mi2014-640746

Research ArticleImmunomodulatory Effect of Red Onion(Allium cepa Linn) Scale Extract on Experimentally InducedAtypical Prostatic Hyperplasia in Wistar Rats

Ahmed A Elberry12 Shagufta Mufti3 Jaudah Al-Maghrabi3 Essam Abdel Sattar4

Salah A Ghareib5 Hisham A Mosli6 and Salah A Gabr7

1 Department of Clinical Pharmacy Faculty of Pharmacy King Abdulaziz University Jeddah 21589 Saudi Arabia2Department of Pharmacology Faculty of Medicine Beni Suef University Beni Suef Egypt3 Department of Pathology Faculty of Medicine King Abdulaziz University Jeddah 21589 Saudi Arabia4Department of Pharmacognosy Faculty of Pharmacy Cairo University Cairo Egypt5 Department of Pharmacology and Toxicology Faculty of Pharmacy King Abdulaziz University Jeddah 21589 Saudi Arabia6Department of Urology Faculty of Medicine King Abdulaziz University Jeddah 21589 Saudi Arabia7Department of Isotopes Applications Nuclear Research Center Atomic Energy Authority Cairo Egypt

Correspondence should be addressed to Ahmed A Elberry berry ahmedyahoocom

Received 19 November 2013 Revised 21 February 2014 Accepted 26 February 2014 Published 13 April 2014

Academic Editor Yona Keisari

Copyright copy 2014 Ahmed A Elberry et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Red onion scales (ROS) contain large amounts of flavonoids that are responsible for the reported antioxidant activity immuneenhancement and anticancer property Atypical prostatic hyperplasia (APH) was induced in adult castrated Wistar rats by bothsc injection of testosterone (05mgratday) and by smearing citral on shaved skin once every 3 days for 30 days Saw palmetto(100mgkg) as a positive control and ROS suspension at doses of 75 150 and 300mgkgday were given orally every day for 30 daysAll medications were started 7 days after castration and along with testosterone and citral The HPLC profile of ROS methanolicextract displayed two major peaks identified as quercetin and quercetin-41015840-120573-O-D-glucoside Histopathological examination ofAPH-induced prostatic rats revealed evidence of hyperplasia and inflammation with cellular proliferation and reduced apoptosisImmunohistochemistry showed increased tissue expressions of IL-6 IL-8 TNF-120572 IGF-1 and clusterin while TGF-1205731 wasdecreased which correlates with the presence of inflammation Both saw palmetto and RO scale treatment have ameliorated thesechanges These ameliorative effects were more evident in RO scale groups and were dose dependent In conclusion methanolicextract of ROS showed a protective effect against APH induced rats that may be attributed to potential anti-inflammatory andimmunomodulatory effects

1 Introduction

Atypical prostatic hyperplasia (APH) is a pseudoneoplasticlesion that can mimic prostate adenocarcinoma because ofits architectural and cytological features In this prostaticdisease there is an imbalance between prostate cell growthand apoptosis [1] The occurrence rate of APH is notknown and its associationswith benign prostatic hypertrophy(BPH) and latent carcinoma of the prostate (LPC) havenot been completely clarified [2] The fact that APH arisesalways in prostates with concomitant BPH and exhibits

several cancer-like features places APH as an intermediatelesion between BPH and the subset of well-differentiatedcancers in a hypothetical pathway between BPH and LPC[3]

The role of inflammation in prostate diseases is suggestedby the presence of inflammatory cells within the BPH [4]Inflammation is usually a self-limited event with initialproinflammatory cytokines growth factor release and angio-genesis followed by an anti-inflammatory cytokine-mediatedresolution [5] Chronic inflammation produces free radicalsas various reactive oxygen species [6]

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 640746 13 pageshttpdxdoiorg1011552014640746

2 Mediators of Inflammation

There is an interest in the potential health benefits ofAllium vegetables mainly onion (Allium cepa) and garlic(A sativum) Intake of Allium vegetables may be inverselyrelated to cancer at various sites including prostate [7 8]Allium vegetables seem to have antiproliferative action onhuman cancers and to prevent diseases associated with aging[9] Moreover onion and garlic intake has been inverselyassociated with BPH [10] Onions contain high levels ofnonnutrient antioxidant compounds flavonoids and thealk(en)yl cysteine sulfoxides (ACSOs) which have protectiveeffects against different degenerative pathologies such ascardiovascular disease cancer and other dysfunctions basedon oxidative stress [11]

The uses of bulb in food and manufacture introducetons of its scales (red dried onion scales ROS) as a wasteproduct Recently these scales have attracted the atten-tion of many researchers to maximize the benefits of thiswaste material ROS contain large amounts of flavonoidsmainly ferulic gallic kaempferol quercetin quercetin dimerquercetin trimer and quercetin glycosides These flavonoidsare responsible for the reported antioxidant activity of ROSwhich is comparable to that of 120572-tocopherol [12 13] Theyare also reported to have hepatoprotective effect immuneenhancement potential anti-infectious antistress and anti-cancer properties [14] Moreover quercetin was found toameliorate significantly prostatitis symptoms by decreasingprostatic inflammation [15] Most of the studies were doneusing an onion bulb per se bulb extracts or bulb juicewithout studying the protective effect of RO scales the richestpart of the onion with quercetin The aim of this studywas to investigate the protective effect of the ROS extracton enlarged rat prostate and the histopathological changesrelated to inflammation proliferation andor apoptosis inAPH induced experimentally in rats

2 Materials and Methods

21 Chemicals and Reagents Solvents of HPLC grade usedfor HPLC analysis in addition to analytical grade solvents forextraction and thin layer chromatography (TLC) were pur-chased fromSigma-Aldrich (St LouisMOUSA)Antibodiesagainst clusterin phospho-Smad2 and120573-actinwere obtainedfrom Santa Cruz Biotechnology (Santa Cruz CA anticlus-terin forWestern blot analysis) Upstate Biotechnology (LakePlacid NY anticlusterin for immunohistochemistry (IHC))and Cell Signaling Technology Inc (Danvers MA) Antibod-ies against TGF-1205731 ligands were purchased from Dakocy-tomation (Carpinteria CA) Citral was obtained from FlukaChemie AG Buchs Switzerland Testosterone was obtainedfrom Sigma-Aldrich andDakocytomation (Carpinteria CA)while saw palmetto was obtained from Futurebiotics (NYUSA)

22 Collection and Extraction of Dried Red Onion ScalesThe red onion bulbs (Allium cepa L Giza red cultivar) werecollected from a single field cultivated in Sohag city Sohaggovernorate Egypt It was cultivated in mid-November andcollected in the beginning of May 2010 The fully grown

bulbs were peeled and the ROS were stored at 25∘C in a darkplace till extractionThe plant sample was identified by a staffmember of the Horticulture Department Faculty of Agricul-ture Cairo University ROS powder (1250 g) was extractedwith 80 ethanol (3 times 5 liters) by using Ultra-Turrax T50homogenizer (Janke andKunkel IKA-Labortechnik StaufenGermany) at a temperature not exceeding 25∘C The extractwas concentrated by evaporation under reduced pressurelyophilized to give 240 g of reddish semisolid residue andprotected from light at 4∘C to use later

23 Standardization of Red Onion Scale (ROS) Extract Pre-liminary phytochemical tests to identify the main chemicalconstituents were carried out according to the methodsof Trease and Evans [16] Chromatographic TLC profileof the methanolic extract was performed using thin layerchromatography (solvent system ethyl acetate-formic acid-glacial acetic acid-water 100 11 11 26) as per fingerprintingusing a chromatographic condition reported by Wagner andBladt [17] against common reference flavonoids available inthe Pharmacognosy Department Faculty of Pharmacy CairoUniversity Total phenolics were determined as mg gallic acidequivalent per gram dry extract (mg GAEg DE) employingthe Folin-Ciocalteumethod described by Singleton and Rossi[18] Aluminum chloride colorimetric assay described byHertog [19] was applied for determination of total flavonoidscontent as mg quercetin equivalent per gram of dry extract(mgQEgDE)with a slightmodification [20]The free radicalscavenging activity of the extracts based on the scavengingactivity of the stable 11-diphenyl-2-picrylhydrazyl (DPPH)free radical was determined by the method described byBraca et al [21] The capability of the extracts to scavenge theDPPH radical was calculated using the following equationinhibition = (119860Control minus 119860Test) times 100119860Control

24 HPLCAnalysis Quercetin (Q) and quercetin-41015840-120573-O-D-glucoside (QG) were used as standard marker compoundsQGwas separated from theROS extract and identified using aprocedure reported by Abou Zid and Elsherbeiny [22] HPLCanalysis was performed on Agilent HP1200 series HPLC sys-tem equipped with a G1322A quaternary pump and degassera G1314A variable wavelength UV detector and 250 times40mm (particle size 5 120583m) ODS column (Lichrospher 100Merck Darmstadt Germany) Chromatographic separationwas achieved by applying a linear gradient system usingmobile phase A (5 formic acid in water vv) and mobilephase B (methanol) A linear gradient elution was performedusing 50 mobile phase B for 10min rising to 80 B over10min then to 100 B over 11min and back to 50 BThe flow rate was maintained at 10mLmin and UV wasmonitored at 360 nm

25 Experimental Animals Adolescent male Wistar ratsaged 50ndash60 days were obtained from the animal facilityof King Fahd Research Center King Abdulaziz UniversityJeddah Saudi Arabia They were used in the study accordingto the guidelines of the Biochemical and Research EthicsCommittee at King Abdulaziz University in accordance with

Mediators of Inflammation 3

the NIH guidelines Animals were housed in a well-ventilated temperature-controlled room at 22 plusmn 3∘C with a12 h light-dark cycle They were provided with standard ratchow pellets obtained from Grain Silos and Flour Mills orga-nization F-1005 Jeddah Saudi Arabia and tap water ad libi-tum All experimental procedures were performed between8ndash10 am and care was taken to avoid stressful conditions

Orchidectomy was performed aseptically under ethylether anesthesia by a midscrotal incision Following ligationof the spermatic cord and vessels testes and epididymis wereremoved The remaining stump was pushed back throughthe inguinal canal into the abdominal cavity and the scrotalsac was closed by sutures [23] After castration the rats weremaintained under standard laboratory conditions for 7 daysin order to allow a definite involution of the prostatic gland[24]

APH was induced as previously described by Engelsteinet al [25] in castrated rats using citral and testosterone Ratswere subcutaneously injected with testosterone propionatein corn oil (05mg01mLrat) each day for 30 days Citral1M diluted in 70 ethanol was smeared on a differentshaved area of skin each time on the back at a final doseof 185mgkg every 4 days for 30 days Control group ratswere smeared with the solvent (ethanol) alone at the shavedskin Considering the pungent lime fragrance of citral thecontrol groups were kept in a separate location from thecitral-treated animals to avoid possible false results as citralhas some influence via the olfactory tract

26 Animal Treatment Seven days after castration the ani-mals were randomly divided into six groups (119899 = 7) Group Iserved as a control (shamed operation) group and receivedboth corn oil (01mLrat) and 1 CMC-Na (03mL100 gbody weight) daily during the period of the experimentGroups from IIndashVI were castrated and had APH GroupII served as negative control and received CMC-Na 1 aspreviously mentioned Group III served as positive controland received saw palmetto (100mgkg) suspended in 1CMC-NaGroups from IVndashVIwere treatedwithROS at dosesof 75 150 or 300mgkgday respectively by oral gavageAll the ROS extracts were suspended in 1 CMC-Na andwere given to rats once daily by oral gavage along withtestosterone injection and citral smearing for 30 days asdescribed before [26] After euthanization ventral prostateof each rat in each group was removed from the body 24 hafter the last administration and weighed The half of thetissues were frozen in liquid nitrogen and stored at minus75∘Cuntil use The remaining tissues were fixed immediately in01M phosphate-buffered 10 formalin (pH 74) for 48 h andthen embedded in paraffin and were used for histologicalstudies Serial 4 120583m thick sections from each tissue specimenwere prepared and mounted on poly-L-lysine coated glassslides These were used for detection of IL-6 IL-8 TNF-120572IGF-1 clusterin and TGF-1205731 receptors in the prostatic tissueby immunohistochemistry

27 Histopathology and Histoscore A part of the ventrallobes was separated and fixed overnight in Stieversquos solution

Thereafter the tissue was thoroughly rinsed with water andimmersed overnight in ethanol 70Then it was dehydratedembedded in paraffin and 5mm thick sections were cutand stained by Harrisrsquo hematoxylin eosin according to thestandard procedures of Lillie [27]

A score-chart protocol (histoscore) developed by Scolniket al [26] was used to obtain an objective quantitativeassessment The examination description and scoring ofthe slides were performed in a blinded manner The scoringsystem was presented in arbitrary units to make a betterevaluation In a second step the cumulative score in eachgroup was correlated to the final histological diagnosis inorder to establish a score range for normal and hyperplasiaAn additional histological inflammatory score described byde Nunzio et al [28] was used to evaluate the inflammationScore 0 no inflammation score 1 scattered inflammatory cellinfiltrate without nodules score 2 no confluent lymphoidand score 3 large inflammatory areas with confluence

28 Immunohistochemistry and Immunhistoscoring Hema-toxylin and eosin staining was performed to observehistopathology For analysis of IL-6 and TGF-1205731 expressionsections from the paraffin embedded tissue blocks weremounted on charged glass slides and baked at 60∘C for 1 hin the oven then mounted on the Ventana staining machinedewaxed by EZ Prep (Xylene substitute) and rehydratedThetissue sections were heated in Ventana buffer CC1 (pH 6) tofacilitate antigen retrieval and treated with H

2O2to eliminate

endogenous peroxidase This was followed by incubation for60min at room temperature with primary antibodies IL-6and TGF-1205731 The dilutions used were IL-6 (dilution 1 50)and TGF-1205731 (dilution of 1 25) Subsequently the sectionswere incubated with biotinylated secondary antibody usingthe avidin-biotin complex method The immunoreactionwas visualized using diaminobenzidine All sections werelightly counterstained with hematoxylin as a backgroundThe positive control used for IL-6 and TGF-1205731 was from thecolon The negative controls comprised serial sections thatwere stained using equivalent concentrations of nonimmunemouse IgG in place of the primary antibodies The levelof staining was evaluated independently by three observersblinded to experimental conditions

Expression of TGF-1205731 and IL-6 was evaluated accordingto a semiquantitative scale 0 no detectable staining at all 1less than 10 of the cells stained positive 2 10minus50 positivecells and 3 more than 50 of cells positive [29] Stainingintensity was scored as 0 (no detectable stain) 1 (weakstaining detected at intermediate to high power) 2 (moderatedetected at low to intermediate power) to 3 (strong detectedat low power) [30]

29 Reverse Transcription Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from the snap-frozen tissuesamples using total RNA isolation kit (Macherey-Nagel)according to the manufacturerrsquos instructions RT was per-formed in a 10 120583L reaction mixture The RT reaction con-tained 1 120583g RNA 10mM Tris-HCl (pH 83) 50mM KCl15mM MgCl

2 25mM dithiothreitol 500120583molliter each


Table 1 Sequences of oligonucleotides used as primers

Gene Forward primer Reverse primer120573-Actin 51015840-GTCACCCACACTGTGCCCATCT-31015840 51015840-ACAGAGTACTTGCGCTCAGGAG-31015840

IL-6 51015840-GAACTCCTTCTCCACAAGCG-31015840 51015840-TTTTCTGCCAGTGCCTCTTT-31015840

IL-8 51015840-CTGCGCCAACACAGAAATTA-31015840 51015840-ATTGCATCTGGCAACCCTAC-31015840

TNF-120572 51015840-CAGAGGGAAGAGTTCCCCAG-31015840 51015840-CCTTGGTCTGGTAGGAGACG-31015840

TGF-1205731 51015840-GTTCTTCAATACGTCAGACATTCG-31015840 51015840-CATTATCTTTGCTGTCACAAGAGC-31015840

IGF-1 51015840-CACAGGCTATGGCTCCAGCAT-31015840 51015840-TCTCCAGCCTCCTCAGATCACA-31015840

Clusterin 51015840-CTGACCCAGCAGTACAACGA-31015840 51015840-TGACACGAGAGGGGACTTCT-31015840

of dATP dCTP dGTP and dTTP (Bioline) 40U RNasin(Bioline) 25 120583gmL oligo dT pd(T)12ndash18 (Bioline) and100UMoloney murine leukemia virus reverse transcriptase(Bioline) The reaction mixture was incubated at 42∘C for60min and then heated to 80∘C for 5min The resultantcDNA was used for PCR For quantitative real-time RT-PCR we prepared appropriate dilutions of each single-strandcDNA followed by normalizing of the cDNA content using120573-actin as a quantitative control Quantitative PCR amplifi-cation was performed with a 25120583L final volume consistingof 1 120583L RT reaction mixture 3mM MgCl

2 10 pmol of each

sense and antisense primer and 125 120583L (Roche Diagnostics)PCR conditions were as follows initial denaturation at 95∘Cfor 10min and 35 cycles of denaturation at 94∘C for 1minannealing at 55∘C for 1min and elongation at 72∘C for2 sec with a final elongation at 72∘C for 10min Sampleswere migrated in 1 agarose gel using electrophoresis UVvisualized and images were analyzed using total lab120(NonlinearDynamic Ltd) Clusterin TGF-1205731 IGF-1 IL-6 IL-8 and TNF-120572 expression in the test samples were normalizedto the corresponding 120573-actin level and were reported asthe relative band intensity to the 120573-actin gene expressionSequences of oligonucleotides used as primers for120573-actin IL-6 IL-8 TNF120572 TGF-1205731 IGF-1 and clusterin are summarizedin Table 1

210 Statistical Analysis Data were expressed as mean plusmn SEand were analyzed by analysis of variance (ANOVA) fol-lowed by Tukey-Kramer multiple comparisons test Inflam-mation scores and their significance were calculated byChi-square test with Yatersquos corrections Differences wereconsidered significant with a 119875 value less than 005 Statis-tical analyses were performed using the SPSS for Windows(v 100)

3 Results

31 Characterization of ROS Extract Screening of themethanolic onion extract indicated mainly the presence ofsterols andor terpenoids polyphenolic compounds suchas flavonoids tannins and the absence of alkaloids andsaponins Assay of total phenolic content of methanolicextract was determined to be 129mg GAEg DE Assay oftotal flavonoid content was determined to be 119mg QEgDE The antioxidant is expressed as inhibition percentage

corresponding to a reduction of the absorbance of DPPHof 50 (IC

50) Ascorbic acid a commonly used reference

antioxidant elicited 9621 inhibition at 1mMmL Onionextract was able to reduce the stable radical DPPH to theyellow colored diphenylpicrylhydrazine Thus it exhibitedobservable scavenging activity in a dose-related manner withIC50values of 368 120583gmL Figures 1 and 2 showed HPLC and

TLC profile of ROS extract against Q and QG as markercompounds The HPLC profile of ROS methanolic extractdisplayed two major peaks at tR 568 and 1101 at 360 nmidentified as quercetin and quercetin-41015840-120573-O-D-glucosidethrough spiking with standard quercetin and the isolatedquercetin-41015840-120573-O-D-glucoside The amount of quercetin inROS extract was found to be 601mggm of dry extract usingstandard quercetin (15mgmL)

32 Changes in Body Weight (BW) Absolute Prostatic Weight(APW) and Relative Prostatic Weight (RPW) In APH-induced rats BW has increased significantly at the end ofthe experiment compared to the starting weight Moreoverboth APW and RPW were increased significantly comparedto the normal control rats All treatments with saw palmettoor ROS failed to improve the BW at the end of the experimentcompared to the starting weight However saw palmettoinduced small but insignificant reduction in APW andRPW compared to control rats However ROS also inducedsignificant and dose-related reductions in both APW andRPW compared to control rats with APH (Table 2)

33 Effect of ROS on Proinflammatory Cytokines IL-6 IL-8and TNF-120572 Induction of APH in rats significantly increasedthe tissue levels of IL-6 IL-8 and TNF-120572 Saw palmetto treat-ment significantly decreased the IL-6 (203) IL-8 (481)and TNF-120572 (394) ROS induced significant reductionsin IL-6 IL-8 and TNF-120572 which were greater than thatproduced by saw palmetto (Figure 3) These reductions weredose dependant regarding IL-6 (547 59 and 656) IL-8 (93 953 and 979) and TNF-120572 (508 651 and913)

34 Effect of ROS on Gene Expression of TGF-120573R1 IGF-1and Clusterin Induction of APH is accompanied with asignificant increase of the clusterin and IGF-1 expression inthe ventral lobe of rat prostate while the TGF-120573R1 expressionwas significantly decreased Saw palmetto treated rats with


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Figure 1 (a) HPLC profile of red onion scales extract (ROS) and (b) chromatogram of quercetin-41015840-glucoside standard (UVmax 366 nm)

Table 2 Effect of red onion scales (RO scales) extract on absolute prostatic weight (APW) and relative prostatic weight (RPW) of rats withAPH treated for 30 days at different doses

Treatment Body weight (g) APW (g) RPW (mgg)Start End

Normal rats 19914 plusmn 516 229 plusmn 881 0106 plusmn 001 0452 plusmn 005

Rats with APH (negative control) 1876 plusmn 865 2414b plusmn 1255 0756lowast plusmn 005 3136lowast plusmn 019

Rats with APH treated withSaw palmetto (100mgkg) 1928 plusmn 459 2583b plusmn 1022 0698lowast plusmn 003 2705lowast plusmn 021

RO scales (75mgkg) 2275 plusmn 744 2711b plusmn 769 0681lowast plusmn 002 2512lowasta plusmn 012

RO scales (150mgkg) 2255 plusmn 715 2716b plusmn 920 0593lowasta plusmn 0023 2183lowasta plusmn 014

RO scales (300mgkg) 1962 plusmn 739 2627b plusmn 1622 0547lowasta plusmn 0075 2082lowasta plusmn 021lowastSignificantly different from normal rats at 119875 lt 005aSignificantly different from rats with APH at 119875 lt 005bSignificantly different from the value before starting the experiment at 119875 lt 005

Figure 2 TLC profile of red onion scales 1 methanol extract 2quercetin-41015840-glucoside 3 rutin 4 quercetin UV 366 nm

APH showed significant increase in clusterin and TGF-120573R1 expression an effect which was accompanied with thedecrease in IGF-1 expression All the treatment groups withROS extract showed a significant increase in clusterin while

the expression of IGF-1 was significantly decreased in a dose-dependent fashion On the other hand TGF-120573R1 expressionwas not significantly changed compared to the APH group(Figure 4)

35 Histological Changes Ventral prostates from the normalgroup showed normal histology with average histoscorecorresponding to 243 plusmn 12 (Figure 5(a)) Ventral prostatesfrom the APH-induced rats showed increase in the num-ber of acini (hyperplasia) with irregular distribution Theacini were arranged back to back with intraluminal andstromal papillary projections All prostatic acini were linedby tall columnar cells with epithelial pilling and nuclearstratification seen among six of seven prostatic sections inthe group Prostatic sections from two rats in this groupshowed 1-2 mitotic figures The interstitial stroma was scantand showed edema with congested blood vessels and mixedacute and chronic perivascular inflammatory cells Polymor-phonuclear leukocytes with eosinophilic granules were con-sidered as acute inflammatory cells Chronic inflammatorycells seen were lymphocytes which were distinguished bytheir darkly stained ink-dot nucleus and thin rim basophiliccytoplasm These findings were associated with a significant


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RO scales 75mgkgRO scales 150mgkgRO scales 300mgkg

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Figure 3 The expression of IL-6 IL-8 and TNF-120572 target genes in ventral prostate of Wistar rats after treatment with RO scales at differentdose levels All values are expressed as mean plusmn SE of the relative band intensity (RBI) using 120573-actin as a reference lowastSignificantly differentfrom normal shame control rats at 119875 lt 0001 Significantly different from APH control rats received CMC at 119875 lt 0001

increase in the histoscore corresponding to 411 plusmn 18(Figures 5(b)ndash5(d))

The ventral prostate in the saw palmetto group showeddiminution regarding the scale of pathological changes andhistoscore corresponding to 322 plusmn 21 (Figure 5(e)) Theprostatic acini were less crowded However hyperplasticchanges such as acini lined by tall columnar cells withepithelial pilling and nuclear stratification were seen amongall rats in the group The nuclei were round to oval regularand basally placed There was no loss of polarity notedin the epithelium Prostatic sections from two rats in thisgroup also showed 1-2 mitotic figures The interstitial stromawas considerable fibromuscular to edematous and showedvascular congestion Prostatic sections from one rat showedsmall foci of mixed inflammatory cells

Histological changes in the ventral lobe of APH-inducedrats treated with ROS extract (75mgkg) showed irregularand enormous dilatation of prostatic acini with intraluminalsecretions among five of six rats in the group These changeswere more pronounced in the central regions of the prostatictissue Dilated acini showed epithelial changes in the form offlattening or low cuboidal transformation (Figure 6(a)) Fewacini showed loss of lining epithelium although the basementmembrane appeared still intact Acini seen at the peripheral

part of the section were still hyperplastic being lined by tallcolumnar cells showing nuclear stratification (Figure 6(b))Mitotic figures were absent and the surrounding stromashowed edema and congested blood vessels Mixed acute andchronic inflammatory cells were seen among four of six ratsin the group alongwith clustering of stromalmast cells Kary-orrhectic debris was seen within the gland lumina amongfour of six rats in the group These findings corresponded toa histoscore of 353 plusmn 12

Sections examined from ventral prostates of APH-induced rats treated with ROS extract (150mgkg) showedprostatic acini lined by tall columnar epithelium exhibitingnuclear stratification in all five rats The acinar distensionwas slightly diminished Few acini showed scalloped luminalsecretions among three of five rats Mitotic figures wereabsent and surrounding stroma showed edema and congestedblood vessels Small foci of mixed acute and chronic inflam-matory cells were seen among three of five rats in the groupIntraluminal sloughing of karyorrhectic debris was also seenamong three of five rats (Figures 6(c) and 6(d)) Clusteringof stromal mast cells was seen among three of five rats Thesefindings corresponded to a histoscore of 340 plusmn 05

Histological changes in ventral prostates of rats withAPH treated with ROS extract (300mgkg) showed prostatic


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Figure 4 The expression of TGF-120573R1 IGF-1 and clusterin target genes in ventral prostate of Wistar rats after treatment with RO scales atdifferent dose levels All values are expressed as mean plusmn SE of the relative band intensity (RBI) using 120573-actin as a reference lowastSignificantlydifferent from normal shame control rats at 119875 lt 0001 Significantly different from APH control rats receiving CMC at 119875 lt 0001

acini lined by tall columnar epithelium exhibiting nuclearstratification in all six rats Acinar distension was slightlydiminished Scatteredmixed acute and chronic inflammatorycells in the stroma along with intraluminal sloughing of kary-orrhectic debris were seen in three of six rats (Figure 6(e))Mitotic figures were absent and surrounding stroma showededema and congested blood vessels Clustering of stromalmast cells was seen among two rats These findings corre-sponded to a histoscore of 322 plusmn 08

36 Immunohistochemistry Normal control rats showed noexpression of both immunohistochemical markers with pos-itive internal controls (Figures 7(a) and 7(b)) TGFBR-1expression corresponded to score 3 with strong intensityin the secretory epithelial cells of acini with foci of strongexpression among basal cells also in the prostatic sectionsfrom APH-induced rats IL-6 expression corresponded toscore 1 and showed weak intensity in the secretory epithelialcells only No expression was observed in basal or stromalcells (Figures 7(c) and 7(d))

Prostatic sections from saw palmetto treated APH-induced rats revealed TGFBR-1 expression corresponding toscore 3 exhibiting strong intensity in the secretory epithelialcells of acini with foci of strong expression also in the basalcells IL-6 was variable between score 2-3 with a staining

intensity between moderate to strong No expression wasobserved in the basal or stromal cells (Figures 8(a) and 8(b))

Prostatic sections fromRO scale revealed IL-6 expressioncorresponding to score 1 with weak intensity TGFBR-1expression was score 0 No expression was observed in thebasal or stromal cells The immunohistochemical profileexpressed by the prostatic tissue showed no notable dozerelated variation (Figures 8(c) and 8(d))

4 Discussion

It was reported that APH usually arises with concomitantBPH and exhibits several cancer-like features which makesAPH an intermediate lesion between BPH and the subset ofwell-differentiated prostate cancers [31] In the present studyAPH-induced rats revealed prostatic enlargement which wasevidenced by an increase in the APW and RPW of theventral prostate lobe as well as acinar hyperplasia that maybe attributed to the influence of androgen on prostate growth[32] The development of hyperplasia was associated withenhanced proliferation and suppressed apoptosis of prostaticcells [25 33] Citral used in the current study was reported tohave an estrogenic-like effect as it binds to estrogen receptorsthat are located in prostatic epithelial cells of both human[34] and rat [35] Type 2 estrogen-binding sites in rat ventral


(a) (b)

(c) (d)

(e)

Figure 5 (a) Prostate of normal rats with round acini and intact basement membranes Arrow points to acini lined by two layers of lowcuboidal epithelium (b) Prostate of testosterone and citral treated castrated rats with atypical prostatic hyperplasia (APH) reveals increasein the number of prostatic acini (hyperplasia) which are placed back to back with no intervening stroma Thin arrow points to acini linedby tall columnar cells Thick arrow points to nuclear stratification and stromal projections (c) Interstitial stroma shows congested bloodvessels and inflammatory cells (d) Thick vertical down arrow points to polymorphonuclear acute inflammatory cells and the thin slantingarrows point to chronic inlammatory cells (lymphocytes) Very thin arrow points to luminal papillary projections thick long arrow pointsto nuclear stratification in the acini and small arrow points to polymorphonuclear acute inflammatory cells in between prostatic acini (e)Prostate of saw palmeto treated rats showing hyperplastic changes Thick arrow points to acini lined by tall columnar cells showing nuclearstratification Thin arrow points to a small focus of polymorphonuclear acute inflammatory cells in stroma Acinus at the base of the figureshows intraluminal projection (at 60X)

prostate could be responsible for this proliferative effect [35]Moreover the present study revealed an increase in the IGF-1and a decrease in the TGF-1205731 which may cause the prostateenlargement in APH group TGF-1205731 is known to suppresstissue proliferation and induce cell apoptosis [36] Circulatingtestosterone acts locally in the prostate via the productionof growth factors such as IGF and TGF families that act in

a manner which influences prostate cell growth survival orapoptosis [37]

Fruits and vegetables have health benefits and are goodsources of antioxidants therefore a lot of recent literaturehas focused on nutritional and herbal medicine for prostatichyperplasia [38ndash40] In the present study administration ofROS extract induced significant and dose-related reduction


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(c) (d)

(e)

Figure 6 (a)-(b) Prostate of rats treatedwith RO scale extract in a dose of 75mgkg showing (a) Irregularly dilated acini filled with secretionsArrow points to thinning and flattening of lining epithelium (b) Acinus at the periphery of the field is lined bytall columnar epithelial cellsshowing nuclear stratification Arrow points to polymorphonuclear acute inflammatory cell infiltrate in stroma near the acini (c)-(d) Prostateof rats treated with RO scale extract in a dose of 150mgkg showing (c) Arrow pointing to reduced acinar distension which are lined by tallcolumnar cells exhibiting nuclear stratification (d) Arrow pointing to luminal karyorrhectic debris (e) Prostate of rats treated with RO scalesin a dose of 300mgkg showing Thin arrow points to luminal karyorrhectic debris and thick arrow points to hyperplasia as seen by tallcolumnar cells lining the acini with nuclear stratification (at 60X)

in both the absolute and relative weight of prostate inthe APH-induced rats an effect that was greater than thatinduced by saw palmetto These effects may be due to thefact that ROS are rich in flavonols which represent 60 ofthe total active constituents These mainly are kaempferolquercetin quercetin dimer quercetin trimer and otherquercetin glycoside Flavonols were found to be responsiblefor the antioxidant hepatoprotective anticancer antimicro-bial antistress and other biological activities [12 13] Amongthe major constituents of the ROS extract 16 are quercetin

which was found to be mainly responsible for the protectiveeffects against different degenerative pathological diseases[11 14] It was reported that administration of quercetin alongwith finasteride resulted in reduction in prostate weight inrats through a cell cycle-related pathway that may functionindependently of androgens [41]

Moreover the ROS extract has ameliorated the his-topathological changes significantly modulated the expres-sion of the proinflammatory cytokines and decreased the in-flammatory scores in a dosedependentmanner In the current


(a) (b)

(c) (d)

Figure 7 Immunohistochemical expression of TGF120573R-1 and IL-6 in prostates of normal and APH rats (a) Normal rats TGF120573R-1 expressionscore 0 (b) Normal rats IL-6 expression score 0 (c) APH rats TGFBR-I expression score 3 (d) APH rats IL-6 expression score 1 (at 60X)

(a) (b)

(c) (d)

Figure 8 (a)-(b) Immunohistochemical expression of TGF120573R-1 and IL-6 in saw palmetto 100 g treated rats (a) TGF120573R-1 score 3 (b) IL-6expression score 2-3 (c)-(d) Immunohistochemical expression of TGFBR-1 and IL-6 in RO scales 75mg treated rats (c) TGF120573R-1 score 0Note negativity with positive internal control within the stroma in the upper right of the field (d) IL-6 expression score 2 (at 60X)


study ROS significantly decreased IL-6 IL-8 and TNF-120572 inthe ventral lobe of prostatic tissues of rats with APH Thesefindings are in accordance with results that are reported byJung et al [42]

In the context of chronic inflammation and expressionof proinflammatory cytokines IL-6 is one of the majorphysiologic mediators of acute phase reaction that influenceimmune responses and inflammatory reactions [43] IL-6 isalso secreted by both normal and prostatic epithelial cellsand acts as a growth factor for normal prostatic epithelialcells [44] TNF-120572 a proinflammatory cytokine may induceinflammation by induction of COX-2 superoxide radicaland hydrogen peroxide in both human and rat mesangialcells [45] TGF-120573 an inflammatory cytokine has been shownto regulate stromal proliferation and differentiation in BPHand it is a key factor in the androgen control of prostaticgrowth Recently Descazeaud et al [46] investigated theTGF-120573 receptor II protein (TGFBRII) expression in BPHpatients They observed a significant association betweenTGFBRII stromal staining and prostatic volume

The ROS extract contains large amounts of antioxidantflavonoids mainly ferulic gallic kaempferol and quercetinor their glycosides [12 13] Jung et al [42] found thathepatic expressions of TNF-120572 and IL-6 in diabetic rats weresuppressed by quercetin These results are in agreementwith previous reports in which quercetin was found tohave antioxidative and anti-inflammatory activities [47]Quercetin significantly decreases prostatitis symptoms bydecreasing prostatic inflammation [15] Moreover quercetincan affect the prostate cancer biology by inhibiting arachi-donic acid metabolism through the blocking of phospho-lipase A2 and 5 as well as 12-lipooxygenase enzymes [48]and inhibiting androgen receptor mutations [49] Quercetinhas also demonstrated the ability to interrupt the spreadof prostate cancer (metastases) and to promote cell deathIt was able to decrease the activity of specific enzymesknown to be involved in tumor invasion and metastases [50]Furthermore quercetin was found to have antiproliferativeactivity in vitro against several cancer cells [51] and arrestedcell cycle progression either at the G1S phase [50] or at theG2M transitional boundary [52] Quercetin is also a possibleoption to relieve symptoms for men who have prostateproblems and it has been identified as being beneficial incases of prostatitis [53]

The ROS extract significantly reduced the absolute andrelative weight of ventral lobe prostate of rats with APH com-pared with the negative control rats This effect may be par-tially due to that ROS extract may have antiproliferative andapoptotic action on the ventral lobe of prostatic tissues TheROS extract significantly decreased the expression of IGF-1and increased clusterin expressionThe prostate enlargementand the increased net weight of the ventral lobe in case ofAPHmay be partly due to the increased IGF-1 and decreasedTGF-1205731 expression This modulation in expression of thesecytokines may play a crucial role in prostate cell proliferationand apoptosis These findings are in agreement with that ofWu et al [54] IGF-1 a mitogenic factor interacts with IGF-1 receptor stimulating cell proliferation [55] and inducing

proliferative prostatic diseases [56] Moreover Senthilkumaret al [57] suggested that quercetin can decrease the survivalof androgen-independent prostate cancer cells by changingthe expression of IGF-1 signaling and inducing apoptosis incancer patients

The antiproliferative effect of quercetin is probably medi-ated by interactionwith the type II estrogen binding sites [58]Quercetin also inhibits cell invasion and induces apoptosisthrough a pathway involving heat shock proteins [59] Theability of RO extract to decrease the prostate weight in ratswith APH may also be due to that it has some active con-stituents with a specific ability to inhibit phosphodiesterasesubtype 5A (PDE-5A) These findings are in agreement withthat which was reported by Lines and Ono [60] who foundthat the improvement in sexual function might be due toquercetin 1 Quercetin 1 was reported to have specific PDE-5Ainhibitory activity [55] Chronic administration of NO donordrugs and PDE5-inhibitors may also induce antiproliferativeandor apoptotic effects in the prostate [61]

In conclusion the results of the present study showed thatthe methanolic extract of ROS have the ability to decreasethe prostate weight in APH-induced rats This effect may bedue to the anti-inflammatory and immunomodulatory effectsof the extract All of these effects are dose-dependent andmay not only be related to the antioxidant activities of theflavonoids or their glycosides but also due to its phenoliccontent in ROS

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by Sheikh Ahmed H FetaihiChair for Research on Prostatic Diseases King AbdulazizUniversity Jeddah Saudi ArabiaThe authors would also liketo thankMr IslamFarouk Department of Pharmacology andToxicology Faculty of Pharmacy King Abdulaziz UniversityJeddah Saudi Arabia for his great effort and help in theexperimental study

References

[1] A Sciarra G Mariotti S Salciccia et al ldquoProstate growth andinflammationrdquo Journal of Steroid Biochemistry and MolecularBiology vol 108 no 3ndash5 pp 254ndash260 2008

[2] K Stamatiou A AlevizosM Natzar et al ldquoAssociations amongbenign prostate hypertrophy atypical adenomatous hyperplasiaand latent carcinoma of the prostaterdquo Asian Journal of Androl-ogy vol 9 no 2 pp 229ndash233 2007

[3] D G Bostwick J Srigley D Grignon et al ldquoAtypical ade-nomatous hyperplasia of the prostate morphologic criteriafor its distinction from well-differentiated carcinomardquo HumanPathology vol 24 no 8 pp 818ndash832 1993

[4] G Novara A Galfano R B Berto V Ficarra R V Navarreteand W Artibani ldquoInflammation apoptosis and BPH what isthe evidencerdquo European Urology vol 5 no 4 pp 401ndash4092006


[5] J S Sandhu ldquoProstate cancer and chronic prostatitisrdquo CurrentUrology Reports vol 9 no 4 pp 328ndash332 2008

[6] B Rigas and Y Sun ldquoInduction of oxidative stress as amechanismof action of chemopreventive agents against cancerrdquoBritish Journal of Cancer vol 98 no 7 pp 1157ndash1160 2008

[7] K Rahman andGM Lowe ldquoGarlic and cardiovascular diseasea critical reviewrdquo Journal of Nutrition vol 136 3 pp 736Sndash740S2006

[8] C Galeone C Pelucchi F Levi et al ldquoOnion and garlic use andhuman cancerrdquo American Journal of Clinical Nutrition vol 84no 5 pp 1027ndash1032 2006

[9] A de Martino G Filomeni K Aquilano M R Ciriolo and GRotilio ldquoEffects of water garlic extracts on cell cycle and viabilityof HepG2 hepatoma cellsrdquo Journal of Nutritional Biochemistryvol 17 no 11 pp 742ndash749 2006

[10] F Bravi C Bosetti L dal Maso et al ldquoFood groups and risk ofbenign prostatic hyperplasiardquo Urology vol 67 no 1 pp 73ndash792006

[11] G Griffiths L Trueman T Crowther BThomas and B SmithldquoOnionsmdashaglobal benefit to healthrdquoPhytotherapyResearch vol16 no 7 pp 603ndash615 2002

[12] N L Tram C Hazama M Shimoyamada H Ando K Katoand R Yamauchi ldquoAntioxidative compounds from the outerscales of onionrdquo Journal of Agricultural and Food Chemistry vol53 no 21 pp 8183ndash8189 2005

[13] A Gulsen D P Makris and P Kefalas ldquoBiomimetic oxidationof quercetin isolation of a naturally occurring quercetin het-erodimer and evaluation of its in vitro antioxidant propertiesrdquoFood Research International vol 40 no 1 pp 7ndash14 2007

[14] B N Singh B R Singh R L Singh et al ldquoPolyphenolicsfrom various extractsfractions of red onion (Allium cepa) peelwith potent antioxidant and antimutagenic activitiesrdquo Food andChemical Toxicology vol 47 no 6 pp 1161ndash1167 2009

[15] D A Shoskes S I Zeitlin A Shahed and J Rajfer ldquoQuercetinin men with category III chronic prostatitis a preliminaryprospective double-blind placebo-controlled trialrdquo Urologyvol 54 no 6 pp 960ndash963 1999

[16] G E Trease and W C Evans Pharmacognosy Bailliere TindallPress London UK 1983

[17] H Wagner and S Bladt Plant Drug Analysis A Thin LayerChromatography Atlas Springer London UK 1996

[18] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology and Viticulture vol 16 pp 144ndash158 1965

[19] M G L Hertog ldquoContent of potentially anticarcinogenicflavonoids of 28 vegetables and 9 fruits commonly consumedin the Netherlandsrdquo Journal of Agricultural and Food Chemistryvol 40 no 12 pp 2379ndash2383 1992

[20] E A Abdel-Sattar S M Mouneir G F Asaad and HM Abdallah ldquoProtective effect of Calligonum comosum onhaloperidol-induced oxidative stress in ratrdquo Toxicology andIndustrial Health vol 30 no 2 pp 147ndash153 2014

[21] A Braca D Nunziatina D B Lorenzo P Cosimo PMateo andM Ivano ldquoAntioxidant principles from Bauhinia terapotensisrdquoJournal of Natural Products vol 64 pp 892ndash895 2001

[22] S F Abou Zid and G M Elsherbeiny ldquoIncrease in flavonoidscontent in red onion peel bymechanical shreddingrdquo Journal ofMedicinal Plants Research vol 2 no 9 pp 258ndash260 2008

[23] E Golomb A Kruglikova D Dvir and A AbramovicildquoInduction of atypical prostatic hyperplasia in rats by sympath-omimetic stimulationrdquoThe Prostate vol 34 pp 214ndash221 1998

[24] L Sandford J W Searle and J F Kerr ldquoSuccessive wavesof apoptosis in the rat prostate after repeated withdrawal oftestosterone stimulationrdquo Pathology vol 16 no 4 pp 406ndash4101984

[25] D Engelstein J Shmueli S Bruhis C Servadio and AAbramovici ldquoCitral and testosterone interactions in inducingbenign and atypical prostatic hyperplasia in ratsrdquo ComparativeBiochemistry and Physiology vol 115 no 2 pp 169ndash177 1996

[26] M D Scolnik C Servadio and A Abramovici ldquoComparativestudy of experimentally induced benign and atypical hyperpla-sia in the ventral prostate of different rat strainsrdquo Journal ofAndrology vol 15 no 4 pp 287ndash297 1994

[27] R D LillieHistopathologic Techniques and Practical Histochem-istry McGraw-Hill New York NY USA 1965

[28] C de Nunzio G Kramer M Marberger et al ldquoThe contro-versial relationship between benign prostatic hyperplasia andprostate cancer the role of inflammationrdquo European Urologyvol 60 no 1 pp 106ndash117 2011

[29] AHobischH Rogatsch andAHittmair ldquoImmunohistochem-ical localization of interleukin-6 and its receptor in benignpremalignant and malignant prostate tissuerdquo The Journal ofPathology vol 191 pp 239ndash244 2000

[30] S K Gounder V T Chang D Hoover et al ldquoProstate cancerimmunohistochemical (IHC) stains and survival in stage D3patientsrdquo J Clin Oncol vol 26 22187 no 15 2008

[31] D G Bostwick W H Cooner L Denis G W Jones PT Scardino and G P Murphy ldquoThe association of benignprostatic hyperplasia and cancer of the prostaterdquo Cancer vol70 no 1 pp 291ndash301 1992

[32] A Abramovici C Servadio J Shmuely and U SandbankldquoExperimental induction of atypical hyperplasia in rat ventralprostaterdquo Progress in clinical and biological research vol 243 pp559ndash568 1987

[33] AAGeldof C Engel andB R Rao ldquoEstrogenic action of com-monly used fragrant agent citral induces prostatic hyperplasiardquoUrological Research vol 20 no 2 pp 139ndash144 1992

[34] J Brolin L Skoog and P Ekman ldquoImmunohistochemistryand biochemistry in detection of androgen progesterone andestrogen receptors in benign and malignant human prostatictissuerdquo Prostate vol 20 no 4 pp 281ndash295 1992

[35] S M Ho I Leav F B Merk M Yu P W L Kwan and JZiar ldquoInduction of atypical hyperplasia apoptosis and type IIestrogen- binding sites in the ventral prostates of noble ratstreated with testosterone and pharmacologic doses of estradiol-17120573rdquo Laboratory Investigation vol 73 no 3 pp 356ndash365 1995

[36] N Soulitzis I Karyotis D Delakas and D A SpandidosldquoExpression analysis of peptide growth factors VEGF FGF2TGFB1 EGF and IGF1 in prostate cancer and benign prostatichyperplasiardquo International Journal of Oncology vol 29 no 2 pp305ndash314 2006

[37] DMarinese R Patel and P DWalden ldquoMechanistic investiga-tion of the adrenergic induction of ventral prostate hyperplasiain micerdquo Prostate vol 54 no 3 pp 230ndash237 2003

[38] I S Shin M Y Lee H K Ha C S Seo and H K ShinldquoInhibitory effect of Yukmijihwang-tang a traditional herbalformula against testosterone-induced benign prostatic hyper-plasia in ratsrdquo BMC Complementary and Alternative Medicinevol 20 pp 12ndash48 2012

[39] C C Peng J H Liu C H Chang et al ldquoAction mechanismof Ginkgo biloba leaf extract intervened by exercise therapyin treatment of benign prostate hyperplasiardquo Evidence-Based


Complementary and Alternative Medicine vol 2013 Article ID408734 12 pages 2013

[40] C H Ma W L Lin S L Lui et al ldquoEfficacy and safety ofChinese herbal medicine for benign prostatic hyperplasia sys-tematic review of randomized controlled trialsrdquo Asian Journalof Andrology vol 15 pp 471ndash482 2013

[41] Z Ma T H Nguyen T H Huynh P T Do and H HuynhldquoReduction of rat prostate weight by combined quercetin-finasteride treatment is associated with cell cycle deregulationrdquoJournal of Endocrinology vol 181 no 3 pp 493ndash507 2004

[42] J Y Jung Y Lim M S Moon J Y Kim and O Kwon ldquoOnionpeel extracts ameliorate hyperglycemia and insulin resistancein high fat dietstreptozotocin-induced diabetic ratsrdquo Nutritionand Metabolism vol 8 article 18 2011

[43] B Djavan E Eckersberger G Espinosa et al ldquoComplexMechanisms in Prostatic Inflammatory Responserdquo EuropeanUrology vol 8 no 13 pp 872ndash878 2009

[44] DGiriMOzen andM Ittmann ldquoInterleukin-6 is an autocrinegrowth factor in human prostate cancerrdquoThe American Journalof Pathology vol 159 no 6 pp 2159ndash2165 2001

[45] H H Radeke B Meier N Topley J Floge G G Habermehland K Resch ldquoInterleukin 1-120572 and tumor necrosis factor-120572induce oxygen radical production in mesangial cellsrdquo KidneyInternational vol 37 no 2 pp 767ndash775 1990

[46] A Descazeaud N Weinbreck G Robert et al ldquoTransform-ing growth factor 120573-receptor II protein expression in benignprostatic hyperplasia is associated with prostate volume andinflammationrdquo BJU International vol 108 no 2 pp E23ndashE282011

[47] O Coskun M Kanter A Korkmaz and S Oter ldquoQuercetina flavonoid antioxidant prevents and protects streptozotocin-induced oxidative stress and 120573-cell damage in rat pancreasrdquoPharmacological Research vol 51 no 2 pp 117ndash123 2005

[48] J S Bland Clinical Nutrition A Functional Approach TheInstitute for Functional Medicine Gig Harbor Wash USA1999

[49] N Xing Y Chen S H Mitchell and C Y F Young ldquoQuercetininhibits the expression and function of the androgen receptorin LNCaP prostate cancer cellsrdquo Carcinogenesis vol 22 no 3pp 409ndash414 2001

[50] M R Vijayababu A Arunkumar P Kanagaraj P Venkatara-manGKrishnamoorthy and J Arunakaran ldquoQuercetin down-regulates matrix metalloproteinases 2 and 9 proteins expressionin prostate cancer cells (PC-3)rdquo Molecular and Cellular Bio-chemistry vol 287 no 1-2 pp 109ndash116 2006

[51] N Bhatia C Agarwal and R Agarwal ldquoDifferential responsesof skin cancer-chemopreventive agents silibinin quercetinand epigallocatechin 3-gallate on mitogenic signaling and cellcycle regulators in human epidermoid carcinoma A431 cellsrdquoNutrition and Cancer vol 39 no 2 pp 292ndash299 2001

[52] J A Choi J Y Kim J Y Lee et al ldquoInduction of cell cyclearrest and apoptosis in human breast cancer cells by quercetinrdquoInternational journal of oncology vol 19 no 4 pp 837ndash8442001

[53] D A Shoskes J C Nickel and M W Kattan ldquoPhenotypicallydirected multimodal therapy for chronic prostatitischronicpelvic pain syndromepp A prospective study using UPOINTrdquoUrology vol 75 no 6 pp 1249ndash1253 2010

[54] S Wu H Sun X Qi and Z Tu ldquoEffect of epristeride on theexpression of IGF-1 and TGF-120573 receptors in androgen-inducedcastrated rat prostaterdquo Experimental Biology and Medicine vol226 no 10 pp 954ndash960 2001

[55] R W Furlanetto J N DiCarlo and C Wisehart ldquoThe type IIinsulin-like growth factor receptor does not mediate deoxyri-bonucleic acid synthesis in human fibroblastsrdquo Journal ofClinical Endocrinology and Metabolism vol 64 no 6 pp 1142ndash1149 1987

[56] Z Culig A Hobisch M V Cronauer et al ldquoRegulation ofprostatic growth and function by peptide growth factorsrdquoProstate vol 28 pp 392ndash405 1996

[57] K Senthilkumar P Elumalai R Arunkumar et al ldquoQuercetinregulates insulin like growth factor signaling and inducesintrinsic and extrinsic pathway mediated apoptosis in andro-gen independent prostate cancer cells (PC-3)rdquo Molecular andCellular Biochemistry vol 344 no 1-2 pp 173ndash184 2010

[58] FO Ranelletti R Ricci LM Larocca et al ldquoGrowth-inhibitoryeffect of quercetin and presence of type-II estrogen-bindingsites in human colon-cancer cell lines and primary colorectaltumorsrdquo International Journal of Cancer vol 50 no 3 pp 486ndash492 1992

[59] Y QWei X Zhao Y Kariya H Fukata K Teshigawara and AUchida ldquoInduction of apoptosis by quercetin involvement ofheat shock proteinrdquo Cancer Research vol 54 no 18 pp 4952ndash4957 1994

[60] T C Lines and M Ono ldquoFRS 1000 an extract of red onionpeel strongly inhibits phosphodiesterase 5A (PDE 5A)rdquo Phy-tomedicine vol 13 no 4 pp 236ndash239 2006

[61] C H Deng H R Chen S P Qiu J Z Liu K L Zhengand H Mei ldquoEffect of nitric oxide donor and alpha1-receptorantagonist on proliferationapoptosis of hyperplastic prostaticstromal cells in vitrordquo Zhonghua Wai Ke Za Zhi vol 42 no 4pp 201ndash204 2004


There is an interest in the potential health benefits ofAllium vegetables mainly onion (Allium cepa) and garlic(A sativum) Intake of Allium vegetables may be inverselyrelated to cancer at various sites including prostate [7 8]Allium vegetables seem to have antiproliferative action onhuman cancers and to prevent diseases associated with aging[9] Moreover onion and garlic intake has been inverselyassociated with BPH [10] Onions contain high levels ofnonnutrient antioxidant compounds flavonoids and thealk(en)yl cysteine sulfoxides (ACSOs) which have protectiveeffects against different degenerative pathologies such ascardiovascular disease cancer and other dysfunctions basedon oxidative stress [11]

The uses of bulb in food and manufacture introducetons of its scales (red dried onion scales ROS) as a wasteproduct Recently these scales have attracted the atten-tion of many researchers to maximize the benefits of thiswaste material ROS contain large amounts of flavonoidsmainly ferulic gallic kaempferol quercetin quercetin dimerquercetin trimer and quercetin glycosides These flavonoidsare responsible for the reported antioxidant activity of ROSwhich is comparable to that of 120572-tocopherol [12 13] Theyare also reported to have hepatoprotective effect immuneenhancement potential anti-infectious antistress and anti-cancer properties [14] Moreover quercetin was found toameliorate significantly prostatitis symptoms by decreasingprostatic inflammation [15] Most of the studies were doneusing an onion bulb per se bulb extracts or bulb juicewithout studying the protective effect of RO scales the richestpart of the onion with quercetin The aim of this studywas to investigate the protective effect of the ROS extracton enlarged rat prostate and the histopathological changesrelated to inflammation proliferation andor apoptosis inAPH induced experimentally in rats

2 Materials and Methods

21 Chemicals and Reagents Solvents of HPLC grade usedfor HPLC analysis in addition to analytical grade solvents forextraction and thin layer chromatography (TLC) were pur-chased fromSigma-Aldrich (St LouisMOUSA)Antibodiesagainst clusterin phospho-Smad2 and120573-actinwere obtainedfrom Santa Cruz Biotechnology (Santa Cruz CA anticlus-terin forWestern blot analysis) Upstate Biotechnology (LakePlacid NY anticlusterin for immunohistochemistry (IHC))and Cell Signaling Technology Inc (Danvers MA) Antibod-ies against TGF-1205731 ligands were purchased from Dakocy-tomation (Carpinteria CA) Citral was obtained from FlukaChemie AG Buchs Switzerland Testosterone was obtainedfrom Sigma-Aldrich andDakocytomation (Carpinteria CA)while saw palmetto was obtained from Futurebiotics (NYUSA)

22 Collection and Extraction of Dried Red Onion ScalesThe red onion bulbs (Allium cepa L Giza red cultivar) werecollected from a single field cultivated in Sohag city Sohaggovernorate Egypt It was cultivated in mid-November andcollected in the beginning of May 2010 The fully grown

bulbs were peeled and the ROS were stored at 25∘C in a darkplace till extractionThe plant sample was identified by a staffmember of the Horticulture Department Faculty of Agricul-ture Cairo University ROS powder (1250 g) was extractedwith 80 ethanol (3 times 5 liters) by using Ultra-Turrax T50homogenizer (Janke andKunkel IKA-Labortechnik StaufenGermany) at a temperature not exceeding 25∘C The extractwas concentrated by evaporation under reduced pressurelyophilized to give 240 g of reddish semisolid residue andprotected from light at 4∘C to use later

23 Standardization of Red Onion Scale (ROS) Extract Pre-liminary phytochemical tests to identify the main chemicalconstituents were carried out according to the methodsof Trease and Evans [16] Chromatographic TLC profileof the methanolic extract was performed using thin layerchromatography (solvent system ethyl acetate-formic acid-glacial acetic acid-water 100 11 11 26) as per fingerprintingusing a chromatographic condition reported by Wagner andBladt [17] against common reference flavonoids available inthe Pharmacognosy Department Faculty of Pharmacy CairoUniversity Total phenolics were determined as mg gallic acidequivalent per gram dry extract (mg GAEg DE) employingthe Folin-Ciocalteumethod described by Singleton and Rossi[18] Aluminum chloride colorimetric assay described byHertog [19] was applied for determination of total flavonoidscontent as mg quercetin equivalent per gram of dry extract(mgQEgDE)with a slightmodification [20]The free radicalscavenging activity of the extracts based on the scavengingactivity of the stable 11-diphenyl-2-picrylhydrazyl (DPPH)free radical was determined by the method described byBraca et al [21] The capability of the extracts to scavenge theDPPH radical was calculated using the following equationinhibition = (119860Control minus 119860Test) times 100119860Control

24 HPLCAnalysis Quercetin (Q) and quercetin-41015840-120573-O-D-glucoside (QG) were used as standard marker compoundsQGwas separated from theROS extract and identified using aprocedure reported by Abou Zid and Elsherbeiny [22] HPLCanalysis was performed on Agilent HP1200 series HPLC sys-tem equipped with a G1322A quaternary pump and degassera G1314A variable wavelength UV detector and 250 times40mm (particle size 5 120583m) ODS column (Lichrospher 100Merck Darmstadt Germany) Chromatographic separationwas achieved by applying a linear gradient system usingmobile phase A (5 formic acid in water vv) and mobilephase B (methanol) A linear gradient elution was performedusing 50 mobile phase B for 10min rising to 80 B over10min then to 100 B over 11min and back to 50 BThe flow rate was maintained at 10mLmin and UV wasmonitored at 360 nm

25 Experimental Animals Adolescent male Wistar ratsaged 50ndash60 days were obtained from the animal facilityof King Fahd Research Center King Abdulaziz UniversityJeddah Saudi Arabia They were used in the study accordingto the guidelines of the Biochemical and Research EthicsCommittee at King Abdulaziz University in accordance with










2O2to eliminate















2 10 pmol of each



3 Results










(min)

(mAU

)

400

300

200

100

0

0 5 10 15 20

2129

2990

11011

16116

18209

19131


5682

(a)

0 2 4 6 8 10 12 14

(min)

200

150

100

50

0

(mAU

)


5115

(b)















00

01

02

03

04

000

002

004

006

008

010IL

-6120573

-act

in (R

BI)

IL-8

120573-a

ctin

(RB

I)

lowast

lowast

00

01

02

03

04



TNF-120572

120573-a

ctin

(RB

I)

lowast









00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References










































































2O2to eliminate















2 10 pmol of each



3 Results










(min)

(mAU

)

400

300

200

100

0

0 5 10 15 20

2129

2990

11011

16116

18209

19131


5682

(a)

0 2 4 6 8 10 12 14

(min)

200

150

100

50

0

(mAU

)


5115

(b)















00

01

02

03

04

000

002

004

006

008

010IL

-6120573

-act

in (R

BI)

IL-8

120573-a

ctin

(RB

I)

lowast

lowast

00

01

02

03

04



TNF-120572

120573-a

ctin

(RB

I)

lowast









00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References











































































2 10 pmol of each



3 Results










(min)

(mAU

)

400

300

200

100

0

0 5 10 15 20

2129

2990

11011

16116

18209

19131


5682

(a)

0 2 4 6 8 10 12 14

(min)

200

150

100

50

0

(mAU

)


5115

(b)















00

01

02

03

04

000

002

004

006

008

010IL

-6120573

-act

in (R

BI)

IL-8

120573-a

ctin

(RB

I)

lowast

lowast

00

01

02

03

04



TNF-120572

120573-a

ctin

(RB

I)

lowast









00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































(min)

(mAU

)

400

300

200

100

0

0 5 10 15 20

2129

2990

11011

16116

18209

19131


5682

(a)

0 2 4 6 8 10 12 14

(min)

200

150

100

50

0

(mAU

)


5115

(b)















00

01

02

03

04

000

002

004

006

008

010IL

-6120573

-act

in (R

BI)

IL-8

120573-a

ctin

(RB

I)

lowast

lowast

00

01

02

03

04



TNF-120572

120573-a

ctin

(RB

I)

lowast









00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































00

01

02

03

04

000

002

004

006

008

010IL

-6120573

-act

in (R

BI)

IL-8

120573-a

ctin

(RB

I)

lowast

lowast

00

01

02

03

04



TNF-120572

120573-a

ctin

(RB

I)

lowast









00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































00

02

04

06

08

00

05

10

15

lowast

lowast

TGF-120573

R1120573

-act

in (R

BI)

IGF-1

120573-a

ctin

(RB

I)

00

05

10

15

lowast

Clus

terin

120573-a

ctin

(RB

I)









4 Discussion



(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































(a) (b)

(c) (d)

(e)






(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References


































































(a) (b)

(c) (d)


(a) (b)

(c) (d)












Acknowledgments


References











































































Acknowledgments


References

































































mi2014-640746

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