Rev. bras. farmacogn. vol.26 número5

(1)

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Flax

lignan

concentrate

attenuate

hypertension

and

abnormal

left

ventricular

contractility

via

modulation

of

endogenous

biomarkers

in

two-kidney-one-clip

(2K1C)

hypertensive

rats

Sameer

Hanmantrao

Sawant,

Subhash

Laxmanrao

Bodhankar

∗

DepartmentofPharmacology,PoonaCollegeofPharmacy,BharatiVidyapeethDeemedUniversity,Erandwane,Pune,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2December2015 Accepted10May2016 Availableonline14June2016

Keywords:

Flaxlignanconcentrate Antihypertensiveactivity 2K1Chypertension Endothelin-1 Renalangiotensin-II

a

b

s

t

r

a

c

t

ThepresentinvestigationwasdesignedtostudytheeffectofflaxlignanconcentrateobtainedfromLinum

usitatissimumL.,Linaceae,intwo-kidney,oneclip(2K1C)hypertensionmodelinWistarrats.2K1C

Gold-blattmodelratsweredividedrandomlyintosixgroups:sham,2K1Ccontrol,captopril(30mg/kg),flax

lignanconcentrate(200,400and800mg/kg).Flaxlignanconcentrateandcaptoprilwereadministered

dailyforeightconsecutiveweeks.Sham-operated,and2K1Ccontrolratsreceivedthevehicle.

Treat-mentwithflaxlignanconcentrate(400and800mg/kg)significantlyanddose-dependentlyrestoredthe

hemodynamicparameterssystolicbloodpressure,diastolicbloodpressure,meanarterialbloodpressure

andleftventricularfunctions.Theflaxlignanconcentratesignificantlyrestoredtheelevatedhepatic,

renalandcardiacmarkerenzymesintheserum.Italsorestoredtheorgansweights(kidneyandheart),

serumelectrolytelevelandhistologicalabnormalities.Furthermore,flaxlignanconcentratesignificantly

elevatedthelevelofbiochemicalmarkersthatisenzymaticantioxidantssuperoxidedismutase,

glu-tathioneanddecreasedmalondialdehydeintheheartandkidneytissues.Meanwhile,wefoundthat

plasmanitricoxideandplasmanitricoxidesynthasecontentsweresignificantlyincreasedintheflax

lignanconcentrate-treatedgroup,andplasmaendothelin-1andrenalangiotensin-IIlevelswere

signif-icantlylowerthan2K1Chypertensivegroup.Inconclusion,theantihypertensiveandantioxidanteffect

offlaxlignanconcentrateweredose-dependentandatthehighestdose(i.e.800mg/kg)similartothose

ofcaptopril(30mg/kg).Itissuggestedthatflaxlignanconcentratereducedbloodpressurebyreduction

ofrenalangiotensin-IIlevel,inhibitionofplasmaendothelin-1production,inductionofthenitricoxide,

nitricoxidesynthaseandinvivoantioxidantdefensesystem.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Theone-thirdadultpopulationoftheworldisaffectedby

hyper-tension, and it can beconsidered as one of themost common

chronicdiseasenowadays(Leeetal., 2009;Gosaviet al.,2011;

Ghoshetal.,2012;Shivakumaretal.,2014).Theworld’sadult

pop-ulationwithhypertensionislikelytoincreasefromonebillionin

2000to1.56billionby2025(Kearneyetal.,2005).

Renin–angiotensin–aldosteronesystem(RAAS)playsan

impor-tantroleinhypertension.Renalischemialeadstosecretereninby

kidneytissue whichis responsiblefor catalyzingthehydrolysis

of angiotensin-I (Ang-I) from the N-terminus of

angiotensino-gen.Ang-IisconvertedintoAng-IIbytheangiotensin-converting

enzyme (ACE). Ang-II is the primary product of RAAS, which

∗ Correspondingauthor.

E-mail:subhash.bodhankar@bharatividyapeeth.edu(S.L.Bodhankar).

leadstovasoconstrictionandhypertensionthroughbindingtothe

Ang-IIreceptorandstimulatingsynthesisofaldosterone(Kamble

et al., 2013; Badole et al., 2015). It is well known that Ang-II

alsostimulatethegenerationofsuperoxideanionradical(O2−•)

(Griendling et al., 1994; Rajagopalan et al., 1996), which

con-tributetodecreasednitricoxide(NO)bioavailabilityandimpaired

endothelium-dependentvasorelaxation(Gryglewskietal.,1986).

SyntheticACEinhibitorstherapyiscommonlyusedtodaytotreat

hypertension.However,this advanced antihypertensivetherapy

hasserioussideeffectssuchasangioedemaanddrycough(Coulter

andEdwards,1987).Therefore,theuseofanantioxidantmaybe

thepossibletherapyforthepreventionandtreatmentof

hyper-tensionwiththeestablishedantihypertensivedrug(Gosavietal.,

2011,2014;Visnagrietal.,2013).

Flaxseedorlinseed(LinumusitatissimumL.,Linaceae)isbrown

or yellowcoloredseed harvested fromtheblue flowersofflax

crop. It has been used as food in India and around theworld

for a long time. Flaxseed mainly contains omega-3 fatty acid,

http://dx.doi.org/10.1016/j.bjp.2016.05.005

(2)

␣-linolenicacid,dietaryfiberandsecoisolariciresinoldiglucoside

(SDG)lignan(Bassettetal.,2009).Flaxseedcontainstento

hun-dredtimesmorelignanthanmostotheredibleplantsseeds.Itis

reportedthatflaxseedalsocontainsotherlignanslikematairesinol,

lariciresinol, hinokinin, arctigenin, pinoresinol and demethoxy

secoisolariciresinolin smallquantitywithseveralphenolicacid

compounds(Prasadetal.,1998;Johnssonetal.,2000).Antidiabetic

(Prasadetal.,2000), antihyperlipidemic(Raygudeetal.,2012a),

cardioprotective(Zanwaretal.,2011,2013),renoprotective(Ghule

etal., 2011,2012,2015), antiatherogenic(Prasad,1997; Prasad

etal.,1998),antioxidant,anticancer,antiviral,bactericidaland

anti-inflammatory(Chenetal.,2002;Collinsetal.,2003;Kinniryetal.,

2006;Rajeshaetal.,2006;Zanwaretal.,2010)potentialsofflaxseed

havebeenalreadyreported.

Clinicalstudiesinvolvingpatientswithperipheralartery

dis-easeandhighbloodpressure(Rodriguez-Leyvaetal.,2013;Khalesi

etal.,2015)reportedthatconsumptionofflaxseedsinthedietfor

thedurationofmorethanthreemonthsloweredbloodpressure.

Theantihypertensivepotentialofflaxlignaninchronic

hyperten-siveconditionhasnotbeenwellexplainedinanimals.Theobjective

ofthepresentworkwastostudytheeffectofflaxlignan

concen-trate(FLC)intwo-kidney,one-clip(2K1C)inducedhypertension

inWistarrats.

Materialsandmethods

Experimentalprotocol

MaleWistarratsweighing(200–250g)werepurchasedfrom

NationalToxicology Centre,Pune, India.They weremaintained

at 25±1◦_C _temperature _and _45–55% _relative _humidity _under

12h light/dark cycle. The animals had access to food pellets

(manufacturedbyPranavAgroIndustriesLtd.,Sangli,India)and

wateradlibitum.Theexperimentalprotocolwasapprovedbythe

InstitutionalAnimalEthics Committee(IAEC)constitutedasper

guidelinesofCommitteeforthePurposeofControland

Supervi-sionofExperimentsonAnimal(CPCSEA),India.TheIAECapproval

numberisCPCSEA/PCL/08/2014-15.

Drugsandchemicals

Captopril,sulfanilicacid,N-(1-naphthyl)ethylenediaminewas

purchasedfromSigma–AldrichCorporation,USA.Absolute

alco-hol(manufacturedbyChangshuYangyuanChemicals,China)was

purchasedfromtherespectivevendor.Analyticalgrade hexane,

hydrochloricacid, and sodium hydroxide werepurchased from

QualigenesFineChemicalsPvt.Ltd.,Mumbai,India.

Collectionandauthenticationofplantseeds

Seeds of Linum usitatissimum L., Linaceae (flaxseeds) were

obtainedfromPunjabraoDeshmukhKrishiVidyapeeth,Collegeof

Agriculture,Nagpur,India.Aftertheauthenticationoftheseeds,

avoucherspecimenwasdepositedatourInstitute,PoonaCollege

ofPharmacy,Pune,India.Theflaxseedswerestoredinacoldroom

beforeprocessingforoilextractionatourRealWorldNutritionLab,

BharatiVidyapeethDeemedUniversity,Pune,India.

Preparationofflaxlignanconcentrate(FLC)

Preparation of FLC was carried out as described previously

(Zanwaretal.,2013).Theflaxseedcakewasdefattedbyhexane

toremove residual oil. Thedefatted cakewas thenhydrolyzed

withaqueoussodiumhydroxidefor1hatroomtemperaturewith

intermittentshakingfollowedbyextractionwith50%ethanol.The

filtratewasacidifiedtopH3using1Mhydrochloricacid.The

fil-tratewasdriedusingrotavacapparatusat50◦_C._The_dry_powder_of

hydroalcoholicextractwaslabeledasFLC.

PreparationofdrugsolutionandselectionofFLCdose

CaptoprilandFLCweredissolvedindistilledwater.Thisstudy

was carried out using three doses of FLC (i.e. 200, 400 and

800mg/kg,p.o.)andonedoseofcaptopril(i.e.30mg/kg,p.o.).

Experimentalinductionofhypertension

Wistar rats weighing 200–250g were anesthetized with

50mg/kgintraperitonealadministrationofthiopentalsodium.The

furonthebackofeach ratwasshaved,andtheskinwas

disin-fected.Aflankincisionwasmadeintheleftlumbarareaparallel

tothelongaxisoftherat.Therenal pedicelwasexposed with

thekidneyretractedtotheabdomen.Leftrenalarterywas

con-strictedtoinducetwo-kidney,one-clip hypertension(2K1C),as

previouslydescribedbyKharinandKrandycheva(2004).Briefly,

a loop of the left renal artery was pulled into a segment of

polyurethane tube [MRE 040-S20, Braintree Scientific; internal

diameter(ID)=0.50mm,length2mm].Themuscleandskinlayer

(incisionsite)weresuturedwithahighlysterilesutureneedle.After

oneweekoftherecoveryperiod,theanimalswereusedforthe

furtherexperiment.Ratsinsham-operatedgroupunderwentthe

exposureoftheleftrenalartery,butthearterywasnotconstricted.

Themuscleandskinlayer(incisionsite)weresuturedwithasterile

sutureneedle.Afteroneweekoftherecoveryperiod,theanimals

wereusedforthefurtherexperiment.

Experimentaldesign

Theratswererandomlydividedintosixgroups,eachcontaining

sixrats:

GroupI:Sham-operated(vehicledistilledwaterp.o.)

GroupII:2K1Ccontrol(vehicledistilledwaterp.o.)

GroupIII:2K1C+captopril(30mg/kgp.o.)

GroupIV:2K1C+FLC(200mg/kgp.o.)

GroupV:2K1C+FLC(400mg/kgp.o.)

GroupVI:2K1C+FLC(800mg/kgp.o.)

FLCandcaptoprilwereandadministeredtotheratsorallyusing

anoralfeedingneedledailyforeightconsecutiveweeks.The

sham-operatedand2K1Ccontrolratsreceivedvehicledistilledwater.At

theendofthestudyperiod,bloodwascollectedbyaretro-orbital

punctureforthemeasurementofbiochemicalparameters.

Assessmentofhemodynamicchanges

Each rat was anesthetized with intraperitoneal injection of

urethane(1.25g/kg).Thetracheawascannulatedtoassist

respi-ration.Thesystolicbloodpressure(SBP),diastolicbloodpressure

(DBP)and meanarterialbloodpressure (MABP)weremeasured

byinvasivetechniqueattheendoftheeighthweek.A

polyethyl-enecannula(PE50)filledwithheparinizedsaline(100IU/ml)was

insertedintotherightcarotidartery.Thecannulawasconnected

toatransducerandthesignalwasamplified.Theleft

ventricu-larhemodynamicchangesweremeasuredusingaMillarmikro-tip

transducercatheter(ModelSRP-320;MillarInstrument,Inc.

320-7051,Houston, TX 77023-5417) inserted intothe left ventricle

viatherightcarotidarteryandconnectedtoabioamplifier(Adil

etal.,2015,2016a;Visnagrietal.,2015).Maximumfirst

deriva-tiveofventricularpressure(dP/dtmax),minimumfirstderivativeof

(3)

pressure(EDP)signals wereobtainedfromprimarysignals (left

ventricularsystolicpressureandbloodpressure)bymeansof

Pow-erlab8-channeldataacquisitionsystem(ADInstrumentsPvt.Ltd.,

withLabChart7.3Prosoftware,Australia).

Samplecollectionanddeterminationofbiomarkers

Serumandplasmasamplecollection

Attheendofthestudyperiodand1hafterthetestsubstance

administration,thebloodwascollectedbyretro-orbitalpuncture

underanesthesia.Serumsampleswerecollectedwithoutadded

anticoagulant.Serumsampleswerecollectedaftercentrifugation

for10minat845×gand4◦_C._The_blood_was_collected_into

anti-coagulantcontainingtubesandimmediatelycentrifuged(10min

at845×g and4◦_C _{temperatures)}_for _plasma_sample_collection.

Theserumandplasmasampleswerestoredat−80◦_C_until_being

analyzed.

Heartandrenaltissuesamples

At the end of the experimental period, all the rats were

humanely euthanized. The heart and kidneys were removed

for furtherexperiments. The portions of the heart and clipped

renal tissues were individually homogenized in 10% cold

Tris–hydrochloridebuffer(10mmol/l,pH7.4)usingtissue

homog-enizer(Remi,India)andcentrifugedat5283×gfor15minat0◦_C.

Theclearsupernatantcollectedafterthecentrifugationwasused

forbiochemicalandmolecularestimations.

Measurementofbiologicalserummarkers

TheserumelectrolytessuchasNa+_,_K+_and_Cl−_were_estimated

usingcommerciallyavailablemeasurementkits(CoralClinical

Sys-tem,Goa,India).Creatinekinase(CK-MB),lactatedehydrogenase

(LDH),aspartateaminotransferase(AST),alanineaminotransferase

(ALT),alkalinephosphatase(ALP),totalprotein,bloodureanitrogen

(BUN),uricacidandcreatininewerealsomeasuredbyusing

com-merciallyavailablemeasurementkits(AccurexPvt.Ltd.,Mumbai,

India).

Estimationofendogenousantioxidantenzyme

Thesuperoxidedismutase(SOD)concentrationwasdetermined

bythemethodpreviouslydescribedelsewhere(Kambleetal.,2013;

Adiletal.,2014;Aswaretal.,2015;Honmoreetal.,2015).The

SODactivitywasexpressedasU/mgofprotein.Theglutathione

(GSH)assaywasperformedaccordingtothemethodpreviously

describeelsewhere(Moronet al.,1979;Kandhareet al.,2011a,

2015a;Kumaret al.,2014; Ketkaretal., 2015;Goswami etal.,

2016;Adiletal.,2016b).Theamountofreducedglutathionewas

expressedas␮g/mgofprotein.Malondialdehyde(MDA)levelinthe

kidneyandhearttissuesweremeasuredbythemethodpreviously

describedelsewhere(SlaterandSawyer,1971;Patiletal.,2011,

2015;Raygudeetal.,2012a,b;Saraswathietal.,2014;Kandhare

et al., 2016a), and thevalues wereexpressed in nanomoles of

MDA/mgofprotein.

Determinationofnitricoxide(NO),nitricoxidesynthase(NOS),

endothelin-1(ET-1),Ang-IIlevel

NOishighlyunstablefreeradical,whichisconvertedinto

sta-blemetabolitesnitrateandnitriteintheequimolarratio(Schlaich

etal.,2007;Visnagrietal.,2014;Kandhareetal.,2016a,2015b;

Sarkaretal.,2015).TheplasmaNOlevelwasdeterminedasnitrite

bytheacidicGriessreaction.Theassaywasperformedbyarapid,

simplespectrophotometricmethoddescribedelsewhere(Miranda

etal.,2001;Gosavietal.,2012a,b;Kandhareetal.,2013a,2014a).

Theprincipleofthisassayisareductionofnitratebyvanadium.

ThenitritereactswithsulfonamideandN-(1-naphthyl)

ethylenedi-aminetoproduceapinkazo-productwithmaximumabsorbanceat

543nm.Theconcentrationswerecalculatedusingastandardcurve

ofsodiumnitrateandtheresultswereexpressedin␮mol/l.ET-1,

NOS[GenxbioHealthSciencesLtd.,India]intheplasmandAng-II

level[RayBiotech,Inc.,USA]intherenaltissuehomogenatewere

measuredusingElisakitsaspertheinstructionsaregivenbythe

manufacturer.

Histopathologicalexamination

The excised heart and kidney samples were cleaned and

immediatelyfixedinneutralbuffered10%formalinsolution.The

specimenswereroutinelyprocessedandembeddedinparaffin.The

specimenswerecutinsectionsof5␮mthicknessbymicrotome

andstainedwithMasson’strichromeformicroscopicexamination.

Thesectionswereobservedunderthemicroscopeand

photomicro-graphsofthetissuesectionweretakenusingamicroscopecamera

(NikonCoolpix).Theparametersofhistopathologicalassessment

ofthekidneysectionsweremainlyperivascularedema,fibrosis,

glomerular necrosisand collagendeposition.The parametersof

histopathologicalassessmentoftheheartsectionsweremyocardial

degeneration,collagendeposition,andfibrosis.

Statisticalanalysis

The data wereexpressed as mean±standard errorof mean

(SEM)andstatisticalanalysiswascarriedoutbyone-wayANOVA

followedbyposthocDunnett’stestusingGraphPadPrism5.0

soft-ware(GraphPadSoftware,SanDiego,CA,USA).Differenceswitha

valueofp<0.05wereconsideredstatisticallysignificant.

Results

EffectofFLConhemodynamicparametersandleftventricular

contractilefunctionofheart

Fig.1presentstheeffectofthreedifferentconcentrations(200,

400and800mg/kg)onhemodynamicparameters andleft

ven-tricularcontractilefunctioninalltheI–VIgroupsafter8weeks.

Comparedtoshamoperatedgroup,theratsin2K1Ccontrolgroup

showedsignificant(p<0.001each)increase inSBP, DBP,MABP,

EDP,dP/dtmax and dP/dtmin after 8weeks. Captopril (30mg/kg)

and FLC (800mg/kg) treatment showed a significant (p<0.001

each)decreaseintheSBP,DBP,MABP,EDP,dP/dtmaxanddP/dtmin.

FLC(400mg/kg)treatmentalsoshowedsignificant(p<0.01each)

decreaseinSBP,DBP,MABP,EDP,dP/dtmaxanddP/dtmincompared

to2K1C-controlgroup.TreatmentwithFLC(200mg/kg)showed

significant(p<0.05)decreaseinSBP,anddP/dtmin;butdidnotshow

anysignificantdecreaseinDBP,MABP,EDPanddP/dtmaxvalues

(Fig.1).

EffectofFLConorgansweightandelectrolyte

The2K1C-controlhypertensive(group-II)ratsshowed

signifi-cant(p<0.001)increaseintheweightsofkidneyandheart.The

serumsodiumion(Na+₎_and_chloride_ion_(Cl−₎_levels_{significantly}

increasedwhilethatofserumpotassiumion(K+₎_level_decreased

compared to sham. The treatments with captopril (30mg/kg)

and FLC (400, 800mg/kg) reduced organs weight and restored

thoseions(sodium,chloride,andpotassium)leveltonearnormal

(Table1).

EffectofFLConserumcardiac,hepaticandrenalmarkersand

serumtotalproteinlevel

The activities of CK-MB, LDH, AST, ALT, ALP, total protein,

(4)

0

15

10

EDP (mmHg)

dp/dt

max

(mmHg/s)

dp/dt

min

(mmHg/s)

DBP(mmHg) _MABP(mmHg)

SBP (mmHg)

5

Treatment

0 0 –6000

–4000 –2000 0 0 50 100 150 200

2000 4000 6000 0 50 100 150

Sham

2k1c control_Captopr il (30)

FLC (200) FLC (400) FLC (800)

Treatment Sham

FLC (200) FLC (400) FLC (800)

Treatment Sham

FLC (200) FLC (400) FLC (800) 50

100 150

###

### ***

***

*** ***

* **

** **

**

* _**

** 200

A

B

C

F

E

D

Fig.1. EffectofadministrationofFLCandcaptoprilonhemodynamicparameters(A)SBP,(B)DBP,(C)MABP,(D)EDP,(E)dP/dtmaxand(F)dP/dtmininmodified2K1C

hypertensiveWistarrats(n=6).Dataareexpressedasmean±S.E.M.andstatisticalanalysiswascarriedoutbyone-wayANOVAfollowedbyposthocDunnett’stest; ns=non-significant,*p<0.05,**p<0.01,***p<0.001ascomparedwith2K1Ccontrolgroup-II,#_p_<_0.05,##_p_<_0.01,###_p_<_0.001_compared_to_sham_group-I.

Table1

EffectofFLC(200,400and800mg/kg)andcaptopril(30mg/kg)onorgans(heartandkidney)weightsandserumelectrolytesin2K1ChypertensiveWistarrats.

Parameter Treatment

Sham 2K1Ccontrol 2K1C+captopril

(30mg/kg)

2K1C+FLC (200mg/kg)

2K1C+FLC (400mg/kg)

2K1C+FLC (800mg/kg)

Organweight(g)

Heartweight 1.18±0.03 1.54±0.04d _1.20_±_0.02c _1.44_±_0.04ns _1.38_±_0.05b _1.27_±_0.02c

Kidneyweight 1.41±0.03 2.18±0.05d _1.53

±0.04c _2.02

±0.05ns _1.94

±0.05b _1.62

±0.04c

Electrolyte(mEq/l)

Sodium 144±1.57 183±6.29d ₁₅₂_±_3.39c ₁₇₉_±_5.45ns ₁₆₁_±_5.03b ₁₅₅_±_3.75c

Potassium 6.22±0.22 3.67±0.19d _5.73

±0.27c _3.81

±0.19ns _4.65

±0.20b _5.4

±0.15c

Chloride 104±2.12 131±4.21d ₁₁₅

±1.66c ₁₂₆

±2.5ns ₁₂₀

±2.1a ₁₁₆

±1.79b

Valuesareexpressedasmean±SEMforn=6rats.Dataareexpressedasmean±S.E.M.andstatisticalanalysiswascarriedoutbyone-wayANOVAfollowedbyposthoc

Dunnett’stest;ns=non-significant.

a_p_<_0.05_as_compared_with_2K1C_control_(group-II). b _p_<_0.01_as_compared_with_2K1C_control_(group-II). c _p_<_0.001_as_compared_with_2K1C_control_(group-II). d _p_<_0.001_compared_to_sham_group_(I).

Table2

EffectofFLC(200,400and800mg/kg)andcaptopril(30mg/kg)onserumcardiac,hepatic,renalmarkersandserumtotalproteinin2K1ChypertensiveWistarrats.

Parameter Treatment

Sham 2K1Ccontrol 2K1C+captopril (30mg/kg)

2K1C+FLC (200mg/kg)

2K1C+FLC (400mg/kg)

2K1C+FLC (800mg/kg)

Aspartateaminotransferase(AST)(IU/l) 90.6±3.21 162±5.69d ₁₀₈

±5.53c ₁₄₄

±4.09ns ₁₃₆

±4.96b ₁₂₁

±3.97c

Alanineaminotransferase(ALT)(IU/l) 37.5±2.13 75.3±1.82d _50.8

±2.75c _68.6

±2.37ns _62.5

±1.38b _59.9 ±3.05c

Alkalinephosphatase(ALP)(IU/l) 73.1±4.54 186±6.98d ₁₂₉

±13.6c ₁₆₆

±8.22ns ₁₄₀

±9.48b ₁₄₀

±10b

Totalprotein(mg/dl) 5.31±0.22 8.50±0.33d _6.01_±_0.27c _7.54_±_0.24ns _7.00_±_0.26b _5.98_±_0.29c

Bloodureanitrogen(BUN)(mg/dl) 17.80±0.70 36.10±1.81d _23.80_±_1.38c _31.10_±_1.05a _29.40_±_1.27b _27.20_±_1.05c

Uricacid(mg/dl) 1.93±0.08 3.90±0.15d _2.22_±_0.07c _3.52_±_0.10a _3.38_±_0.10b _2.98_±_0.09c

Creatinine(mg/dl) 0.77±0.07 1.83±0.04d _0.90

±0.05c _1.49

±0.12ns _1.36

±0.14b _1.12 ±0.09c

Creatininekinase(CK-MB)(IU/l) 345±13.9 563±16.6d ₄₆₄

±14.1c ₅₃₁

±11.4ns ₅₀₀

±10.6b ₄₈₄

±11.5c

Lactatedehydrogenase(LDH)(IU/l) 655±10.6 770±10.7d ₆₇₂

±8.1c ₇₃₂

±12.6a ₇₁₂

±9.72b ₆₈₀

±5.71c

(5)

Table3

EffectofFLC(200,400and800mg/kg)andcaptopril(30mg/kg)onendogenousantioxidantenzymesin2K1ChypertensiveWistarrats.

Parameter Treatment

Sham 2K1Ccontrol 2K1C+captopril

(30mg/kg)

2K1C+FLC (200mg/kg)

2K1C+FLC (400mg/kg)

2K1C+FLC (800mg/kg)

SOD(Unit/mgprotein)

Kidney 12.47±0.27 5.58±0.21d _9.16_±_0.34c _6.61_±_0.34ns _7.21_±_0.44b _8.62_±_0.32c

Heart 4.6d7±0.39 3.04±0.08d _4.25_±_0.11c _3.32_±_0.17ns _4.13_±_0.11b _4.22_±_0.08c

MDA(nmolofMDA/mgprotein)

Kidney 3.42±0.23 7.93±0.31d _4.33

±0.28c _6.95

±0.17a _6.44

±0.20b _5.45

±0.29c

Heart 4.35±0.24 8.06±0.23d _5.86_±_0.09c _7.80_±_0.16ns _6.91_±_0.13b _6.05_±_0.25c

GSH(g/mgprotein)

Kidney 8.11±0.26 4.58±0.26d _6.41

±0.21c _4.94

±0.28ns _5.66

±0.18b _5.99

±0.15c

Heart 7.76±0.36 3.91±0.24d _6.74_±_0.20c _3.86_±_0.23ns _5.14_±_0.15b _5.34_±_0.18c

a_p_<_0.05_as_compared_with_2K1C_control_(group-II). b_p_<_0.01_as_compared_with_2K1C_control_(group-II). c _p_<_0.001_as_compared_with_2K1C_control_(group-II). d _p_<_0.001_compared_to_sham_group_(I).

hypertensiverats(2K1C-controlgroup-II).Thetreatmentwith

cap-topril(30mg/kg)andFLC(200,400,800mg/kg)showedareduction

intheactivitiesofthesecardiac,hepaticandrenalmarkerstoward

near normal. Captopril (30mg/kg) showed the highest activity

than the test drug FLC (200, 400, 800mg/kg). FLC (800mg/kg)

showedsignificant(p<0.001)reductioninserumlevelofall

mark-ers,exceptinALP(p<0.01).TreatmentwithFLC(400mg/kg)also

showedsignificant(p<0.01)reductioninserumlevelofallmarkers.

However,FLCinlowdose(200mg/kg)didnotshowanysignificant

inhibitioninthelevelofserumCK-MB,LDH,AST,ALT,ALP,total

pro-tein,andcreatinine;butshowedasignificantreductioninserum

levelofBUNanduricacidcomparedtothe2K1Ccontrolanimals

(Table2).

Endogenousantioxidantenzymes

TheSOD and GSHactivityin thetissues (kidneyand heart)

of2K1Chypertensiveratsweredecreasedsignificantly(p<0.001)

after8weeks.Captopril(30mg/kg),aswellasFLC(400,800mg/kg),

restoredtheSODandGSHactivityinthetissues(kidneyandheart)

aftereight weeks. On the other hand,FLC (200mg/kg) didnot

showanysignificantrestorationoftheSOD andGSHactivityin

thetissues.MDAlevelinthetissues(kidneyandheart)of2K1C

hypertensiveratsincreasedsignificantlyafter8weeks.Captopril

(30mg/kg)andFLC(800mg/kg)treatedgroupshadsignificantly

decreasethe levelof MDAthan in theFLC(400mg/kg) treated

group.However,FLC(200mg/kg)treatedgroupdidnotshowany

significantrestorationofMDAlevel(Table3).

EffectofFLConNO,NOS,Ang-IIandET-1level

The2K1C-controlratsshowedasignificantdecreaseinplasma

NOandNOSlevelascomparedtotheshamoperatedgrouprats.The

ratstreatedwithcaptopril(30mg/kg)andFLC(400and800mg/kg)

significantlyelevatedplasmaNOandplasmaNOSlevels.However,

2K1ChypertensiveratstreatedwithFLC(200mg/kg)didnotshow

anysignificanteffect(Fig.2AandB).

Theincreased blood pressure in the2K1C hypertensiverats

wasalsolinkedwithsignificantlyincreasedkidneyAng-IIlevels

compared withsham-operated group.Captopril(30mg/kg) and

FLC (400 and 800mg/kg) treatment in 2K1C hypertensive rats

showeddecreasesinAng-IIlevelsofthekidney(Fig.2C).Plasma

ET-1level waselevatedsignificantlyin the2K1C-controlgroup

compared withshamgroup.Captopril (30mg/kg)and FLC(400

and 800mg/kg) significantly decreased the plasma ET-1 level

dose-dependentlytowardnearnormallevel(Fig.2D).

EffectofFLConhistopathologyofkidney

The histopathological examination of kidney tissues in the

sham-operatedratsshowednormalglomeruluscellandtubuliwith

theabsenceofperivascularedema,fibrosis,andcollagen

deposi-tion.Ontheotherhand,thehypertensive2K1Cgroupratsshowed

a significantincrease inperivascular edema,fibrosis, and

colla-gendeposition.Captopril(30mg/kg)andFLC(400and800mg/kg)

showedadecreaseinperivascularedema,fibrosis,collagen

depo-sition, and necrosis. However, FLC (200mg/kg) did not show

significantprotectionfromhypertensivedamage(Fig.3).

EffectofFLConhistopathologyofheart

The 2K1C hypertensive group rats showed severe

myocar-dialdegeneration,hypertrophy,andfibrosis.Captopril(30mg/kg)

treatedgroupshowedminimalmyocardialdegenerationand

col-lagendepositionandfibrosis.FLC(400and800mg/kg)treatment

alsoshowedadecreaseinmyocardialdegenerationandcollagen

depositionand fibrosis.However,FLC(200mg/kg)didnotshow

anysignificantprotection(Fig.4).

Discussion

The2K1Cisaclassicalmethodtoinducehypertensioninrats

similartohuman,whichisprimarilybasedonRAAS(Thurstonetal.,

1980;PonchonandElghozi,1996;Kandhareetal.,2011b).Themain

mechanismbehindthe2K1ChypertensionisRAASratherthana

disturbanceinkidneyfunction(Nogueiraetal.,2012).Unilateral

renalarteryocclusiondecreasesperfusionpressureinsidethe

kid-neyandstimulatesreninsynthesis,whichthenproduceAng-IIand

increasestheperipheralresistanceandbloodpressure(Pickering,

1989).

Our studydemonstrated that higher doses of FLC (400 and

800mg/kg) significantly decreased systolic, diastolic and mean

arterial blood pressures in the 2K1C hypertensive Wistar rats.

Secoisolariciresinoldiglucoside(SDG),amainconstituentofFLC,

isreportedtohaveasimilartypeofresultsinnormotensiveand

(6)

100

A

B

D

C

Plasma nitr

ite and nitr

ate

concentr

ation (

µ

mol/l)

Kidne

y Ang-II

(pg/mg protein)

Plasma ET

-1 (pg/ml)

Plasma NOS (U/ml)

80

60

40

20

0

200

150

###

### ### #

***

*** ***

***

*** ***

** **

* **

**

100

50

Treatment 0

Sham

FLC (200) FLC (400) FLC (800)

Treatment 0

20 40 60 80 100 0 5 10 15 20

Sham

FLC (200) FLC (400) FLC (800)

Fig.2.EffectofadministrationofFLCandcaptoprilonplasmaNOlevels(A),plasmaNOSactivity(B),Ang-IIofclippedkidney(C)andplasmaET-1(D)in2K1Chypertensive Wistarrats(n=6).Dataareexpressedasmean±S.E.M.andstatisticalanalysiswascarriedoutbyone-wayANOVAfollowedbyposthocDunnett’stest;ns=non-significant, *p<0.05,**p<0.01,***p<0.001ascomparedwith2K1Ccontrolgroup-II,#_p_<_0.05,##_p_<_0.01,###_p_<_0.001_compared_to_sham_group-I.

(7)

Fig.4. EffectofadministrationofFLCandcaptoprilonhearthistologyin2K1ChypertensiveWistarrats.Photomicrographofsectionsoflungsof(A)sham-operatedrats (group-I)showednormalcardiacmusclefiberswiththeabsenceofmyocardialfibrosis.(B)2K1Ccontrol(group-II)showedseveremyocardialdegeneration,hypertrophy,andfibrosis. (C)2K1C+Captopril(30mg/kg)(group-III)showedminimalmyocardialdegenerationandcollagendepositionandmyocardialfibrosis.(D)2K1C+FLC(200mg/kg)(group-IV) showednosignificantdecreaseincollagendepositionandmyocardialfibrosis.(E)2K1C+FLC(400mg/kg)(GroupV)showedadecreaseinmyocardialdegenerationand collagendepositionandfibrosis.(F)2K1C+FLC(800mg/kg)(GroupVI)showedasignificantreductioninmyocardialdegeneration,collagendeposition,andfibrosis(Masson’s trichrome20×).

theactivityofSDGmaybedue tothestimulationof guanylate

cyclase-nitric oxide pathway and by inhibition of angiotensin

converting enzyme (ACE) (Prasad, 2004; Kamble et al., 2013).

Earlier,we havedeterminedSDGcontentinFLCbyusing

high-performanceliquidchromatography(HPLC)analysisandreported

bloodpressureloweringeffectofFLCinDOCA-salt-inducedrenal

hypertensionmodelinrats(SawantandBodhankar,2016).Inthe

presentstudy,theeffectofFLContherenin–angiotensinsystem

dependent2K1Chypertensiverat, similartothose of captopril,

leadus toconsiderthatSDG a mainconstituent fromFLCmay

possess ACE-inhibitor-like properties. However, the possibility

of a potentiating effect of SDG by other flavonoids and minor

constituentspresentinFLCcannotberuledout.

Thehemodynamicdatashowedthatleftventricleenddiastolic

pressure,maxdP/dtandmindP/dtincreasedin2K1Chypertensive

rats,whichareaclearsignofincreasedpreloadandafterloadinthe

heart.Thealteredleftventricularparametersin2K1Chypertensive

ratsalsoshoweddecreasecontractility,diastoliccomplianceand

dysfunctionintheheart(Wangetal.,2007;Junhongetal.,2008).

FLCandcaptoprilarerestoredEDP,maxdP/dtandmindP/dt

sig-nificantlyindicatedthatFLCandcaptoprildecreasedtheburdenon

theheart,increasedtheconformityofmyocardiumandimproved

cardiacfunction.ThesefindingsthussupportthatFLCcontaining

SDGasmainconstituenthasantihypertensivepotentialin2K1C

hypertensiverats.

Chronic hypertension leads to continuous accumulation of

interstitialcollagenfibersandanincreaseinheartweight(Rossi

andPeres,1992).ItisprovedthatAng-IIofRAASisalsoinvolved

inthetissue hypertrophyorhyperplasia.Therefore,RAASplays

animportantrole intheweightincreaseofheartandkidneyin

the2K1Chypertensivemodel(Kobayashietal.,1999).Thecurrent

results showed thecaptopril and FLC significantly prevent the

increaseinkidneyandheartweightassociatedwithhypertrophy,

whichmaybeduetoantihypertensiveeffectsofcaptoprilandFLC.

It is wellknownthatintracellular sodiumion concentration

increasesandpotassiumionconcentrationdecreasessignificantly

in hypertension (Adrogue and Madias, 2007). Our results are

thus,inaccordance withthepreviousstudyandsuggestedthat

restoration ofserum sodiumandpotassium ion maybedue to

antihypertensiveeffectsofFLC.

Theliverplaysanimportantroleinmetabolism,toxicityand

eliminationofendogenousandexogenouselements.Liverdamage

leadstoincreasedactivityofAST,ALT,ALPandtotalproteininthe

plasma(Navarroetal.,1993;Bhattacharjeeetal.,2009;Visnagri

etal.,2012;Kandhareetal.,2013b,c,2015d;Sarkateetal.,2015;

Devkaretal.,2016).ThereasonbehindtheelevationofAST,ALT,

ALPandtotalproteininthe2K1Chypertensionmaybeoxidative

stressthatcausedleakageoftheseenzymesfromlivertissuesdueto

membranedamage.AftertheadministrationofFLCandcaptopril,

therewasasignificantdecreaseintheserumactivitiesofAST,ALT,

ALPandtotalproteinthatclearlysignifiesthatcaptoprilandFLC

protectedthefunctionalcapacityofliverandpreventedoxidative

damageduetohypertension.

Inhypertension,volumeandpressureloadsonthekidneyslead

tothedysfunctionanddamagetotherenaltissues(Möhringetal.,

1975;Kandhareetal.,2015d).Bloodureanitrogen(BUN),serum

creatinine,anduricacidareconsideredasmarkersoftherenal

func-tion.Theyareproducedduetodisturbanceofproteinandnucleic

acidmetabolisminthehypertensivestress.Severalanimal

stud-iesof2K1Chypertensionhavereportedthathypertensionelevates

thelevelsofbloodureanitrogen,serumcreatinineanduricacid

(8)

duetohighblood pressuremaybethereason fortheelevated

levelsofBUN,creatinine,and uricacidintheserum(Kandhare

etal.,2012a,b,c,d,2015c).Thecurrentstudyshowedareduction

intheelevatedserumlevelofrenalmarkersbyFLCandexplainits

protectiveeffectin2K1Chypertensiverats.Hypertensioninduces

myocardialdamage,andtheserumlevelsofCK-MBandLDHare

consideredasstandardmarkers fortheidentificationofcardiac

damage(Mairetal., 1994).The decreased serumlevel ofthese

enzymesintheFLC-treatedgroupsofferedprotectiontoheartin

the2K1Chypertension.

ThepresentstudyshowedthattheactivityofSODandGSHin

theheartandkidneytissuesof2K1Ccontrolgroupwaslowerthan

thatinthesham-operatedgroup.Ontheotherhand,MDAactivity

inthe2K1Ccontrolgroupwassignificantlyhigherthaninthe

sham-operatedgroup.Ourresultsthussuggestedthathypertensionledto

increasedoxidativestress,whichisinagreementwiththeprevious

study(Caoetal.,2013).ThecurrentstudyshowedthatFLCand

captopriladministrationscavengingtheoxygenfreeradicalinthe

bloodleadingtotheiranti-oxidanteffects.

Bloodflow-inducedshearstressonendothelialcellsplayskey

roleintheproductionofNObytheendothelialNOS(Pohletal.,

1986;Kandhareetal.,2012d,2014b,2016b).NOisvasorelaxant

substanceandphysiologicalantagonist oftheAng-IIofRAASat

vascular levels.Ang-II reduces NObioavailability by promoting

superoxideanion, whichis responsibleforvasoconstriction and

increaseinbloodpressure(DeNicolaetal.,1992;Cosentinoetal.,

1994;Zhouetal.,2014).

TheantagonisticeffectsofNOandAng-IIcomeoutalsoin

inter-actionswithothervasoactivesubstanceET-1,whichisthepotent

vasoconstrictorreleased fromtheendothelium (Rubanyi, 1994;

Zhouetal.,2014).ET-1isoverexpressedinthevasculatureand

bloodinvariousmodelsofhypertension,includingthe2K1Cmodel,

whichincreasessystemicbloodpressure(IglarzandSchiffrin,2003;

Cao et al., 2013).In the present study, these biomarkers were

altered in the 2K1C control hypertensive rats, where

simulta-neouslythebloodpressureincreasedwithAng-IIandET-1,asa

synthesisofNOandNOSdecreased.Allthesechangeswereopposed

significantlybytheACEinhibitordrugcaptopriland FLC.

Dose-dependenteffectsofFLContherenin–angiotensinsystemmarkers

weresimilartothoseofcaptoprilandsuggestACEinhibitorlike

propertyofFLC.

Histologicalstudyoftheratheart andkidneys revealedthat

theFLCtreatmentinthe2K1Chypertensiveratsreducedcardiac

andrenaldamagecorrelatewiththevarioushemodynamic,

bio-chemicalobservations.Theseresultssupporttheantihypertensive

activityofFLCinthe2K1Chypertensiverats.

Conclusion

AntihypertensiveandantioxidanteffectsofFLCin2K1C

hyper-tensionweredose-dependentandatthehighestdose(800mg/kg)

similartothoseofcaptopril,whichismainlycharacterizedbythe

reductioninbloodpressure,restorationofalteredleftventricular

functionsandendogenousbiomarkers.ItisconcludedthatFLCmay

reducebloodpressurebyreductionofkidneyAng-IIlevel,

inhibi-tionofET-1productionandinductionoftheNO,NOSandinvivo

antioxidantdefensesystem.

Ethicalstatement

Theanimalexperimentsinthepresentworkwerecarriedout

strictlyfollowingtheguidelinesgivenbyCPCSEA,India.The

pro-tocolwasapprovedbytheInstitutionalAnimalEthicalCommittee

(IAEC)constitutedasperguidelinesofCPCSEA.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe

regula-tionsoftherelevantclinicalresearchethicscommitteeandwith

thoseoftheCodeofEthicsoftheWorldMedicalAssociation

(Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

SScontributedincollectingplantsampleandidentification,a

confectionoftheherbarium,runningthelaboratorywork,analysis

ofthedataanddraftedthepaper.SLBsupervisedthelaboratory

workandcontributedtocriticalreadingofthemanuscript.Allthe

authorshavereadthefinalmanuscriptandapprovedthe

submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

References

Adil, M., Kandhare, A., Dalvi, G., Ghosh, P., Venkata, S., Raygude, K., Bod-hankar,S.,2016a.Ameliorativeeffectofberberineagainstgentamicin-induced nephrotoxicityinratsviaattenuationofoxidativestress,inflammation, apo-ptosis andmitochondrial dysfunction.Ren.Fail., http://dx.doi.org/10.3109/ 0886022X.2016.1165120.

Adil,M.,Kandhare,A.,Ghosh,P.,Venkata,S.,Raygude,K.,Bodhankar,S.,2016b. Ameliorativeeffectofnaringininacetaminophen-inducedhepaticandrenal toxicityinlaboratoryrats:roleofFXRandKIM-1.Ren.Fail.,http://dx.doi.org/ 10.3109/0886022X.2016.1163998.

Adil,M.,Kandhare,A.D.,Visnagri,A.,Bodhankar,S.L.,2015.Naringinameliorates sodiumarsenite-inducedrenalandhepatictoxicityinrats:decisiveroleof KIM-1,Caspase-3,TGF-beta,andTNF-alpha.Ren.Fail.37,1396–1407.

Adil,M.,Visnagri,A.,Kumar,V.S.,Kandhare,A.D.,Ghosh,P.,2014.Protectiveeffect ofnaringinonsodiumarseniteinducedtesticulartoxicityviamodulationof biochemicalperturbationsinexperimentalrats.Pharmacologia5,222–234. Adrogue,H.J.,Madias,N.E.,2007.Sodiumandpotassiuminthepathogenesisof

hypertension.N.Engl.J.Med.356,1966–1978.

Amat, N., Amat, R., Abdureyim,S., Hoxur, P., Osman, Z., Mamut,D., Kijjoa, A., 2014. Aqueous extract of Dioscorea opposita thunb. normalizes the hypertension in2K1C hypertensive rats. BMC Complement.Altern. Med., http://dx.doi.org/10.1186/1472-6882-14-36.

Aswar,U.M.,Kandhare,A.D.,Mohan,V.,Thakurdesai,P.A.,2015.Anti-allergiceffect ofintranasaladministrationoftype-Aprocyanidinpolyphenolsbased standard-izedextractofcinnamonbarkinovalbuminsensitizedBALB/cmice.Phytother. Res.29,423–433.

Badole,S.L.,Chaudhari,S.M.,Jangam,G.B.,Kandhare,A.D.,Bodhankar,S.L.,2015. CardioprotectiveactivityofPongamiapinnatainstreptozotocin–nicotinamide induceddiabeticrats.Biomed.Res.Int.2015,403291.

Bassett,C.M.,Rodriguez-Leyva,D.,Pierce,G.N.,2009.Experimentalandclinical researchfindingsonthecardiovascularbenefitsofconsumingflaxseed.Appl. Physiol.Nutr.Metab.34,965–974.

Bhattacharjee,N.,Pathak,S.,Khuda-Bukhsh,A.R.,2009.Ameliorationof carcinogen-inducedtoxicityinmicebyadministrationofapotentizedhomeopathicdrug, natrumsulphuricum200.Evid.BasedComplement.Alternat.Med.6,65–75. Cao,Y.J.,He,X.,Wang,N.,He,L.C.,2013.Effectsofimperatorin,theactivecomponent

fromRadixAngelicae(Baizhi),onthebloodpressureandoxidativestressin2K,1C

hypertensiverats.Phytomedicine20,1048–1054.

Chen,J.,Stavro,P.M.,Thompson,L.U.,2002.Dietaryflaxseedinhibitshumanbreast cancergrowthandmetastasisanddownregulatesexpressionofinsulin-like growthfactorandepidermalgrowthfactorreceptor.Nutr.Cancer43,187–192. Collins,T.F.,Sprando,R.L.,Black,T.N.,Olejnik,N.,Wiesenfeld,P.W.,Babu,U.S.,Bryant, M.,Flynn,T.J.,Ruggles,D.I.,2003.Effectsofflaxseedanddefattedflaxseedmeal onreproductionanddevelopmentinrats.FoodChem.Toxicol.41,819–834. Cosentino,F.,Sill,J.C.,Katusic,Z.S.,1994.Roleofsuperoxideanionsinthemediation

ofendothelium-dependentcontractions.Hypertension23,229–235. Coulter,D.M.,Edwards,I.R.,1987.Coughassociatedwithcaptoprilandenalapril.

(9)

DeNicola,L.,Blantz,R.C.,Gabbai,F.B.,1992.NitricoxideandangiotensinII. Glomeru-larandtubularinteractionintherat.J.Clin.Invest.89,1248–1256.

Devkar,S.T.,Kandhare,A.D.,Zanwar,A.A.,Jagtap,S.D.,Katyare,S.S.,Bodhankar, S.L.,Hegde,M.V.,2016.Hepatoprotectiveeffectofwithanolide-richfractionin acetaminophen-intoxicatedrat:decisiveroleofTNF-alpha,IL-1beta,COX-IIand iNO.Pharm.Biol.,1–10,http://dx.doi.org/10.3109/13880209.2016.1157193. Ghosh, P.,Kandhare, A.D.,Raygude,K.S.,Kumar, V.S.,Rajmane, A.R.,Adil,M.,

Bodhankar,S.L.,2012.Determinationofthelongtermdiabetesrelated compli-cationsandcardiovasculareventsusingUKPDSriskengineandUKPDSoutcomes modelinarepresentativewesternIndianpopulation.AsianPac.J.Trop.Dis.2, S642–S650.

Ghule,A.E.,Jadhav,S.S.,Bodhankar,S.L.,2011.RenoprotectiveeffectofLinum

usitatis-simumseedsthroughhaemodynamicchangesandconservationofantioxidant

enzymes in renal ischaemia-reperfusion injury in rats. Arab. J. Urol. 9, 215–221.

Ghule,A.E.,Jadhav,S.S.,Bodhankar,S.L.,2012.Effectofethanolicextractofseedsof

Linumusitatissimum(Linn.)onhemodynamicchangesandleftventricular

func-tioninrenalarteryoccludedrenovascularhypertensioninrats.Pharmacologia 3,283–290.

Ghule,A.E.,Kandhare,A.D.,Jadhav,S.S.,Zanwar,A.A.,Bodhankar,S.L.,2015. Omega-3-fattyacidaddstotheprotectiveeffectofflaxlignanconcentrateinpressure overload-inducedmyocardialhypertrophyinratsviamodulationofoxidative stressandapoptosis.Int.Immunopharmacol.28,751–763.

Gosavi,T.,Kandhare,A.,Raygude,K.,Ghosh,P.,Bodhankar,S.,2011.Acomparative studyontheefficacy,safetyandcosteffectivenessofViscumalbumand

Rau-wolfiaserpentinamothertinctureinhypertensivepatients.Deccan.J.Nat.Prod.

2,29–35.

Gosavi,T.P.,Ghosh,P.,Kandhare,A.D.,Kumar,V.S.,Adil,M.,Rajmane,A.R., Bod-hankar,S.L., 2012a.TherapeuticeffectofH. pylorinosode,a homeopathic preparationinhealingofchronicH.pyloriinfectedulcersinlaboratoryanimals. AsianPac.J.Trop.Dis.2,S603–S611.

Gosavi,T.P.,Kandhare,A.D.,Ghosh,P.,Bodhankar,S.L.,2012b.Anticonvulsant

activ-ityofArgentummetallicum,ahomeopathicpreparation.DerPharm.Lett.4,

626–637.

Gosavi,T.P.,Kumar,V.S.,Kandhare,A.D.,Zanwar,A.A.,Hegde,M.V.,Bodhankar,S.L., 2014.Acomprehensivemetaanalysisandsystematicreviewoneffectof genis-teinonmetabolicsyndrome.Pharmacologia5,120–126.

Goswami,S.,Kandhare,A.,Zanwar,A.A.,Hegde,M.V.,Bodhankar,S.L.,Shinde,S., Deshmukh,S.,Kharat,R.,2016.Orall-glutamineadministrationattenuated cutaneouswoundhealinginWistarrats.Int.WoundJ.13,116–124.

Griendling,K.K.,Minieri,C.A.,Ollerenshaw,J.D.,Alexander,R.W.,1994.Angiotensin IIstimulatesNADHandNADPHoxidaseactivityinculturedvascularsmooth musclecells.Circ.Res.74,1141–1148.

Gryglewski,R.J.,Palmer,R.M.J.,Moncada,S.,1986.Superoxideanionisinvolvedin thebreakdownofendothelium-derivedvascularrelaxingfactor.Nature320, 454–456.

Honmore,V.,Kandhare,A.,Zanwar,A.A.,Rojatkar,S.,Bodhankar,S.,Natu,A.,2015.

Artemisiapallensalleviatesacetaminopheninducedtoxicityviamodulationof

endogenousbiomarkers.Pharm.Biol.53,571–581.

Iglarz,M.,Schiffrin,E.L.,2003.Roleofendothelin-1inhypertension.Curr.Sci.Inc.5, 144–148.

Johnsson,P.,Kamal-Eldin,A.,Lundgren,L.N.,Aman,P.,2000.HPLCmethodfor anal-ysisofsecoisolariciresinoldiglucosideinflaxseeds.J.Agric.FoodChem.48, 5216–5219.

Junhong,W.,Jing,Y.,Jizheng,M.,Shushu,Z.,Xiangjian,C.,Hengfang,W.,Di,Y.,Jinan, Z.,2008.Proteomicanalysisofleftventriculardiastolicdysfunctionheartsin renovascularhypertensiverats.Int.J.Cardiol.127,198–207.

Kamble,H.,Kandhare,A.D.,Bodhankar,S.,Mohan,V.,Thakurdesai,P.,2013.Effectof lowmolecularweightgalactomannansfromfenugreekseedsonanimalmodels ofdiabetesmellitus.Biomed.AgingPathol.3,145–151.

Kandhare,A.,Raygude,K.,Ghosh,P.,Bodhankar,S.,2011a.Theameliorativeeffectof fisetin,abioflavonoid,onethanol-inducedandpylorusligation-inducedgastric ulcerinrats.Int.J.GreenPharm.5,236–243.

Kandhare,A.D.,Alam,J.,Patil,M.V.,Sinha,A.,Bodhankar,S.L.,2016a.Wound heal-ingpotentialofnaringinointmentformulationviaregulatingtheexpression ofinflammatory,apoptoticandgrowthmediatorsinexperimentalrats.Pharm. Biol.54(3),419–432.

Kandhare,A.D.,Bodhankar,S.L.,Mohan,V.,Thakurdesai,P.A.,2015a.Acuteand repeateddoses(28days)oraltoxicitystudy ofglycosidesbased standard-izedfenugreekseedextractinlaboratorymice.Regul.Toxicol.Pharmacol.72, 323–334.

Kandhare,A.D.,Bodhankar,S.L., Mohan,V., Thakurdesai,P.A., 2015b.Effectof glycosidesbasedstandardizedfenugreekseedextractinbleomycin-induced pulmonaryfibrosisinrats:decisiveroleofBax,Nrf2,NF-kappaB,Muc5ac, TNF-alphaandIL-1beta.Chem.Biol.Interact.237,151–165.

Kandhare,A.D.,Bodhankar,S.L.,Mohan,V.,Thakurdesai,P.A.,2015c. Pharmacoki-netics,tissuedistributionandexcretionstudyofafurostanolglycoside-based standardizedfenugreekseedextractinrats.Ren.Fail.37,1208–1218. Kandhare,A.D.,Bodhankar,S.L.,Singh,V.,Mohan,V.,Thakurdesai,P.A.,2013a.

Anti-asthmaticeffectsoftype-Aprocyanidinepolyphenolsfromcinnamonbarkin ovalbumin-inducedairwayhyperresponsivenessinlaboratoryanimals.Biomed. AgingPathol.3,23–30.

Kandhare,A.D.,Ghosh,P.,Bodhankar,S.L.,2014a.Naringin,aflavanoneglycoside, promotesangiogenesisandinhibitsendothelialapoptosisthroughmodulation ofinflammatoryandgrowthfactorexpressionindiabeticfootulcerinrats. Chem.Biol.Interact.219,101–112.

Kandhare, A.D., Ghosh, P., Ghule, A.E., Bodhankar, S.L., 2013b. Elucidationof molecularmechanisminvolvedinneuroprotectiveeffectofcoenzymeQ10in alcohol-inducedneuropathicpain.Fundam.Clin.Pharmacol.27,603–622. Kandhare,A.D.,Ghosh,P.,Ghule,A.E.,Zambare,G.N.,Bodhankar,S.L.,2013c.

Protec-tiveeffectofPhyllanthusamarusbymodulationofendogenousbiomarkersand DNAdamageinaceticacidinducedulcerativecolitis:roleofphyllanthinand hypophyllanthin.ApolloMed.10,87–97.

Kandhare,A.D.,Kumar,V.S.,Adil,M.,Rajmane,A.R.,Ghosh,P.,Bodhankar,S.L.,2012a. InvestigationofgastroprotectiveactivityofXanthiumstrumariumL.by modula-tionofcellularandbiochemicalmarker.OrientalPharm.Exp.Med.12,287–299. Kandhare,A.D.,Patil,A.,Guru,A.,Mukhrjee,A.,Sarkar,A.,Sengupta,A.,Parmar, H.M.,Muthal,A.P.,Wangikar,P.,Bodhankar,S.L.,2016b.Ameliorativeeffectof ferulicacidagainstaceticacidinducedulcerativecolitis:RoleofHO-1andNrf2. Pharmacologia7,114–124.

Kandhare,A.D.,Patil,M.V.,Bodhankar,S.L.,2015d.l-Arginineattenuatesthe ethyl-eneglycolinducedurolithiasisinininephrectomizedhypertensiverats:roleof KIM-1,NGAL,andNOs.Ren.Fail.37,709–721.

Kandhare,A.D.,Raygude,K.S.,Ghosh,P.,Ghule,A.E.,Bodhankar,S.L.,2012b. Neu-roprotectiveeffectofnaringinbymodulationofendogenousbiomarkersin streptozotocininducedpainfuldiabeticneuropathy.Fitoterapia83,650–659. Kandhare,A.D.,Raygude,K.S.,Ghosh,P.,Ghule,A.E.,Gosavi,T.P.,Badole,S.L.,

Bod-hankar,S.L.,2012c.EffectofhydroalcoholicextractofHibiscusrosasinensisLinn. leavesinexperimentalcolitisinrats.AsianPac.J.Trop.Biomed.2,337–344. Kandhare, A.D., Raygude,K.S.,Ghosh, P., Gosavi, T.P., Bodhankar,S.L., 2011b.

Patentabilityofanimalmodels:Indiaandtheglobe.Int.J.Pharm.Biol.Arc.2, 1024–1032.

Kandhare,A.D.,Raygude,K.S.,ShivaKumar,V.,Rajmane,A.R.,Visnagri,A.,Ghule,A.E., Ghosh,P.,Badole,S.L.,Bodhankar,S.L.,2012d.Ameliorativeeffectsquercetin againstimpairedmotornervefunction,inflammatorymediatorsandapoptosis inneonatalstreptozotocin-induceddiabeticneuropathyinrats.Biomed.Aging Pathol.2,173–186.

Kandhare,A.D.,Shivakumar,V.,Rajmane,A.,Ghosh,P.,Bodhankar,S.L.,2014b. Eval-uationoftheneuroprotectiveeffectofchrysinviamodulationofendogenous biomarkersinaratmodelofspinalcordinjury.J.Nat.Med.68,586–603. Kang,D.H.,Nakagawa,T.,Feng,L.,Watanabe,S.,Han,L.,Mazzali,M.,Truong,L.,Harris,

R.,Johnson,R.J.,2002.Aroleforuricacidintheprogressionofrenaldisease.J. Am.Soc.Nephrol.13,2888–2897.

Kearney,P.,Whelton,M.,Reynolds,K.,Muntner,P.,Whelton,P.,He,J.,2005.Global burdenofhypertension:analysisofworldwidedata.Lancet365,217–223. Ketkar,S.,Rathore,A.,Kandhare, A.,Lohidasan,S.,Bodhankar,S.,Paradkar,A.,

Mahadik,K.,2015.Alleviatingexercise-inducedmuscularstressusingneatand processedbeepollen:oxidativemarkers,mitochondrialenzymes,and myo-statinexpressioninrats.Integr.Med.Res.4,147–160.

Khalesi,S.,Irwin,C.,Schubert,M.,2015.Flaxseedconsumptionmayreduceblood pressure:asystematicreviewandmeta-analysisofcontrolledtrials.J.Nutr.145, 758–765.

Kharin,S.N.,Krandycheva,V.V.,2004.Methodofexperimentalconstrictionofrenal arteryformodelingofrenovascularhypertensioninrats.Bull.Exp.Biol.Med. 138,103–105.

Kinniry,P.,Amrani,Y.,Vachani,A.,Solomides,C.C.,Arguiri,E.,Workman,A.,Carter, J.,Christofidou-Solomidou,M.,2006.Dietaryflaxseedsupplementation ame-lioratesinflammationandoxidativetissuedamageinexperimentalmodelsof acutelunginjuryinmice.J.Nutr.136,1545–1551.

Kobayashi, S.,Ishida,A., Moriya,H.,Mori, N.,Fukuda, T.,Takamura, T.,1999. AngiotensinIIreceptorblockadelimitskidneyinjuryintwo-kidney,one-clip Goldblatthypertensiveratswithspecialreferencetophenotypicchanges.J.Lab. Clin.Med.133,134–143.

Kumar,V.S.,Rajmane,A.R.,Adil,M.,Kandhare,A.D.,Ghosh,P.,Bodhankar,S.L.,2014. Naringinamelioratesaceticacidinducedcolitisthroughmodulationof endoge-nousoxido-nitrosativebalanceandDNAdamageinrats.J.Biomed.Res.28, 132–145.

Lee,J.C.,Krochak,R.,Blouin,A.,Kanterakis,S.,Chatterjee,S.,Arguiri,E.,Vachani,A., Solomides,C.C.,Cengel,K.A.,Christofidou-Solomidou,M.,2009.Dietaryflaxseed preventsradiation-inducedoxidativelungdamage,inflammationandfibrosis inamousemodelofthoracicradiationinjury.CancerBiol.Ther.8,47–53. Mair,J.,Wagner,I.,Jakob,G.,Lechleitner,P.,Dienstl,F.,Puschendorf,B.,Michel,G.,

1994.Differenttimecoursesofcardiaccontractileproteinsafteracute myocar-dialinfarction.Clin.Chim.Acta231,47–60.

Miranda,K.M.,Espey,M.G.,Wink,D.A.,2001.Arapid,simplespectrophotometric methodfor simultaneous detection ofnitrate and nitrite.Nitric Oxide5, 62–71.

Möhring,J.,Möhring,B.,Haack,D.,Lazar,J.,Oster,P.,Schömig,A.,Gross,F.,1975. ThirstandSaltAppetiteinExperimentalRenalHypertensionofRats.In:Control MechanismsofDrinking.SpringerScience+BusinessMedia,pp.155–164. Moron,M.S.,Depierre,J.W.,Mannervik,B.,1979.Levelsofglutathione,glutathione

reductaseandglutathioneS-transferaseactivitiesinratlungandliver.Biochim. Biophys.Acta582,67–78.

Navarro,M.,Montilla,M.,Martín,A.,Jiménez,J.,Utrilla,M.,1993.Freeradical scav-engerandantihepatotoxicactivityofRosmarinustomentosus.PlantaMed.59, 312–314.

Nogueira,B.V.,Palomino,Z.,Porto,M.L.,Balarini,C.M.,Pereira,T.M.,Baldo,M.P., Casarini,D.E.,Meyrelles,S.S.,Vasquez,E.C.,2012.Granulocytecolony stimulat-ingfactorpreventskidneyinfarctionandattenuatesrenovascularhypertension. CellPhysiol.Biochem.29,143–152.

(10)

Morininpylorusligationinducedgastriculcerviamodulationofoxidative stress.DerPharm.Lett.7,131–139.

Patil,M.,Kandhare,A.,Bhise,S.,2011.Pharmacologicalevaluationofameliorative effectofaqueousextractofCucumissativusL.fruitformulationonwoundhealing inWistarrats.ChroniclesYoungSci.2,207–213.

Pickering,T.G.,1989.Renovascularhypertension:etiologyandpathophysiology. Semin.Nucl.Med.19,79–88.

Pohl,U.,Holtz,J.,Busse,R.,Bassenge,E.,1986.Crucialroleofendotheliuminthe vasodilatorresponsetoincreasedflowinvivo.Hypertension8,37–44. Ponchon, P., Elghozi, J.L., 1996. Contribution of the renin–angiotensin and

kallikrein–kininsystemstoshort-termvariabilityofbloodpressurein two-kidney,one-cliphypertensiverats.Eur.J.Pharmacol.297,61–70.

Prasad,K.,1997.Dietaryflaxseedinpreventionofhypercholesterolemic atheroscle-rosis.Atherosclerosis132,69–76.

Prasad,K.,2004.Antihypertensiveactivityofsecoisolariciresinoldiglucoside(SDG) isolatedfromflaxseed:roleofguanylatecyclase.Int.J.Angiol.13,7–14. Prasad,K.,Mantha,S.V.,Muir,A.D.,Westcott,N.D.,1998.Reductionof

hypercholes-terolemicatherosclerosisbyCDC-flaxseedwithverylowalpha-linolenicacid. Atherosclerosis136,367–375.

Prasad,K.,Mantha,S.V.,Muir,A.D.,Westcott,N.D.,2000.Protectiveeffectof sec-oisolariciresinoldiglucosideagainststreptozotocin-induceddiabetesandits mechanism.Mol.CellBiochem.206,141–149.

Rajagopalan,S.,Kurz,S.,Munzel,T.,Tarpey,M.,Freeman,B.A.,Griendling,K.K., Har-rison,D.G.,1996.AngiotensinII-mediatedhypertensionintheratincreases vascularsuperoxideproductionviamembraneNADH/NADPHoxidase acti-vation. Contributionto alterations of vasomotor tone. J. Clin. Invest. 97, 1916–1923.

Rajesha,J.,Murthy,K.N.,Kumar,M.K.,Madhusudhan,B.,Ravishankar,G.A.,2006. Antioxidantpotentialsofflaxseedbyinvivomodel.J.Agric.FoodChem.54, 3794–3799.

Raygude,K.S.,Kandhare,A.D.,Ghosh,P.,Bodhankar,S.L.,2012a.Anticonvulsant effectoffisetinbymodulationofendogenousbiomarkers.Biomed.Prev.Nutr. 2,215–222.

Raygude,K.S.,Kandhare,A.D.,Ghosh,P.,Ghule,A.E.,Bodhankar,S.L.,2012b. Eval-uationofameliorativeeffectofquercetininexperimentalmodelofalcoholic neuropathyinrats.Inflammopharmacology20,331–341.

Rodriguez-Leyva,D.,Weighell,W.,Edel,A.L.,LaVallee,R.,Dibrov,E.,Pinneker,R., Maddaford,T.G.,Ramjiawan,B.,Aliani,M.,Guzman,R.,Pierce,G.N.,2013.Potent antihypertensiveactionofdietaryflaxseedinhypertensivepatients. Hyperten-sion62,1081–1089.

Rossi,M.A.,Peres,L.C.,1992.Effectofcaptoprilonthepreventionandregressionof myocardialcellhypertrophyandinterstitialfibrosisinpressureoverloadcardiac hypertrophy.Am.HeartJ.124,700–709.

Rubanyi,G.,1994.Endothelins:molecularbiology,biochemistry,pharmacology, physiology,andpathophysiology.Pharmacol.Rev.46,325–415.

Saraswathi,K.Y.,Muthal,A.,Kandhare,A.,Rojatkar,S.,Bodhankar,S.,2014.Studyof methanolicextractofArtemisiapallensWallonenduranceoflaboratoryanimals. Pharmacologia5,298–309.

Sawant,S.H.,Bodhankar,S.L.,2016.Flaxlignanconcentratereversealterationsin bloodpressure,leftventricularfunctions,lipidprofileandantioxidantstatusin DOCA-saltinducedrenalhypertensioninrats.Ren.Fail.38,411–423.

Sarkar,A.,Sengupta,A.,Mukhrjee,A.,Guru,A.,Patil,A.,Kandhare,A.D.,Bodhankar, S.L.,2015.Antiulcerpotentialofmorininaceticacid-inducedgastriculcervia modulationofendogenousbiomarkersinlaboratoryanimals.Pharmacologia6, 273–281.

Sarkate,A.P.,Murumkar,P.R.,Lokwani,D.K.,Kandhare,A.D.,Bodhankar,S.L.,Shinde, D.B.,Bothara,K.G.,2015.DesignofselectiveTACEinhibitorsusingmolecular dockingstudies:synthesisandpreliminaryevaluationofanti-inflammatoryand TACEinhibitoryactivity.SARQSAREnviron.Res.26,905–923.

Schlaich,M.P.,Delles,C.,Schmieder,R.E.,2007.Involvementofendothelial mecha-nismsinl-arginine-inducedalterationsofrenalhaemodynamicsinhumans.J. Hypertens.25,1515–1516(authorreply1516–1517).

Shivakumar,V.,Kandhare,A.,Rajmane,A.,Adil,M.,Ghosh,P.,Badgujar,L.,Saraf,M., Bodhankar,S.,2014.Estimationofthelong-termcardiovasculareventsusing UKPDSriskengineinmetabolicsyndromepatients.IndianJ.Pharm.Sci.76, 174–178.

Slater,T.F.,Sawyer,B.C.,1971.Thestimulatoryeffectsofcarbontetrachlorideand otherhalogenoalkanesonperoxidativereactionsinratliverfractionsinvitro. Generalfeaturesofthesystemsused.Biochem.J.123,805–814.

Thurston,H.,Bing,R.F.,Swales,J.D.,1980.Reversaloftwo-kidneyoneclip renovas-cularhypertensionintherat.Hypertension2,256–265.

Visnagri,A.,Kandhare,A.D.,Bodhankar,S.L.,2015.Renoprotectiveeffectof berber-ineviaintonationonapoptosisandmitochondrial-dependentpathwayinrenal ischemiareperfusion-inducedmutilation.Ren.Fail.37,482–493.

Visnagri,A.,Kandhare,A.D.,Chakravarty,S.,Ghosh,P.,Bodhankar,S.L.,2014. Hes-peridin, aflavanoglyconeattenuates experimentaldiabeticneuropathy via modulationofcellularandbiochemicalmarkertoimprovenervefunctions. Pharm.Biol.52,814–828.

Visnagri,A.,Kandhare,A.D.,Ghosh,P.,Bodhankar,S.L.,2013.Endothelinreceptor blockerbosentaninhibitshypertensivecardiacfibrosisinpressure overload-inducedcardiachypertrophyinrats.Cardiovasc.Endocrinol.2,85–97. Visnagri,A.,Kandhare,A.D.,ShivaKumar,V.,Rajmane,A.R.,Mohammad,A.,Ghosh,

P.,Ghule,A.E., Bodhankar,S.L.,2012. Elucidationofameliorativeeffectof co-enzymeQ10instreptozotocin-induceddiabeticneuropathicperturbation bymodulationofelectrophysiological,biochemicalandbehavioralmarkers. Biomed.AgingPathol.2,157–172.

Wang,P.,Tang,F.,Li,R.,Zhang,H.,Chen,S.,Liu,P.,Huang,H.,2007.Contributionof differentNoxhomologuestocardiacremodelingintwo-kidneytwo-clip reno-vascularhypertensiverats:effectofvalsartan.Pharmacol.Res.55,408–417. Zanwar,A., Hegde,M.V., Bodhankar, S.L.,2011. Ethanolic extract ofseeds of

Linumussitattimum(Flaxlignanconcentrate)preventsdoxorubicin-induced

car-diotoxicityinrats.AtherosclerosisSuppl.12,146.

Zanwar,A.A.,Hegde,M.,Bodhankar,S.,2010.Invitroantioxidantactivityofethanolic extractofLinumusitatissimum.Pharmacol.Online1,683–696.

Zanwar,A.A.,Hegde,M.V.,Bodhankar,S.L.,2013.Protectiveroleofconcomitant administrationofflaxlignanconcentrateandomega-3-fattyacidon myocar-dialdamageindoxorubicin-inducedcardiotoxicity.FoodSci.Hum.Wellness2, 29–38.