Research Article Flavonoids Extraction from Propolis ...downloads.hindawi.com/journals/ecam/2016/6281376.pdfResearch Article Flavonoids Extraction from Propolis Attenuates Pathological

Research ArticleFlavonoids Extraction from Propolis Attenuates PathologicalCardiac Hypertrophy through PI3KAKT Signaling Pathway

Guang-wei Sun12 Zhi-dong Qiu3 Wei-nan Wang3 Xin Sui3 and Dian-jun Sui13

1China-Japan Union Hospital of Jilin University Changchun 130033 China2Chinese Traditional Medicine Institute of Ji Lin Province Changchun 130021 China3Changchun University of Chinese Medicine Changchun 130117 China

Correspondence should be addressed to Dian-jun Sui 834321375qqcom

Received 13 January 2016 Revised 24 March 2016 Accepted 30 March 2016

Academic Editor Yoshiji Ohta

Copyright copy 2016 Guang-wei Sun et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Propolis a traditional medicine has been widely used for a thousand years as an anti-inflammatory and antioxidant drug Theflavonoid fraction is themain active component of propolis which possesses awide range of biological activities including activitiesrelated to heart disease However the role of the flavonoids extraction from propolis (FP) in heart disease remains unknown Thisstudy shows that FP could attenuate ISO-induced pathological cardiac hypertrophy (PCH) and heart failure in mice The effectof the two fetal cardiac genes atrial natriuretic factor (ANF) and 120573-myosin heavy chain (120573-MHC) on PCH was reversed by FPEchocardiography analysis revealed cardiac ventricular dilation and contractile dysfunction in ISO-treated mice This finding isconsistent with the increased heart weight and cardiac ANF protein levels massive replacement fibrosis and myocardial apoptosisHowever pretreatment of mice with FP could attenuate cardiac dysfunction and hypertrophy in vivo Furthermore the cardiacprotection of FP was suppressed by the pan-PI3K inhibitor wortmannin FP is a novel cardioprotective agent that can attenuateadverse cardiac dysfunction hypertrophy and associated disorder such as fibrosis The effects may be closely correlated withPI3KAKT signaling FP may be clinically used to inhibit PCH progression and heart failure

1 Introduction

Cardiac hypertrophy is frequently observed in various clin-ical conditions including hypertension and aortic stenosisIt can progressively lead to heart failure (HF) HF is themost frequent cause of cardiovascular deathworldwide HF iscaused by a pathological state that results in insufficient car-diac output [1ndash6] Although there has been great progress intherapy the mortality and morbidity of HF place it in the top5 lethal diseases Currently the high mortality of HF imposesa significant economic burden in both China and Westerncountries [7]Thus identifying novel preventive andor ther-apeutic strategies to counteract this deadly disease is ofimportance

Cardiac hypertrophy which is characterized by increasedcell volume and metabolic and biochemical disorders canalso lead to reactivation of fetal cardiac genes such as atrialnatriuretic factor (ANF) and 120573-myosin heavy chain (120573-MHC) [8ndash10] Currently cardiac hypertrophy is attracting

attention because it can progressively lead to HF [11ndash15]Some specific factors with cardioprotective properties havebeen identified through the preventativemechanisms ofmin-imizing cardiac hypertrophy Some cellularmolecular path-ways including the mitochondrial pathway phosphoinosi-tide 3-kinase (PI3K) AKT andmammalian target of rapamy-cin (mTOR) are involved in the progression of cardiac hyper-trophy [1 4 16]

Propolis a complex mixture of naturally sticky gummyand resinous components is produced by honeybees fromplant materials [17] Propolis contains various compoundssuch as flavonoids terpenes 120573-steroids aromatic aldehydesand alcohols [18] Propolis exhibits a wide range of biologicalactivities including antibacterial antifungal anti-inflamma-tory immunostimulatory antitumor and antioxidant effects[18]Therefore propolis is extensively used in food and bever-ages to improve health and prevent diseases

In recent years considerable efforts have been focusedon identifying naturally occurring herbal medicines [19ndash22]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 6281376 11 pageshttpdxdoiorg10115520166281376

2 Evidence-Based Complementary and Alternative Medicine

The flavonoid fraction is the main component of propolisMany flavonoids exhibit strong antioxidant activity [17]Our research has focused on studying the cardioprotectiveproperties of the flavonoids extraction from propolis (FP)Our preliminary results showed that FP exerts an effectiverole in cardiac protectionHowever the protective effect of FPon pathological cardiac hypertrophy (PCH) and HF remainsunknown This study shows that FP is a novel cardiopro-tective agent First FP significantly attenuates adverse car-diac dysfunction hypertrophy and associated disorder suchas fibrosis Secondly FP significantly attenuates apoptoticdamage and cardiac remodeling which is correlated withthe PI3KAKT signaling Thus FP may be clinically used toinhibit PCH progression and prevent HF

2 Materials and Methods

21 Preparation of FP Propolis was purchased from theAn Guo herbal medicine market in Hebei province ChinaThe propolis was authenticated by Professor Dacheng Jiangfrom the Changchun University of Chinese Medicine and avoucher specimen (number 1019907) was deposited at theJilin museum of Materia Medica Jilin province China Thesample was frozen at 4∘C before being ground into a powderThen 10x of 75 ethanolwater was added to extract theactive fractions This process was conducted twice and thefractions were filtered through a 045 120583m membrane afterbeing combined The filtrate was evaporated to dryness at80∘C under reduced pressure One g of the powder wasdissolved in 100mL chloroform-ethanol (10 1) and wasextracted twice by the same volume of 1 NaOH solutionThe aqueous alkali was adjusted to pH 6 by 1 HCl and thenleft at room temperature for another 24 h for precipitationThe FP were then obtained through filtration rinsed in waterto neutral pH and dried under reduced pressure

22 High Performance Liquid Chromatography-High-Resolu-tion Mass Spectrometry (HPLC-HRMS) Analysis of FP TheFP was dissolved in pure methanol and filtered through a022120583m membrane before HPLC-Q-TOF-MS analysis Theliquid chromatography separation was performed on anAgilent SB-Aq column (250mm times 46mm 45 120583m 400 bar)at 30∘C For HPLC analysis 01 formic acidwater (vv) and01 formic acidmethanol were used as the mobile phases Aand B respectively The gradient elution was programmed asfollows 0ndash110min (35ndash90 B) and 110ndash120min (90 B)Theflow rate was 10mLmin and the injected sample volumewas10 120583L The Q-TOF-MS scan range was set at119898119911 100ndash1200 inpositive modesThe dry gas (N

2) flow rate was 90 Lmin the

dry gas temperature was 350∘C the nebulizer gas was set at30 psi the capillary voltage was 3500V the fragmentor was175V and the skimmer was 65V

Standard pinocembrin kaempferol isosakuranetinpinobanksin-3-O-acetate 12-acetoxyviscidone galanginand chrysin (purity gt 98 by HPLC) were purchased fromthe National Institutes for Food and Drug Control of ChinaThey were mixed and dissolved in pure methanol at theconcentration of 2120583M per compound prior to use

Data analysis was performed on an Agilent MassHunterWorkstation Software-Qualitative Analysis (version B0400Build 404795 Service Pack 3 Agilent Technologies Inc2011)

23 Animals and In Vivo Pharmacological Treatment Themice used in this study were handled in compliance withthe guidelines for the care and use of laboratory animalsestablished by the Chinese Council on Animal Care andall animal protocols were approved by the Jilin UniversityAnimal Care and Use Committee Eight-week-old male micewere anesthetized with 15 isoflurane The adult mice wereintragastrically given different doses of FP (1ndash50mgsdotkgminus1sdotdminus1)for 7 d Alzet osmotic minipumps containing PBS or iso-proterenol (ISO) were surgically implanted subcutaneouslyin the interscapular region of the mouse ISO was cali-brated to release the drug at a rate of 25mgsdotkgminus1sdotdminus1 for7 d to experimentally induce heart hypertrophy The dose-dependent effect of FP on ISO-induced gene reactivationwas determined FP (50mgsdotkgminus1sdotdminus1) did not exert an addi-tional benefit to reduce heart hypertrophy thus we selected25mgsdotkgminus1sdotdminus1 for the following experiments In a separateexperiment mice were pretreated with the selective PI3Kantagonist wortmannin (WM) (1mgsdotkgminus1) at 1 h before ISOadministrationThe PI3K inhibitor doses were selected basedon the results of previous studies

24 Determination of Cardiac Dysfunction through Echocar-diography The animals were euthanized and the hearts wereremoved for hypertrophic evaluationThe analysis showed noeffect on cardiac function Cardiac function was examinedthrough echocardiography using a Vevo 770 microultra-sound system (VisualSonics Toronto Ontario Canada) asdescribed previously [17] Briefly an in vivo transthoracicechocardiography of the left ventricle was performed using a30MHz scan head interfaced with a Vevo 770 An ultrasoundbeamwas placed on the heart and near the papillary musclesHigh-resolution two-dimensional electrocardiogram-basedkilohertz visualization was achieved The parameters ofcardiac function were digitally measured on the M-modetracings and then averaged from three to five cardiac cycles

25 Histological Analyses The animals were euthanizedand the hearts were removed for hypertrophic evaluationSerial sections (4mm) of heart tissues were stained withhematoxylin-eosin [20] or Massonrsquos trichrome and thenvisualized using a light microscope as previously described

26 Transmission Electron Microscopy The animals wereeuthanized and the hearts were removed for hypertrophicevaluation Heart tissue sections were collected and observedby transmission electron microscopy

27 Real-Time RT-PCR Total RNA was extracted usingTRIzol (Invitrogen Carlsbad CA) Briefly 2mg of total RNAwas reverse transcribed using the SuperScript first-strandsynthesis system (Invitrogen Carlsbad CA USA) cDNAwas synthesized from the isolated RNA Cycle time valueswere obtained using real-time RT-PCR with the Power SYBR

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers used for real-time RT-PCRs

Target genes Primer pairs (51015840-31015840)Forward Reverse

ANF AGCATGGGCTCCTTCTCCAT TGGCCTGGAAGCCAAAAG120573-MHC CCTACAAGTGGCTGCCTGTGT ATGGACTGATTCTCCCGATCTG120572-SKA GGAGAAGATCTGGCACCATACATT AGCAGGGTTGGGTGTTCCTMMP-9 GGACGACGTGGGCTACGT CACGGTTGAAGCAAAGAAGGAp53 CAAAAGAAAAAACCACTTGATGGA CGGAACATCTCGAAGCGTTTATNFR CTCAGGTACTGCGGTGCTGTT GCACATTAAACTGATGAAGATAAAGGAFas GCTGCGCCTCGTGTGAA GCGATTTCTGGGACTTTGTTTCGAPDH AACGACCCCTTCATTGAC TCCACGACATACTCAGCAC

Counts versus acquisition time (min)

0

02

04

06

08

1

12

14

16

18

times102

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70 74 78 82 86 90 94 98 102 106 110 114 118

+ESI BPC Fragmentor = 1750V

7

6

542

1

3

Figure 1 LC-MS base peak chromatograms of FP The peak numbers refer to Table 1 FP (0ndash120min)

green PCR master mix (Applied Biosystems Foster CityCA USA) the iQ5 real-time PCR detection system andanalysis software (Bio-Rad Hercules CA USA) as previouslydescribed [23] Primers were designed using the AppliedBiosystems Primer Express Software (version 20) (Table 1)

28 Western Blot Analysis Heart tissues were lysed on icewith T-PER tissue or cell protein extraction reagent (PierceChemical Co Rockford IL) containing 01mMdithiothreitoland proteinase inhibitor cocktail Lysate preparation andWestern blot analysis were performed as previously described[21] Protein concentration was determined using a Bio-RadDC protein determination kit with BSA as the standardImmunoblots were developed using an ECL kit

29 Caspase-3 Caspase-8 and Caspase-9 Activity AssayCaspase-3 caspase-8 and caspase-9 activities were measuredusing a fluorometric assay kit (BioVision Mountain ViewCA USA) according to the manufacturerrsquos instructions Thesamples were subjected to a Fluoroskan Ascent FL fluorom-eter (Thermo Fisher Scientific Waltham MA USA) with400 nm excitation and 505 nm emission wavelengths Theresultswere expressed as fold change compared to the control

210 Biochemical Measurements The protein levels of ANFand 120573-MHC were quantified using ELISA according to

the manufacturerrsquos instructions (RampD Systems WiesbadenGermany)

211 Data Analysis The data are expressed as the mean plusmnSE The significance of the differences between the meanswas assessed using Studentrsquos 119905-test and 119901 values lower than005 were considered significant One-way ANOVA and Bon-ferroni corrections were used to determine the significanceformultiple comparisons Calculationswere performed usingSPSS (version 110) statistical software

212 Materials All chemicals were purchased from Sigma(St LouisMO) and all antibodies were purchased from SantaCruz Biotechnology (Santa Cruz CA)

3 Results

31 Chemical Profiling of FP By HPLC-Q-TOF-MS anal-ysis 7 well-separated chromatographic peaks in propolisflavonoids were identified in the BPC of FP (Figure 1) Sixof the peaks were characterized as pinocembrin kaempferolisosakuranetin pinobanksin-3-O-acetate 12-acetoxyvisci-done galangin and chrysin by comparison to the externalstandards and the mass data relative abundance and frag-mentation rules according to reported references [24ndash27](Table 2) And the extraction efficiency of the flavonoids


Table 2 Characteristics of chemical components of TFP by HPLC-HRMS

Peak number Retention time (min) 119898119911 for [M + H]+ MS119899 [M + H]+ Formula Identification Relative abundance ()1 48499 2570824 215 153 131 C

15

H12

O4

Pinocembrin 6742 50621 2870950 165 153 121 C

15

H10

O6

Kaempferol 6013 52497 2870931 245 217 C

16

H14

O5

Isosakuranetin 1714 53480 3150867 273 255 C

17

H14

O6

Pinobanksin-3-O-acetate 415 56231 2770852 232 216 C

15

H16

O5

12-Acetoxyviscidone 6516 58527 2710615 165 153 105 C

15

H10

O5

Galangin 1777 60755 2550676 209 153 129 C

15

H10

O4

Chrysin 447

Rela

tive t

rans

crip

t lev

els

FP + ISO151252550ISOCT

ANF mRNA

lowast

lowast

lowastlowast

0

2

4

6

8

10

(mgmiddotkgminus1middotdminus1)

(a)

Rela

tive t

rans

crip

t lev

elslowast lowast

0

1

2

3

4

5

151252550ISOCT

120573-MHC mRNA

FP + ISO(mgmiddotkgminus1middotdminus1)

(b)

Figure 2 FP represses isoproterenol-induced fetal gene reactivation in a dose-dependent manner Intragastric injection of various doses ofFP (1ndash50mgsdotkgminus1sdotdminus1) for 7 days was followed by continuous infusion with isoproterenol (ISO 25mgsdotkgminus1sdotdminus1) for 7 days to experimentallyinduce heart hypertrophy Heart tissues were collected and assayed for ANF (a) and 120573-MHC expression (b) The results are expressed as themeans plusmn SE 119899 = 10mice per group (lowast119901 lt 005 compared with isoproterenol group)

extraction from propolis is 3132 as determined by col-orimetric methods The purity of flavonoids is 5613 inchloroform-ethanol solution and increased to 7988 afterextracted by NaOH solution

32 FP Dose-Dependently Repressed ISO-Induced Fetal GeneReactivation Figure 2(a) shows that intragastric injectionof various doses of FP (1ndash50mgsdotkgminus1sdotdminus1) for 7 days wasfollowed by continuous infusion with isoproterenol (ISO25mgsdotkgminus1sdotdminus1) for 7 days to experimentally induce hearthypertrophy Heart tissues were collected and assayed todetermine ANF and 120573-MHC expression FP (50mgsdotkgminus1sdotdminus1)exerted no additional benefit to reduce heart hypertrophythus we selected 25mgsdotkgminus1sdotdminus1 for the following experi-ments

33 FP Attenuated ISO-Induced Cardiac Dysfunction in MiceTo determine whether FP could inhibit cardiac dysfunctionin vivo we performed echocardiography in the mice treatedwith various drugs The representative echocardiographicparameters are depicted in Table 3Themice infusedwith ISOshowed an enlarged left ventricular chamber size indicatedby the significant increase in the left ventricular internal

dimension values at the end diastole (LVIDd) and end systole(LVIDs) compared to those treated with saline (Table 3)Consistently these structural alterations were accompaniedwith marked impairment in cardiac contractile functionrepresented by the decrease in the left ventricular fractionalshortening (FS) and ejection fraction (EF) The treatmentwith FP significantly reversed the changes stimulated by ISOin these parameters (Table 3)

34 Reduced Cardiac Hypertrophy in Mice Treated with FPTo further verify the antihypertrophic effect of FP we directlymeasured the heart weight (HW) and heart-to-body weight(HWBW) ratio after the animals were euthanized As shownin Figure 3(a) after 7 d of ISO infusion HW significantlyincreased in ISO-treated mice compared to saline-treatedmice However FP treatment attenuated the increased HWand HWBW ratio

Histological sections were also analyzed using hematoxy-lin-eosin staining (Figures 3(b)ndash3(d)) whereas ANF mRNAand protein levels were quantified through qPCR (Fig-ure 3(e)) and ELISA assay (Figure 3(e)) As shown in Fig-ure 3(d) theCMcross-sectional areawas larger in ISO-treatedmice than in saline-treated mice FP treatment attenuated


CT FP ISO FP + ISO

lowast

40

45

50

55

60

65

HW

BW

(mg

g)

Average

lowast

08

09

10

11

12

13

Average

HW

(g)

CT FP ISO FP + ISO

(a)

FP

CT

ISO

Saline

(b)

FP

CT

ISO

Saline

(c)

lowast

CT ISO FP + ISO FP

Cardiomyocyte area

0

200

400

600

800

1000

Card

iom

yocy

te ar

ea (120583

m2)

(d)

CT ISO FP + ISO FP

Plas

mat

ic A

NF

leve

ls (n

gm

l)

ANF protein

lowastlowast lowast

CT ISO FP + ISO FP

Rela

tive t

rans

crip

t lev

els

ANF mRNA

0 00

05

10

15

20

25

5

10

15

20

25

(e)

Figure 3 Cardiac hypertrophy is reduced in mice treated by FP Intragastric injection of FP (25mgsdotkgminus1sdotdminus1) for 7 days followed bycontinuous infusion with isoproterenol (ISO 25mgsdotkgminus1sdotdminus1) for 7 days was sued to experimentally induce heart hypertrophy (a) Animalswere euthanized and the hearts were removed for hypertrophic evaluation A comparison of HW (g) and HWBW ratios (mgg) for theanimals is shown (b) Representative histological sections of hearts stained with HampE (c) Magnified images of histological sections in (b)used to determine CM cross-sectional area (d) Quantification of CM cross-sectional area in the left ventricular wall (e) mRNA expressionand plasma protein levels of the hypertrophy marker ANF The results are expressed as the means plusmn SE 119899 = 10 mice per group (lowast119901 lt 005compared to the control group 119901 lt 005 compared to the isoproterenol group)


Table 3 Echocardiography data after ISO-induced hypertrophy

Saline TFPCT (119899 = 5) ISO (119899 = 5) CT (119899 = 5) ISO (119899 = 5)

LVIDs (mm) 209 plusmn 031 301 plusmn 028lowast 211 plusmn 021 225 plusmn 035

LVIDd (mm) 360 plusmn 056 434 plusmn 034lowast 372 plusmn 042 395 plusmn 051

LVVs (120583L) 1429 plusmn 317 353 plusmn 804lowast 1835 plusmn 420 1726 plusmn 512

LVVd (120583L) 5428 plusmn 1063 8474 plusmn 1928 6389 plusmn 1311 7644 plusmn 1451EF () 7523 plusmn 834 5834 plusmn 591lowast 7128 plusmn 1155 7742 plusmn 1001

FS () 4320 plusmn 603 3058 plusmn 467lowast 3994 plusmn 698 4576 plusmn 920

SV (120583L) 3999 plusmn 1156 4944 plusmn 1264 4555 plusmn 1311 5918 plusmn 1003LVIDs left ventricular internal diameter at systolic phase LVIDd left ventricular internal diameter at diastolic phase LVVs left ventricle end-systolic volumeLVVd left ventricle end-diastolic volume EF ejection fraction FS fractional shortening SV stroke volume All measurements are means plusmn SE The data wereanalyzed by one-way ANOVA (lowast119901 lt 005 compared to saline controls 119901 lt 005 compared to ISO-induced saline-treated animals)

the increased cross-sectional area in ISO-treated mice Theexpression of the hypertrophic marker ANF also increased inISO-treatedmice compared to saline-treatedmiceMoreoverFP treatment reduced the increasedmRNA and protein levelsof ANF However FP alone had no significant effect on theseprocesses

35 FP Treatment Reduced Associated Disorder Such as Fibro-sis To evaluate fibrosis we evaluated the expression of 120572-skeletal actin (120572-SKA) mRNA andmatrix metalloproteinase-9 by using Massonrsquos trichrome staining (Figure 4) Consis-tently the ISO-treated hearts presented massive cell deathwith replacement fibrosis (Figure 4(b)) In contrast FPtreatment abbreviated ISO-elicited cardiac cell deathfibrosisUsing both measures ISO substantially increased the mRNAlevels of 120572-SKA (Figure 4(a)) Moreover FP treatment atten-uated the ISO-induced 120572-SKA levels However ISO and FPdid not significantly affect the MMP-9 levels Collectively FPtreatment attenuated cardiac hypertrophy and the associateddisorder such as fibrosis

36 Effects of ISO and FP onApoptotic Damage and Apoptosis-Related Gene Expression in Heart Tissues Apoptotic damagehas been implicated in cardiac hypertrophy To determinethe relationship between the cardioprotection of FP againstISO-induced cardiac hypertrophy and apoptosis we assessedthe morphology of mouse heart tissues using transmissionelectronmicroscopy (Figure 5(a))The results showed evidentheart tissue abnormalities including cytoplasmic vacuoliza-tion myofibrillar loss mitochondrial edema chromatincondensation and cardiomyocyte necrosis in the cardiacsections of ISO-induced mice The structural abnormalitiesin the hearts were partially prevented by cotreatment with FP(Figure 5(a)) Moreover the activation of caspase-3 resultedin the cleavage of PARP Caspase-3 activation is one ofthe key processes involved in apoptosis and contributes tomyocardial dysfunction and cardiac hypertrophy Figure 5(b)shows that myocardial caspase-3 activation was reduced inthe mice cotreated with FP and ISO compared to the micetreated with ISO alone as indicated by the increased activityFigure 5(c) shows that apoptotic damage was activated in themyocardium of ISO-infused mice This finding was depictedby the elevated caspase-8 caspase-9 and caspase-3 activities

(Figure 5(b)) and the increasedmRNA levels in p53 TNF-R1and Fas whichwere attenuated by FP treatment (Figure 5(c))

37 FP Prevented ISO-Induced Cardiac Remodeling In VivoWhich Is Correlated with the PI3KAKT Signaling As shownin Figure 6(a) Western blot analysis revealed significantlyenhancedAKTphosphorylation in the FP-treated hearts evenif the total AKT protein was similar in all animal groups FP-ISO cotreatedmice exhibited higher p-AKT in the hearts thanthose treated with FP alone however the difference was notsignificant These results indicated that the cardioprotectiveeffect of FP could be attributed to the antihypertrophic andantiapoptotic activities which possibly involved the PI3K-AKT signaling pathway

We also determined the functional consequences ofdisrupting the PI3K-AKT signaling pathway in pathologicalhypertrophy induction WM a selective PI3K inhibitor wasused to suppress PI3Ks The results were consistent with thefindings shown in Figure 2 ISO immediately upregulatedthe expression of ANF and 120573-MHC (Figure 6(b)) whereasFP treatment markedly antagonized ISO-induced fetal geneexpression (Figure 6(b)) However the ISO-induced increasein the ANF and 120573-MHCmRNAs was attenuated by FP in themice pretreatedwithWM (Figure 6(b)) whereas FP andWMshowed no significant effect compared to the control Fur-thermore as is shown in supplementary figure (see Supple-mentaryMaterial available online at httpdxdoiorg10115520166281376) cardiac hypertrophy phenotype is not alteredby WM These results suggested that the inhibition of PI3Kswith WM could suppress the FP-induced antihypertrophiceffect

4 Discussion

Natural drugs that exert cardioprotective properties havegained worldwide attention [9 13] Propolis a traditionalmedicine has been widely used for a thousand years asan anti-inflammatory and antioxidant drug However therole of propolis in cardiovascular diseases remains unknownPropolis possesses a wide range of biological activities Totalflavonoids are the main active component of propolis andexhibit strong antioxidant activities Therefore this studyassessed the cardioprotective effects of FP in the ISO-induced


120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)

Figure 4 FP treatment reduced associated disorder such as fibrosis (a) mRNA expression levels of the hypertrophy marker a-SKA and thefibrosis marker matrix metalloproteinase-9 (MMP-9) (b) Determination of fibrosis in histological sections by Massonrsquos trichrome stainingScale bar 50 120583m The arrows show fibrotic areas The results are expressed as the means plusmn SE 119899 = 10 mice per group (lowast119901 lt 005 comparedwith control group 119901 lt 005 compared with isoproterenol group)

C57BL6 mouse model as well as the mechanisms thatmediate the therapeutic activities of this drugThe significantfindings of this study were as follows (1) FP treatment signif-icantly attenuated adverse cardiac dysfunction hypertrophyand associated disorder such as fibrosis (2) FP treatment sig-nificantly attenuated apoptotic damage and cardiac remodel-ing which were correlated with the PI3KAKT signaling

Cardiac hypertrophy can be divided into two categoriesphysiological hypertrophy and pathological hypertrophy It isgenerally recognized that ANP level is increased in hyper-trophic hearts However some results also supported thatfactors can protect against pathological cardiac hypertrophyby activating PI3KAKT signaling [28] In accordance withour study factors can also attenuate pathological cardiac

hypertrophy by increasing AKT and effects can be blockedwith PI3K inhibition [23 29 30] Furthermore some fac-tors such as Cav-3 protect against cardiac hypertrophy andischemia by increasing p-AKT signaling and ANP partiallydue tomimicking ischemia-induced preconditioning [31 32]These seemingly opposite results may be partially ascribedto multiple functions of PI3K and complicated experimentalconditions varied in the settings of cardiac hypertrophyAlthough PI3K is a well-known survival factor which caninhibit cardiac apoptosis and cell death in some conditionssuch as exercise it can activate PI3KAKT signaling andleads to cardiac hypertrophy with little effect on cell deathPrevious experiments have shown that insulin or IGF-I mayregulate heart development [33] PI3K of the IA group could


FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)

Figure 5 Effects of isoproterenol and FP on apoptotic damage and apoptosis-related gene expression in heart tissues (a) Transmissionelectron microscopy of heart tissues (b) The activities of caspase-8 caspase-9 and caspase-3 were measured using a fluorometric assay andexpressed as the fold change over the control (c) mRNA levels of p53 TNFR1 and Fas were determined by real-time RT-PCR The levels ofmRNAwere normalized to GAPDH Relative mRNA levels are shown using arbitrary units and the value of the control group (CT) is definedas 1 The results are expressed as the means plusmn SE 119899 = 10mice per group (lowast119901 lt 005 compared to the control group 119901 lt 005 compared tothe isoproterenol group)


lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM

FP FPWM WMFP + ISO FP + ISO0

2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)

Figure 6 FP prevents isoproterenol-induced cardiac remodeling in vivo which is correlated with PI3KAKT signaling (a-b) Intragastricinjection of FP (25mgsdotkgminus1sdotdminus1) or saline (CT) for 7 days was followed by continuous infusion with isoproterenol (ISO 25mgsdotkgminus1sdotdminus1) for7 days to experimentally induce heart hypertrophy The animals were euthanized and the hearts were removed for hypertrophic evaluation(a) Representative Western blotting assays and quantitative analysis of phosphorylated AKT in the hearts by drug treatment (lowast119901 lt 005compared to the control group 119901 lt 005 compared to the isoproterenol group) (b) Effect of a selective PI3K antagonist wortmannin(WM) on isoproterenol-induced gene reactivationThe heart tissues were collected and assayed for ANF and 120573-MHC expressionThe resultsare expressed as the means plusmn SE 119899 = 10 mice per group (lowast119901 lt 005 compared to the isoproterenol group 119901 lt 005 compared to theFP-isoproterenol cotreated group)

be activated by IGF-I through the binding of ligands toPI3K120572 [34 35] After activation of PI3K plasma membranelipid phosphatidylinositol-45-bisphosphate is converted tophosphatidylinositol-345-triphosphate which may lead tothe activation of a series of signaling components To thebest of our knowledge our present study indicates that FPpreserves cardiac function with a decrease in levels of ANFand 120573-MHC and effects can be inhibited by PI3K antagonistwortmannin And FP induces activation in PI3KAKT sig-naling and induction in antiapoptotic and antioxidative prop-erties may also have complex effects on cardiac hypertrophysignaling

Pathological cardiac hypertrophy (PCH) is a vital andindependent predictor of cardiovascular mortality and isalso a key target in the treatment of HF [36ndash39] Studieshave shown that ISO-induced HF is associated with anarray of metabolic and biomedical disorders in experimentalanimals [2 23] ISO infusion causes myocardial ischemia andcardiomyocyte necrosis and progressively leads to diastolic

and systolic dysfunction in the heart which eventually resultsin PCH and HF [3] In the present study we also observeda significant cardiac dysfunction caused by ISO infusion asindicated by the enlarged left ventricular chamber size andmarked impairment in cardiac contractile function whichcan be partially attenuated by FP This finding was closelycorrelated with our data that FP can attenuate PCH andfibrosismarker expression PCH is characterized by increasedcell volume andmetabolic and biochemical disorders accom-panied by reactivation of fetal cardiac genes such as ANFand 120573-MHC In this study we first investigated the effectsof FP on the mRNA expression of these two fetal cardiacgenes PCH is a maladaptive response that could result inHF when unmatched cardiac cell death and fibrosis occurCollectively our study revealed that FP treatment attenuatedcardiac hypertrophy and associated disorder such as fibrosis

Apoptotic damage has been implicated in cardiac hyper-trophy [40 41] Apoptosis of cardiac cells is caused bybiomedical stimuli that eventually lead to morphological


changes in these cells The signals involved in cardiac hyper-trophy may finally result in cell death Because cardiomy-ocytes are nondividing cells the apoptotic cardiomyocytemay be largely replaced by fibrous tissues [42] Hence cardiaccell death has a close association with the transition ofhypertrophy to irreversible HF [41 42] Our present studyclearly showed that FP attenuated the apoptotic damage acti-vated in the myocardium of ISO-induced mice as evidencedby attenuating structural abnormities and decreasing theelevated caspase-8 caspase-9 and caspase-3 activities and theincreasedmRNA levels of p53 TNF-R1 and FasThese resultsrevealed that the recovery of PCH and cardiac dysfunctioninduced by ISO infusion may be closely associated withdiminished cardiac cell apoptosis after FP pretreatmentTo the best of our knowledge this study is the first todemonstrate the beneficial effects of FP on ISO-induced PCHin mice

5 Conclusions

In summary this study demonstrates that FP has potentcardioprotective activities against ISO-induced HF in theC57BL6mouse and these activities are correlatedwith PI3KAKT signaling Additional experiments should be performedto investigate the cardiac protection properties of FP FP maybe a novel therapeutic agent for inhibiting the progression ofPCH and HF

Competing Interests

The authors declare that there is no conflict of interests

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (no 81173543) The authors are gratefulto Professor Dacheng Jiang from Changchun University ofChinese Medicine for kindly authenticating the propolis

References

[1] E D Abel and T Doenst ldquoMitochondrial adaptations to phys-iological vs pathological cardiac hypertrophyrdquo CardiovascularResearch vol 90 no 2 pp 234ndash242 2011

[2] M A Allwood R T Kinobe L Ballantyne et al ldquoHemeoxygenase-1 overexpression exacerbates heart failure with agingand pressure overload but is protective against isoproterenol-induced cardiomyopathy in micerdquo Cardiovascular Pathologyvol 23 no 4 pp 231ndash237 2014

[3] M Anderson D Moore and D F Larson ldquoComparison ofisoproterenol and dobutamine in the induction of cardiachypertrophy and fibrosisrdquo Perfusion vol 23 no 4 pp 231ndash2352008

[4] T Aoyagi and T Matsui ldquoPhosphoinositide-3 kinase signalingin cardiac hypertrophy and heart failurerdquo Current Pharmaceu-tical Design vol 17 no 18 pp 1818ndash1824 2011

[5] MM Benjamin R L Smith andPAGrayburn ldquoIschemic andfunctional mitral regurgitation in heart failure natural history

and treatmentrdquo Current Cardiology Reports vol 16 no 8 p 5172014

[6] B C Bernardo K L Weeks L Pretorius and J R McMullenldquoMolecular distinction between physiological and pathologicalcardiac hypertrophy experimental findings and therapeuticstrategiesrdquo Pharmacology and Therapeutics vol 128 no 1 pp191ndash227 2010

[7] C Cook G Cole P Asaria R Jabbour and D P Francis ldquoTheannual global economic burden of heart failurerdquo InternationalJournal of Cardiology vol 171 no 3 pp 368ndash376 2014

[8] M G Rosca B Tandler and C L Hoppel ldquoMitochondria incardiac hypertrophy and heart failurerdquo Journal of Molecular andCellular Cardiology vol 55 no 1 pp 31ndash41 2013

[9] A C de Groot ldquoPropolis a review of properties applica-tions chemical composition contact allergy and other adverseeffectsrdquo Dermatitis vol 24 no 6 pp 263ndash282 2013

[10] H S Lim andMTheodosiou ldquoExercise ventilatory parametersfor the diagnosis of reactive pulmonary hypertension in patientswith heart failurerdquo Journal of Cardiac Failure vol 20 no 9 pp650ndash657 2014

[11] T A McKinsey and D A Kass ldquoSmall-molecule therapies forcardiac hypertrophy moving beneath the cell surfacerdquo NatureReviews Drug Discovery vol 6 no 8 pp 617ndash635 2007

[12] L Bacharova H Chen E H Estes et al ldquoDeterminants ofdiscrepancies in detection and comparison of the prognosticsignificance of left ventricular hypertrophy by electrocardio-gram and cardiac magnetic resonance imagingrdquo AmericanJournal of Cardiology vol 115 no 4 pp 515ndash522 2015

[13] A Prathapan V P Vineetha and K G Raghu ldquoProtective effectof Boerhaavia diffusa Lagainst mitochondrial dysfunction inangiotensin II induced hypertrophy in H9c2 cardiomyoblastcellsrdquo PLoS ONE vol 9 no 4 Article ID e96220 2014

[14] P S Saba M Cameli G Casalnuovo et al ldquoVentricular-vascular coupling in hypertension methodological consider-ations and clinical implicationsrdquo Journal of CardiovascularMedicine vol 15 no 11 pp 773ndash787 2014

[15] B Swynghedauw and B Chevalier ldquoBiological characteristics ofthe myocardium during the regression of cardiac hypertrophydue tomechanical overloadrdquo Journal of Heart Valve Disease vol4 supplement 2 pp S154ndashS159 1995

[16] C Tarin B Lavin M Gomez M Saura A Diez-Juanand C Zaragoza ldquoThe extracellular matrix metalloproteinaseinducer EMMPRIN is a target of nitric oxide in myocardialischemiareperfusionrdquo Free Radical Biology and Medicine vol51 no 2 pp 387ndash395 2011

[17] A Mavri H Abramovic T Polak et al ldquoChemical propertiesand antioxidant and antimicrobial activities of slovenian propo-lisrdquoChemistry and Biodiversity vol 9 no 8 pp 1545ndash1558 2012

[18] V C Toreti H H Sato G M Pastore and Y K Park ldquoRecentprogress of propolis for its biological and chemical composi-tions and its botanical originrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 697390 13 pages2013

[19] P K Gupta D J DiPette and S C Supowit ldquoProtective effectof resveratrol against pressure overload-induced heart failurerdquoFood Science amp Nutrition vol 2 no 3 pp 218ndash229 2014

[20] T Wang X Yu S Qu H Xu B Han and D Sui ldquoEffectof ginsenoside Rb3 on myocardial injury and heart functionimpairment induced by isoproterenol in ratsrdquo European Journalof Pharmacology vol 636 no 1ndash3 pp 121ndash125 2010


[21] H Xu X Yu S Qu Y Chen Z Wang and D Sui ldquoProtectiveeffect of Panax quinquefolium 20(S)-protopanaxadiol saponinsisolated from Pana quinquefolium on permanent focal cerebralischemic injury in ratsrdquoExperimental andTherapeuticMedicinevol 7 no 1 pp 165ndash170 2013

[22] H Yang Y Dong H Du H Shi Y Peng and X Li ldquoAntiox-idant compounds from propolis collected in Anhui ChinardquoMolecules vol 16 no 4 pp 3444ndash3455 2011

[23] L Yang X Cai J Liu et al ldquoCpG-ODN attenuates pathologicalcardiac hypertrophy and heart failure by activation of PI3K120572-Akt signalingrdquo PLoS ONE vol 8 no 4 Article ID e62373 2013

[24] C Sun ZWu ZWang and H Zhang ldquoEffect of ethanolwatersolvents on phenolic profiles and antioxidant properties ofBeijing propolis extractsrdquo Evidence-Based Complementary andAlternative Medicine vol 2015 Article ID 595393 9 pages 2015

[25] K Midorikawa A H Banskota Y Tezuka et al ldquoLiquid chro-matography-mass spectrometry analysis of propolisrdquo Phyto-chemical Analysis vol 12 no 6 pp 366ndash373 2001

[26] F Pellati G Orlandini D Pinetti and S Benvenuti ldquoHPLC-DAD and HPLC-ESI-MSMS methods for metabolite profilingof propolis extractsrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 55 no 5 pp 934ndash948 2011

[27] N Volpi and G Bergonzini ldquoAnalysis of flavonoids frompropolis by on-line HPLC-electrospray mass spectrometryrdquoJournal of Pharmaceutical and Biomedical Analysis vol 42 no3 pp 354ndash361 2006

[28] G Y OuditM A Crackower U Eriksson et al ldquoPhosphoinosi-tide 3-kinase 120574-deficientmice are protected from isoproterenol-induced heart failurerdquoCirculation vol 108 no 17 pp 2147ndash21522003

[29] Y SWeng H FWang P Y Pai et al ldquoTanshinone IIA preventsLeu27IGF-II-induced cardiomyocyte hypertrophy mediated byestrogen receptor and subsequentAkt activationrdquoTheAmericanJournal of Chinese Medicine vol 43 no 8 pp 1567ndash1591 2015

[30] P Chang Q Wang H Xu et al ldquoTetrahydrobiopterin reverseleft ventricular hypertrophy and diastolic dysfunction throughthe PI3Kp-Akt pathway in spontaneously hypertensive ratsrdquoBiochemical andBiophysical ResearchCommunications vol 463no 4 pp 1012ndash1020 2015

[31] Y T Horikawa M Panneerselvam Y Kawaraguchi et alldquoCardiac-specific overexpression of caveolin-3 attenuates car-diac hypertrophy and increases natriuretic peptide expressionand signalingrdquo Journal of the American College of Cardiologyvol 57 no 22 pp 2273ndash2283 2011

[32] H Okawa H Horimoto S Mieno Y Nomura M Yoshida andS Sasaki ldquoPreischemic infusion of alpha-human atrial natri-uretic peptide elicits myoprotective effects against ischemiareperfusion in isolated rat heartsrdquo Molecular and CellularBiochemistry vol 248 no 1-2 pp 171ndash177 2003

[33] J Heineke and J D Molkentin ldquoRegulation of cardiac hyper-trophy by intracellular signalling pathwaysrdquo Nature ReviewsMolecular Cell Biology vol 7 no 8 pp 589ndash600 2006

[34] G Y Oudit H Sun B-G Kerfant M A Crackower J MPenninger and P H Backx ldquoThe role of phosphoinositide-3kinase and PTEN in cardiovascular physiology and diseaserdquoJournal of Molecular and Cellular Cardiology vol 37 no 2 pp449ndash471 2004

[35] L CCantley ldquoThephosphoinositide 3-kinase pathwayrdquo Sciencevol 296 no 5573 pp 1655ndash1657 2002

[36] S Unzek andG S Francis ldquoManagement of heart failure a briefreview and selected updaterdquo Cardiology Clinics vol 26 no 4pp 561ndash571 2008

[37] E Braunwald ldquoHeart failurerdquo JACC Heart Failure vol 1 no 1pp 1ndash20 2013

[38] J RMcMullen and G L Jennings ldquoDifferences between patho-logical and physiological cardiac hypertrophy novel therapeu-tic strategies to treat heart failurerdquo Clinical and ExperimentalPharmacology amp Physiology vol 34 no 4 pp 255ndash262 2007

[39] D L Mann R Bogaev and G D Buckberg ldquoCardiac remod-elling and myocardial recovery lost in translationrdquo EuropeanJournal of Heart Failure vol 12 no 8 pp 789ndash796 2010

[40] A Haunstetter and S Izumo ldquoApoptosis basicmechanisms andimplications for cardiovascular diseaserdquo Circulation Researchvol 82 no 11 pp 1111ndash1129 1998

[41] T Kubota MMiyagishima C S Frye et al ldquoOverexpression oftumor necrosis factor-120572 activates both anti- and pro-apoptoticpathways in themyocardiumrdquo Journal ofMolecular and CellularCardiology vol 33 no 7 pp 1331ndash1344 2001

[42] T Hang Z Huang S Jiang et al ldquoApoptosis in pressureoverload-induced cardiac hypertrophy is mediated in partby adenine nucleotide translocator-1rdquo Annals of Clinical ampLaboratory Science vol 36 no 1 pp 88ndash95 2006

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


The flavonoid fraction is the main component of propolisMany flavonoids exhibit strong antioxidant activity [17]Our research has focused on studying the cardioprotectiveproperties of the flavonoids extraction from propolis (FP)Our preliminary results showed that FP exerts an effectiverole in cardiac protectionHowever the protective effect of FPon pathological cardiac hypertrophy (PCH) and HF remainsunknown This study shows that FP is a novel cardiopro-tective agent First FP significantly attenuates adverse car-diac dysfunction hypertrophy and associated disorder suchas fibrosis Secondly FP significantly attenuates apoptoticdamage and cardiac remodeling which is correlated withthe PI3KAKT signaling Thus FP may be clinically used toinhibit PCH progression and prevent HF

2 Materials and Methods

21 Preparation of FP Propolis was purchased from theAn Guo herbal medicine market in Hebei province ChinaThe propolis was authenticated by Professor Dacheng Jiangfrom the Changchun University of Chinese Medicine and avoucher specimen (number 1019907) was deposited at theJilin museum of Materia Medica Jilin province China Thesample was frozen at 4∘C before being ground into a powderThen 10x of 75 ethanolwater was added to extract theactive fractions This process was conducted twice and thefractions were filtered through a 045 120583m membrane afterbeing combined The filtrate was evaporated to dryness at80∘C under reduced pressure One g of the powder wasdissolved in 100mL chloroform-ethanol (10 1) and wasextracted twice by the same volume of 1 NaOH solutionThe aqueous alkali was adjusted to pH 6 by 1 HCl and thenleft at room temperature for another 24 h for precipitationThe FP were then obtained through filtration rinsed in waterto neutral pH and dried under reduced pressure

22 High Performance Liquid Chromatography-High-Resolu-tion Mass Spectrometry (HPLC-HRMS) Analysis of FP TheFP was dissolved in pure methanol and filtered through a022120583m membrane before HPLC-Q-TOF-MS analysis Theliquid chromatography separation was performed on anAgilent SB-Aq column (250mm times 46mm 45 120583m 400 bar)at 30∘C For HPLC analysis 01 formic acidwater (vv) and01 formic acidmethanol were used as the mobile phases Aand B respectively The gradient elution was programmed asfollows 0ndash110min (35ndash90 B) and 110ndash120min (90 B)Theflow rate was 10mLmin and the injected sample volumewas10 120583L The Q-TOF-MS scan range was set at119898119911 100ndash1200 inpositive modesThe dry gas (N

2) flow rate was 90 Lmin the

dry gas temperature was 350∘C the nebulizer gas was set at30 psi the capillary voltage was 3500V the fragmentor was175V and the skimmer was 65V

Standard pinocembrin kaempferol isosakuranetinpinobanksin-3-O-acetate 12-acetoxyviscidone galanginand chrysin (purity gt 98 by HPLC) were purchased fromthe National Institutes for Food and Drug Control of ChinaThey were mixed and dissolved in pure methanol at theconcentration of 2120583M per compound prior to use

Data analysis was performed on an Agilent MassHunterWorkstation Software-Qualitative Analysis (version B0400Build 404795 Service Pack 3 Agilent Technologies Inc2011)

23 Animals and In Vivo Pharmacological Treatment Themice used in this study were handled in compliance withthe guidelines for the care and use of laboratory animalsestablished by the Chinese Council on Animal Care andall animal protocols were approved by the Jilin UniversityAnimal Care and Use Committee Eight-week-old male micewere anesthetized with 15 isoflurane The adult mice wereintragastrically given different doses of FP (1ndash50mgsdotkgminus1sdotdminus1)for 7 d Alzet osmotic minipumps containing PBS or iso-proterenol (ISO) were surgically implanted subcutaneouslyin the interscapular region of the mouse ISO was cali-brated to release the drug at a rate of 25mgsdotkgminus1sdotdminus1 for7 d to experimentally induce heart hypertrophy The dose-dependent effect of FP on ISO-induced gene reactivationwas determined FP (50mgsdotkgminus1sdotdminus1) did not exert an addi-tional benefit to reduce heart hypertrophy thus we selected25mgsdotkgminus1sdotdminus1 for the following experiments In a separateexperiment mice were pretreated with the selective PI3Kantagonist wortmannin (WM) (1mgsdotkgminus1) at 1 h before ISOadministrationThe PI3K inhibitor doses were selected basedon the results of previous studies

24 Determination of Cardiac Dysfunction through Echocar-diography The animals were euthanized and the hearts wereremoved for hypertrophic evaluationThe analysis showed noeffect on cardiac function Cardiac function was examinedthrough echocardiography using a Vevo 770 microultra-sound system (VisualSonics Toronto Ontario Canada) asdescribed previously [17] Briefly an in vivo transthoracicechocardiography of the left ventricle was performed using a30MHz scan head interfaced with a Vevo 770 An ultrasoundbeamwas placed on the heart and near the papillary musclesHigh-resolution two-dimensional electrocardiogram-basedkilohertz visualization was achieved The parameters ofcardiac function were digitally measured on the M-modetracings and then averaged from three to five cardiac cycles

25 Histological Analyses The animals were euthanizedand the hearts were removed for hypertrophic evaluationSerial sections (4mm) of heart tissues were stained withhematoxylin-eosin [20] or Massonrsquos trichrome and thenvisualized using a light microscope as previously described

26 Transmission Electron Microscopy The animals wereeuthanized and the hearts were removed for hypertrophicevaluation Heart tissue sections were collected and observedby transmission electron microscopy

27 Real-Time RT-PCR Total RNA was extracted usingTRIzol (Invitrogen Carlsbad CA) Briefly 2mg of total RNAwas reverse transcribed using the SuperScript first-strandsynthesis system (Invitrogen Carlsbad CA USA) cDNAwas synthesized from the isolated RNA Cycle time valueswere obtained using real-time RT-PCR with the Power SYBR






0

02

04

06

08

1

12

14

16

18

times102

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70 74 78 82 86 90 94 98 102 106 110 114 118


7

6

542

1

3









3 Results





15

H12

O4

Pinocembrin 6742 50621 2870950 165 153 121 C

15

H10

O6

Kaempferol 6013 52497 2870931 245 217 C

16

H14

O5


17

H14

O6


15

H16

O5


15

H10

O5

Galangin 1777 60755 2550676 209 153 129 C

15

H10

O4

Chrysin 447

Rela

tive t

rans

crip

t lev

els


ANF mRNA

lowast

lowast

lowastlowast

0

2

4

6

8

10


(a)

Rela

tive t

rans

crip

t lev

elslowast lowast

0

1

2

3

4

5

151252550ISOCT

120573-MHC mRNA


(b)









CT FP ISO FP + ISO

lowast

40

45

50

55

60

65

HW

BW

(mg

g)

Average

lowast

08

09

10

11

12

13

Average

HW

(g)

CT FP ISO FP + ISO

(a)

FP

CT

ISO

Saline

(b)

FP

CT

ISO

Saline

(c)

lowast

CT ISO FP + ISO FP

Cardiomyocyte area

0

200

400

600

800

1000

Card

iom

yocy

te ar

ea (120583

m2)

(d)

CT ISO FP + ISO FP

Plas

mat

ic A

NF

leve

ls (n

gm

l)

ANF protein

lowastlowast lowast

CT ISO FP + ISO FP

Rela

tive t

rans

crip

t lev

els

ANF mRNA

0 00

05

10

15

20

25

5

10

15

20

25

(e)

















4 Discussion



120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















0

02

04

06

08

1

12

14

16

18

times102

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70 74 78 82 86 90 94 98 102 106 110 114 118


7

6

542

1

3









3 Results





15

H12

O4

Pinocembrin 6742 50621 2870950 165 153 121 C

15

H10

O6

Kaempferol 6013 52497 2870931 245 217 C

16

H14

O5


17

H14

O6


15

H16

O5


15

H10

O5

Galangin 1777 60755 2550676 209 153 129 C

15

H10

O4

Chrysin 447

Rela

tive t

rans

crip

t lev

els


ANF mRNA

lowast

lowast

lowastlowast

0

2

4

6

8

10


(a)

Rela

tive t

rans

crip

t lev

elslowast lowast

0

1

2

3

4

5

151252550ISOCT

120573-MHC mRNA


(b)









CT FP ISO FP + ISO

lowast

40

45

50

55

60

65

HW

BW

(mg

g)

Average

lowast

08

09

10

11

12

13

Average

HW

(g)

CT FP ISO FP + ISO

(a)

FP

CT

ISO

Saline

(b)

FP

CT

ISO

Saline

(c)

lowast

CT ISO FP + ISO FP

Cardiomyocyte area

0

200

400

600

800

1000

Card

iom

yocy

te ar

ea (120583

m2)

(d)

CT ISO FP + ISO FP

Plas

mat

ic A

NF

leve

ls (n

gm

l)

ANF protein

lowastlowast lowast

CT ISO FP + ISO FP

Rela

tive t

rans

crip

t lev

els

ANF mRNA

0 00

05

10

15

20

25

5

10

15

20

25

(e)

















4 Discussion



120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















15

H12

O4

Pinocembrin 6742 50621 2870950 165 153 121 C

15

H10

O6

Kaempferol 6013 52497 2870931 245 217 C

16

H14

O5


17

H14

O6


15

H16

O5


15

H10

O5

Galangin 1777 60755 2550676 209 153 129 C

15

H10

O4

Chrysin 447

Rela

tive t

rans

crip

t lev

els


ANF mRNA

lowast

lowast

lowastlowast

0

2

4

6

8

10


(a)

Rela

tive t

rans

crip

t lev

elslowast lowast

0

1

2

3

4

5

151252550ISOCT

120573-MHC mRNA


(b)









CT FP ISO FP + ISO

lowast

40

45

50

55

60

65

HW

BW

(mg

g)

Average

lowast

08

09

10

11

12

13

Average

HW

(g)

CT FP ISO FP + ISO

(a)

FP

CT

ISO

Saline

(b)

FP

CT

ISO

Saline

(c)

lowast

CT ISO FP + ISO FP

Cardiomyocyte area

0

200

400

600

800

1000

Card

iom

yocy

te ar

ea (120583

m2)

(d)

CT ISO FP + ISO FP

Plas

mat

ic A

NF

leve

ls (n

gm

l)

ANF protein

lowastlowast lowast

CT ISO FP + ISO FP

Rela

tive t

rans

crip

t lev

els

ANF mRNA

0 00

05

10

15

20

25

5

10

15

20

25

(e)

















4 Discussion



120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CT FP ISO FP + ISO

lowast

40

45

50

55

60

65

HW

BW

(mg

g)

Average

lowast

08

09

10

11

12

13

Average

HW

(g)

CT FP ISO FP + ISO

(a)

FP

CT

ISO

Saline

(b)

FP

CT

ISO

Saline

(c)

lowast

CT ISO FP + ISO FP

Cardiomyocyte area

0

200

400

600

800

1000

Card

iom

yocy

te ar

ea (120583

m2)

(d)

CT ISO FP + ISO FP

Plas

mat

ic A

NF

leve

ls (n

gm

l)

ANF protein

lowastlowast lowast

CT ISO FP + ISO FP

Rela

tive t

rans

crip

t lev

els

ANF mRNA

0 00

05

10

15

20

25

5

10

15

20

25

(e)

















4 Discussion



120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































4 Discussion



120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120572-SKA

lowast

CT ISO CT ISO00

05

10

15

20MMP-9

Rela

tive t

rans

crip

t lev

els

CT ISO CT ISO

0

1

2

3

4

Saline SalineFP FP

Rela

tive t

rans

crip

t lev

els

(a)

FP

CT

ISO

Saline

(b)






FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FPSaline

ISO

ISO

FPSaline

CT

CT

(a)

CT FP ISO FP + ISO

lowast

Casp

ase-

8 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

CT FP ISO

lowast

0

1

2

3

Casp

ase-

9 ac

tivity

(fol

d ov

er co

ntro

l)

FP + ISO CT FP ISO

lowast

Casp

ase-

3 ac

tivity

(fol

d ov

er co

ntro

l)

0

1

2

3

4

5

FP + ISO(b)

lowast

0

2

4

6

8

CT FP ISO

Rel

p53

mRN

A

FP + ISO

lowast

00

05

10

15

20

25

CT FP ISO

Rel

TNFR

mRN

A

FP + ISO0

1

2

3

4lowast

CT FP ISO

Rel

Fas m

RNA

FP + ISO(c)



lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

0

005

010

015

020

025

CT ISO FP + ISO FP

p-A

KTA

KT

CT ISO FP + ISO FP

AKT

p-AKT

(a)

CT CTISO ISOFP +

ISO + WM

FP +

ISO + WM


2

4

6

0

2

4

6

8

Rela

tive t

rans

crip

t lev

els

Rela

tive t

rans

crip

t lev

els

120573-MHC mRNA

lowast

lowast

ANF mRNA

(b)








5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Flavonoids Extraction from Propolis ...downloads.hindawi.com/journals/ecam/2016/6281376.pdfResearch Article Flavonoids Extraction from Propolis Attenuates Pathological