to the diabetic control (Figure 4)

37 Effect of Withania coagulans on Proinflammatory Cytoki-nes Figures 5(a)ndash5(c) represent the levels of kidney proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 of

different experimental groups nondiabetic control diabeticcontrol and Withania coagulans treated There was a signif-icant increase in the level of IL-1120573 (119875 lt 0001) IL-6 (119875 lt0001) and TNF-120572 (119875 lt 005) in kidneys of STZ-induceddiabetic rats when compared to nondiabetic control group Asignificant decline in the kidney levels of IL-1120573 (119875 lt 005)IL-6 (119875 lt 005) and TNF-120572 (119875 lt 001) was observedon treatment with Withania coagulans when compared todiabetic control

38 Effect of Withania coagulans on ImmunoregulatoryCytokines The levels of IL-4 and IFN-120574 in kidneys of differ-ent experimental groups are presented in Figure 6 Thoughthe change in IFN-120574 levels was not altered significantly a sig-nificant (119875 lt 005) increase in the IL-4 level was observed inSTZ-induced diabetic rats when compared to the nondiabeticcontrol group However treatment with Withania coagulansextract has significantly reduced the levels of IL-4 (119875 lt 005)and IFN-120574 (119875 lt 001) in kidneys as compared to diabetic rats

4 Discussion

In the present study STZ-injected rats show significantrise in plasma glucose level along with decrease in seruminsulin and body weight and increase in kidney weight incomparison with nondiabetic control rats indicating thedevelopment of diabetes as characterized by chronic andpersistently elevated plasma glucose level Decreased bodyweight in STZ-induced diabetic rats is believed to be due


0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg

mg) lowastlowastlowast

lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)

Figure 5 Effect ofWithania coagulans on kidney levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 Diabetic controls showed significantly elevatedkidney IL-1120573 (a) IL-6 (b) and TNF-120572 (c) cytokines levels compared to nondiabetic controls Withania coagulans treatment significantlydecreased the IL-1120573 (a) IL-6 (b) and TNF-120572 (c) compared to diabetic controls Results are means plusmn SEM 119899 = 6 rats lowast119875 lt 005 lowastlowastlowast119875 lt 0001from nondiabetic controls 119875 005 119875 lt 001 from diabetic controls

0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)

Figure 6 Effect ofWithania coagulans on kidney levels of (a) IL-4 and (b) IFN-120574Withania coagulans treatment significantly decreased thekidney IL-4 and IFN-120574 compared to diabetic controls Results are means plusmn SEM 119899 = 6 rats lowast119875 lt 005 lowastlowastlowast119875 lt 0001 from nondiabeticcontrols 119875 005 119875 lt 001 from diabetic controls

to dehydration breakdown and catabolism of fats and pro-teins Increased catabolic reactions after STZ administrationleads to muscle wasting and decreased body weight STZinduces diabetes by selectively destroying insulin producingpancreatic endocrine cells and damages kidney similar toearly stage diabetic nephropathy [20 21]This is in agreementwith various other observations that STZ-induced animals

exhibit diabetic renal complications [8 9 22] However treat-ment with Withania coagulans restored body weight kidneyweight and reduced hyperglycemia as well as enhancingsurvival and general body growth of diabetic rats Ratio ofkidneybody weight is an index of renal hypertrophy anda significant increase in kidneybody weight indicates renalinjury in STZ administered rats However treatment with


Withania coagulans to the diabetic rats has markedly reducedrenal hypertrophy as evidenced by reduction of kidneybodyweight when compared to the diabetic control These resultsdemonstrate that the extract of Withania coagulans exhibitsantihyperglycemic effects through modulation of insulin andrelated enzyme activities in consonance with other studiesdemonstrated antihyperglycemic as well as protective effectin other organs apart from kidneys [15ndash17]

Pathogenic mechanisms underlying the progressive renaldiseases in diabetics are known to be multifactorial includingoxidative stress inflammation and immune-dysfunction [56] Oxidative stress ultimately triggers inflammation andmodulates immunologic cascade in progression of renaldamage from genesis to progression [2 3] Hyperglycemia-induced oxidative stress and inflammation unleash a cascadeof events that affect cellular proteins gene expression and cellsurface receptor expression ultimately resulting in progres-sive pathologic changes in diabetic kidneys [4] To counteractoxidative stress the first line of defense against reactiveoxygen species (ROS) is GSH an intracellular nonproteinthiols compound which also participate in second lineof defense as a substrate or cofactor for GSH-dependentenzymes to detoxify ROS generated toxic byproducts andprevent propagation of free radicals [23] In the presentstudy decreased levels of GSH in serum of STZ-injectedrats might be explained by depletion or consumption ofGSH in removing the hyperglycemia generated peroxidesFollowing treatment with Withania coagulansthe improve-ment in GSH level demonstrates its antioxidant activity inagreement with other studies whereWithania coagulans wasshown to ameliorate oxidative stress [15ndash17] Although nosignificant change in renal GSH levels was observed in theSTZ administered rats a significant rise in kidney GSH levelswas obtained following treatment with Withania coagulansindicating increased production of GSH

Furthermore ROS by impairing antioxidant defenserenders the kidneys more susceptible to lipid peroxidationROS induced lipid peroxidation is a marker of cellular oxida-tive damage and is an important pathogenic event in renalinjury [24] In our study increased level of lipid peroxidationproduct MDA clearly indicates oxidative stress in diabetickidneys Following treatment with Withania coagulanstheinhibition of lipid peroxidation as evidenced by decreasedalbeit not significant MDA levels in kidney demonstratesthe antioxidant effect of Withania coagulans in agreementwith previous studies which showed its antilipid peroxidationactivity [15ndash17] In addition to reduction of hyperglycemiathe ability of Withania coagulans to prevent GSH depletionand lipid peroxidation seems to be advantageous to mitigatethe oxidative stress and may delay the development andprogression of renal complications in diabetes

In addition change in MPO activity has been demon-strated to play role in degenerative and immunologic changesof the kidney [25] In this study we did not observe asignificant change in MPO activity Changes in renal NOlevels have been linked to the pathogenesis of diabetes andassociated complications [26] The complex oxidative milieuin diabetes triggers several pathophysiologic mechanismsthat simultaneously stimulate or suppress NO production at

a given stage of the disease Many studies demonstrated thatdecrease in renal NO levels are partly results of enhancedoxidative stress and partly of decreased NOS expression [27]However treatment of diabetic rats withWithania coagulanssignificantly increased NO levels in the kidneys This effect issupported by the reduction of oxidative stress and could beascribed to the induction of NOS following a counterbalanceof NOS activity under the oxidative burst in accordance withprevious other studies [21 24]

Recent studies have shown that long-term innateimmune system activation resulting in chronic low gradeinflammation is associated with the risk of developing renalcomplications implying that immunologic and inflammatorymechanisms play a significant role in disease developmentand progression [4ndash6] Studies suggest that proinflammatorycytokines (IL-1120573 IL-6 and TNF-120572) and IFN-120574 (Th1) andIL-4 (Th2) act as pleiotropic polypeptides that are inde-pendently associated and exert an important diversity ofactions in diabetic kidneys from development to progression[2 6] Both infiltrating immune cells (mainly monocytes andmacrophages) and renal resident cells (endothelial mesan-gial dendritic and epithelial) produce proinflammatorycytokines such as IL-1120573 IL-6 and TNF-120572 [28] The releaseof these cytokines may lead to renal injury through severalmechanisms [6] Being chemotactic in nature the producedchemokines recruit more inflammatory cells and activatefibroblasts and matrix production therefore inducing thedevelopment of diabetic renal complications [2 6] FurtherIFN-120574 secreted by activated T cells and NK cells in conjunc-tion with proinflammatory cytokines activates macrophagesand stimulates chemokine production which result in patho-logical lesions of diabetic renal diseases Increased IL-1120573 inkidney is known to increase the subsequent expression ofchemotactic factors and adhesion whereas increased IL-6levels are known to alter endothelial permeability induceproliferation and increase fibronectin expression [6 9] Inthe present study a significant increase in cytokine levelsIL-1120573 IL-4 IL-6 and TNF-120572 in kidneys of rats injectedSTZ are in agreement with previous studies [21] Followingtreatment with Withania coagulans significant reduction inthe level of these cytokines is clearly suggestive of its anti-inflammatory effect in diabetic kidney Thus the attenuationof proinflammatory cytokines and lipid peroxidation alongwith diminution of hyperglycemia and improved antioxi-dants by Withania coagulans treatment is clearly suggestiveof its beneficial effects in diabetic kidney

Recent evidences in alternative medicine have encour-aged that whole herb formulation is an effective therapeu-tic modality in chronic diseases including diabetes due totheir multitudes of synergistic bioactivities and nutritionalproperties [29] The current concept has revealed a new classof agents known as adaptogens which increase resistanceof the organism to aversive stimuli threatening to perturbinternal homeostasis The adaptogens have the potential toreverse stress induced immunity deregulation and organdysfunction by sparing the antioxidants and modulating theimmune system [29] The immunoregulatory cytokines playan essential role in downmodulating adaptive and innateimmune responses leading to chronic inflammation [4]


Several studies have demonstrated the adaptogenic activityofWithania species by inducing immune-surveillance [14] Inthe present study the decreased levels of immunoregulatorycytokines IL-4 and IFN-120574 are strongly suggestive of theimmunomodulatory and associated adaptogenic potential ofWithania coagulans in consonance with therapeutic benefitsof adaptogenic medicines in chronic diseases [30] Withaniadescribed in Indian Ayurvedic medicine as Rasayana drugsis believed to produce its positive health impact throughimmune-enhancing longevity promotion and molecularnutritive effect [29]

Based on the present study findings and supportivedata from ethnomedicinal clinical and preclinical studies[11 15 17ndash19] Withania coagulans holds promise for itspotential in delaying the progression of renal complicationsin diabetes Being a natural agent and due to its time testeduse since ancient time is supportive of its relative safety Thisis encouraging for Withania coagulans to be used in pre-vention and treatment of preventing renal complications indiabetes Coupledwithmultiple pharmacological effects suchas antihypertensive hypolipidemic hypoglycemic immuno-suppressive antioxidant anti-inflammatory and adaptogenicactivity Withania coagulans might be a good therapeuticagent against renal complications of diabetes which involvesmultifactorial aetiopathogenesis

To conclude the results of our study demonstrate thattreatment with Withania coagulans reduces the occurrenceof oxidative stress and inflammation and improves hyper-glycemia owing to its synergistic and polypharmacologicalproperties Further studies are encouraged for the transla-tional application in humans

Conflict of Interests

There are no patents products in development or marketedproducts to declare This study was supported by grants fromCollege of Medicine amp Health Sciences UAE UniversityUAEThe funders had no role in study design data collectionand analysis decision to publish or preparation of thepaper

Authorsrsquo Contribution

Authors who contributed significantly read and approvedthe paper are Shreesh Ojha Juma Alkaabi Naheed AmirAzimullah Sheikh Ahmad Agil Mohamed AbdelmonemFahim and Abdu Adem Authors who conceived anddesigned the experiments are Shreesh Ojha Juma AlkaabiAhmad Agil Mohamed Abdelmonem Fahim and AbduAdem Authors who performed the experiments are NaheedAmir and Azimullah Sheikh Authors who analyzed thedata are Shreesh Ojha Naheed Amir Juma Alkaabi Azimul-lah Sheikh and Abdu Adem Authors who contributedreagentsmaterialsanalysis tools are Juma Alkaabi AbduAdem and Ahmad Agil Authors who wrote the paper areShreesh Ojha Juma Alkaabi Naheed Amir and Abdu AdemShreesh Ojha and Juma Alkaabi contributed equally

References

[1] E A Omara A Kam A Alqahtania et al ldquoHerbal medicinesand nutraceuticals for diabetic vascular complications mecha-nisms of action and bioactive phytochemicalsrdquo Current Phar-maceutical Design vol 16 no 34 pp 3776ndash3807 2010

[2] Y S Kanwar J Wada L Sun et al ldquoDiabetic nephropathymechanisms of renal disease progressionrdquo Experimental Biologyand Medicine vol 233 no 1 pp 4ndash11 2008

[3] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[4] A A Elmarakby and J C Sullivan ldquoRelationship betweenoxidative stress and inflammatory cytokines in diabeticnephropathyrdquo Cardiovascular Therapeutics vol 30 no 1 pp49ndash59 2012

[5] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[6] C C Wu H K Sytwu and Y F Lin ldquoCytokines in diabeticnephropathyrdquo Advances in Clinical Chemistry vol 56 pp 55ndash74 2012

[7] A Y Kang S K Park S Y Park et al ldquoTherapeutictarget achievement in type 2 diabetic patients after hyper-glycemia hypertension dyslipidemia managementrdquo Diabetesand Metabolism Journal vol 35 no 3 pp 264ndash272 2011

[8] S Sen S Chen B Feng Y Wu E Lui and S ChakrabartildquoPreventive effects of NorthAmerican ginseng (Panax quinque-folium) on diabetic nephropathyrdquo Phytomedicine vol 19 no 6pp 494ndash505 2012

[9] K He X Li X Chen et al ldquoEvaluation of antidiabetic potentialof selected traditional Chinese medicines in STZ-induceddiabetic micerdquo Journal of Ethnopharmacology vol 137 no 3 pp1135ndash1142 2011

[10] K M Ramkumar P Ponmanickam S Velayuthaprabhu GArchunan and P Rajaguru ldquoProtective effect of Gymnemamontanum against renal damage in experimental diabetic ratsrdquoFood and Chemical Toxicology vol 47 no 10 pp 2516ndash25212009

[11] R Maurya A B Singh and A K Srivastava ldquoCoagulanolidea withanolide from Withania coagulans fruits and antihyper-glycemic activityrdquo Bioorganic and Medicinal Chemistry Lettersvol 18 no 24 pp 6534ndash6537 2008

[12] H Kataria RWadhwa S C Kaul and G Kaur ldquoWithania som-nifera water extract as a potential candidate for differentiationbased therapy of human neuroblastomasrdquo PLoS ONE vol 8 no1 Article ID e55316 2013

[13] S K Prasad R Kumar D K Patel and S Hemalatha ldquoWoundhealing activity of Withania coagulans in streptozotocin-induced diabetic ratsrdquo Pharmaceutical Biology vol 48 no 12pp 1397ndash1404 2010

[14] S K Bhattacharya and A V Muruganandam ldquoAdaptogenicactivity of Withania somnifera an experimental study using arat model of chronic stressrdquo Pharmacology Biochemistry andBehavior vol 75 no 3 pp 547ndash555 2003

[15] Q Hoda S Ahmad M Akhtar A K Najmi K K Pillaiand S J Ahmad ldquoAntihyperglycaemic and antihyperlipidaemiceffect of poly-constituents in aqueous and chloroform extractsof Withania coagulans Dunal in experimental type 2 diabetesmellitus in ratsrdquo Human and Experimental Toxicology vol 29no 8 pp 653ndash658 2010


[16] B Saxena ldquoAnti-hyperlipidemic activity of Withania coag-ulans in streptozotocin-induced diabetes a potent anti-atherosclerotic agentrdquo Drug Discoveries amp Therapeutics vol 4pp 334ndash340 2010

[17] K Shukla P Dikshit R Shukla and J K Gambhir ldquoTheaqueous extract of withania coagulans fruit partially reversesnicotinamidestreptozotocin-induced diabetes mellitus in ratsrdquoJournal of Medicinal Food vol 15 no 8 pp 718ndash725 2012

[18] B N Upadhyay and V Gupta ldquoA clinical study on the effectof Rishyagandha (Withania coagulans) in the management ofPrameha (Type II Diabetes Mellitus)rdquo Ayu vol 32 no 4 pp507ndash511 2011

[19] S Hemalatha A K Wahi P N Singh and J P N ChansourialdquoHypoglycemic activity of Withania coagulans Dunal in strep-tozotocin induced diabetic ratsrdquo Journal of Ethnopharmacologyvol 93 no 2-3 pp 261ndash264 2004

[20] G H Tesch and T J Allen ldquoRodent models of streptozotocin-induced diabetic nephropathy (methods in renal research)rdquoNephrology vol 12 no 3 pp 261ndash266 2007

[21] E P K Mensah-Brown E N Obineche S Galadari et alldquoStreptozotocin-induced diabetic nephropathy in rats the roleof inflammatory cytokinesrdquo Cytokine vol 31 no 3 pp 180ndash1902005

[22] K Huang W Liu T Lan et al ldquoBerberine reduces Fibronectinexpression by suppressing the S1P-S1P2 receptor pathway inexperimental diabetic nephropathy modelsrdquo PLoS ONE vol 7no 8 Article ID e43874 2012

[23] D Ozkaya M Nazıroglu A Armagan et al ldquoDietary vitaminC and E modulates oxidative stress induced-kidney and lensinjury in diabetic aged male rats through modulating glucosehomeostasis and antioxidant systemsrdquo Cell Biochemistry andFunction vol 29 pp 287ndash293 2011

[24] M Sefi H Fetoui N Soudani Y Chtourou M Makni andN Zeghal ldquoArtemisia campestris leaf extract alleviates earlydiabetic nephropathy in rats by inhibiting protein oxidation andnitric oxide end productsrdquo Pathology Research and Practice vol208 no 3 pp 157ndash162 2012

[25] E Malle T Buch and H Grone ldquoMyeloperoxidase in kidneydiseaserdquo Kidney International vol 64 no 6 pp 1956ndash19672003

[26] S Prabhakar J Starnes S Shi B Lonis and R Tran ldquoDiabeticnephropathy is associated with oxidative stress and decreasedrenal nitric oxide productionrdquo Journal of the American Societyof Nephrology vol 18 no 11 pp 2945ndash2952 2007

[27] A Erdely G Freshour D A Maddox J L Olson L Samselland C Baylis ldquoRenal disease in rats with type 2 diabetesis associated with decreased renal nitric oxide productionrdquoDiabetologia vol 47 no 10 pp 1672ndash1676 2004

[28] M M Speeckaert R Speeckaert M Laute R Vanholder andJ R Delanghe ldquoTumor necrosis factor receptors biology andtherapeutic potential in kidney diseasesrdquo American Journal ofNephrology vol 36 no 3 pp 261ndash270 2012

[29] N N Rege U M Thatte and S A Dahanukar ldquoAdaptogenicproperties of six rasayana herbs used in Ayurvedic medicinerdquoPhytotherapy Research vol 13 pp 275ndash291 1999

[30] B Singh B K Chandan andD K Gupta ldquoAdaptogenic activityof a novel withanolide-free aqueous fraction from the roots ofWithania somnifera Dun (Part II)rdquo Phytotherapy Research vol17 no 5 pp 531ndash536 2003

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


to target these intimately linked cascade oxidative stress-inflammatory cytokine signaling and delay the progressionand development of renal complications in diabetes [7]Therefore in search of newer therapeutic agents medicinalplants considered as a major source of drug discovery fromnatural origin have been extensively explored [1]

Subsequently many plant-derived natural products havethe potential to be effective in diabetic renal complicationsby attenuating oxidative stress and proinflammatory andimmunoregulatory cytokines [8ndash10] The challenge is toidentify the most promising compounds and evaluate theirprotective mechanism The fruits of Withania coagulansbelonging tofamily Solanaceae have received considerableattention for their benefits in chronic degenerative diseasesincluding diabetesTheplantWithania coagulans commonlyknown as Indian Rennet vegetable rennet (English) Panirdodi (Hindi) and Ning gu shui qie (Chinese) has beenreported to possess a variety of ethnomedicinal uses [11]The extract has shown potential activities namely anticancer[12] woundhealing [13] immunomodulating [14] antihyper-glycemic [15] and hypolipidemic [16] activities

Despite several reports of its benefits in diabetes [1115 17ndash19] and considering its potential to target the com-plex interplay of oxidative stress and inflammatory andimmunoregulatory cytokines in diabetic renal complicationit is worthwhile to study the effect of Withania coagulans inkidneys of streptozotocin- (STZ-) induced diabetes In orderto understand the mechanism the present study examinedthe effect ofWithania coagulans on antioxidant defense lipidperoxidation and immunoregulatory and proinflammatorycytokines

2 Material and Methods

21 Chemicals STZ sodium citrate citric acid bovineserum albumin 5-sulfosalicylic acid (SSA) naphthalenediamine dihydrochloride sulphanilamide phosphoric acidHEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid) sucrose 14-dithiothreitol (DTT) CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonatesodium chloride protease inhibitors phenylmethylsulfonylfluoride (PMSF) tween 20 sodium nitrate 33551015840-Te-tramethylbenzidine (TMB) glutathione (GSH) assay kitand all other required chemicals if not specified werepurchased from Sigma-Aldrich (Sigma Chemical Co StLouis MO USA) All chemicals used in the present studywere of analytical grade Malondialdehyde (MDA) assaykit was purchased from Northwest Life Science Specialties(WA USA) Cytokines duo set ELISA kits were purchasedfrom RampD Systems (Minneapolis MN USA) EnzChekmyeloperoxidase (MPO) activity assay kit was purchasedfrom Life Technologies (NY USA)

22 Animals andDiet MaleWistar rats (230 to 250 g) bred inthe animal research facility of College ofMedicine andHealthSciencesUnitedArabEmiratesUniversity AlAinUAEwereused The animals were housed under standard laboratoryconditions (22 plusmn 2∘C and 65 plusmn 5 humidity) and maintained

on a 12-hour lightdark cycle The animals had free accessto food and water and were fed commercially availablestandard rat diet A maximum of four rats were housed percage and acclimatized to the laboratory conditions prior tothe commencement of the experiment The experimentalprotocols were approved by the Institutional Animal EthicsCommittee of College of Medicine and Health Sciences(IAEC CMHS) United Arab Emirates University Al AinUAE and conducted according to the criteria outlined inthe guide for the care and use of laboratory animals by theNational Academy of Sciences

23 Preparation of the Withania coagulans Aqueous FruitExtract A standard protocol was followed for the extractionof Withania coagulans The fruits of Withania coagulans(028 g100mL) were soaked in distilled water overnightfollowed by a mechanical dispersion using a sterile cottonwood (Hardwood Products Company Guilford CT USA)and filtration through cheese clothThe dose of 10mgkg wasselected based on a dose response pilot study in our labora-tory A total of five doses (0 10 125 625 and 1250mgkg)were screened to find out the optimal dose following a doseresponse curve in a postprandial glucose test based doseresponse study Five groups of six STZ diabetic rats eachwere fasted overnight and used in the experiment GroupI served as diabetic control and received vehicle (distilledwater only) Rats of groups II III IV and V received dosesof 10 125 625 and 1250mgkg respectively of aqueous fruitextract suspended in distilled water The level of baselineblood glucose was measured at 0 hr followed by an oraladministration of either distilled water (diabetic controlgroup) or Withania coagulans extract The rats were allowedto have free access to food and waterThe blood samples werecollected from tail vein at 1 2 3 and 4 hrs after giving theextract using anACCU-CHEKperforma glucometer Amongthe doses studied the dose of 10mgkg was found mostpotent in exhibiting the antihyperglycemic activity (resultsnot shown) For further experiments the dose of 10mgkgwas chosen and a detailed study was performed

24 Induction of Experimental Diabetes in Rats A single doseof 60mgkg STZ was dissolved in freshly prepared citratebuffer (pH 45 01M) and injected intraperitoneally to inducediabetes The age matched control rats received an equalamount of citrate buffer andwere used alongwith the diabetescontrol group Diabetes was confirmed by using Accucheckperforma glucometer (Roche Diagnostics NSW Australia)after 48 hours of STZ injection The rats having plasmaglucose levels ofgt350mgdLwere considered as diabetics andwere used in the present study The rats injected with STZprovide a relatively inexpensive and easily accessible rodentmodel that is not extremely obese and simulates the naturalhistory andmetabolic characteristics of patients with diabetesmellitus [20]

25 Experimental Design The rats were divided into threeexperimental groups each consisting of six rats Group 1served as nondiabetic controls group The group 2 and 3


Male Wistar rats

STZ(+)


- Sacrifice- Organ


no ip injection

STZ(minus)


daily (n = 6)

Diabetic treated


Withania coagulans

Nondiabetic group

60mgkg ip

60mgkg ip





















3 Results



0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol


lowastlowastlowast


lowast

treatedW coagulans

leve

l (m

gdL

)Ra

ndom

blo

od g

luco

se







0

50

100

150

200

250

300

350

Serum Kidney


lowast

lowastlowastlowast

W coagulans treated

GSH

(120583M

)

(a)

00

10

20

30

40

50

60

70

Serum Kidney

lowastlowastlowast


lowast


W coagulans treated

Mal

ondi

alde

hyde

(120583M

)

(b)

0

10

20

30

40

50

60

Nondiabeticcontrol

Diabeticcontrol

Kidn

ey M

PO (n

gm

g)

W coagulans treated

(c)



0

5

10

15

20

25

30

35

40

45

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

NO

(120583M

)







4 Discussion



0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg


lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)


0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)




















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Male Wistar rats

STZ(+)


- Sacrifice- Organ


no ip injection

STZ(minus)


daily (n = 6)

Diabetic treated


Withania coagulans

Nondiabetic group

60mgkg ip

60mgkg ip





















3 Results



0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol


lowastlowastlowast


lowast

treatedW coagulans

leve

l (m

gdL

)Ra

ndom

blo

od g

luco

se







0

50

100

150

200

250

300

350

Serum Kidney


lowast

lowastlowastlowast

W coagulans treated

GSH

(120583M

)

(a)

00

10

20

30

40

50

60

70

Serum Kidney

lowastlowastlowast


lowast


W coagulans treated

Mal

ondi

alde

hyde

(120583M

)

(b)

0

10

20

30

40

50

60

Nondiabeticcontrol

Diabeticcontrol

Kidn

ey M

PO (n

gm

g)

W coagulans treated

(c)



0

5

10

15

20

25

30

35

40

45

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

NO

(120583M

)







4 Discussion



0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg


lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)


0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)




















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results



0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol


lowastlowastlowast


lowast

treatedW coagulans

leve

l (m

gdL

)Ra

ndom

blo

od g

luco

se







0

50

100

150

200

250

300

350

Serum Kidney


lowast

lowastlowastlowast

W coagulans treated

GSH

(120583M

)

(a)

00

10

20

30

40

50

60

70

Serum Kidney

lowastlowastlowast


lowast


W coagulans treated

Mal

ondi

alde

hyde

(120583M

)

(b)

0

10

20

30

40

50

60

Nondiabeticcontrol

Diabeticcontrol

Kidn

ey M

PO (n

gm

g)

W coagulans treated

(c)



0

5

10

15

20

25

30

35

40

45

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

NO

(120583M

)







4 Discussion



0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg


lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)


0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)




















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300

350

Serum Kidney


lowast

lowastlowastlowast

W coagulans treated

GSH

(120583M

)

(a)

00

10

20

30

40

50

60

70

Serum Kidney

lowastlowastlowast


lowast


W coagulans treated

Mal

ondi

alde

hyde

(120583M

)

(b)

0

10

20

30

40

50

60

Nondiabeticcontrol

Diabeticcontrol

Kidn

ey M

PO (n

gm

g)

W coagulans treated

(c)



0

5

10

15

20

25

30

35

40

45

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

NO

(120583M

)







4 Discussion



0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg


lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)


0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)




















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

500

1000

1500

2000

2500

3000

3500

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-1120573

(pg


lowastlowastlowast

(a)

0

100

200

300

400

500

600

700

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-6(p

gm

g)

lowastlowastlowast

(b)

0

50

100

150

200

250

300

350

400

450

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF-120572

(pg

mg)

lowast

(c)


0

100

200

300

400

500

600

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey IL

-4(p

gm

g)

lowast

(a)

0

50

100

150

200

250

300

350

Nondiabeticcontrol

Diabeticcontrol

W coagulans treated

Kidn

ey T

NF120574

(pg

mg)

lowastlowastlowast

(b)




















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















researcharticle withania coagulans fruit extract reduces

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