research article immunomodulatory effect of red onion allium … · 2019. 7. 31. · research...

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

Ahmed A. Elberry,1,2 Shagufta Mufti,3 Jaudah Al-Maghrabi,3 Essam Abdel Sattar,4

Salah A. Ghareib,5 Hisham A. Mosli,6 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 [email protected]

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

Academic Editor: Yona Keisari

Copyright © 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 boths.c. injection of testosterone (0.5mg/rat/day) and by smearing citral on shaved skin once every 3 days for 30 days. Saw palmetto(100mg/kg) as a positive control and ROS suspension at doses of 75, 150, and 300mg/kg/day were given orally every day for 30 days.All 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-4-𝛽-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-𝛼, IGF-1, and clusterin, while TGF-𝛽1 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 pageshttp://dx.doi.org/10.1155/2014/640746

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 flavonoids,mainly ferulic, gallic, kaempferol, quercetin, quercetin dimer,quercetin trimer, and quercetin glycosides. These flavonoidsare responsible for the reported antioxidant activity of ROSwhich is comparable to that of 𝛼-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, and/or apoptosis inAPH induced experimentally in rats.

2. Materials and Methods

2.1. 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. Louis,MO,USA).Antibodiesagainst clusterin, phospho-Smad2, and𝛽-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-𝛽1 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 (NY,USA).

2.2. Collection and Extraction of Dried Red Onion Scales.The 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 extraction.The 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 × 5 liters) by using Ultra-Turrax T50homogenizer (Janke andKunkel, IKA-Labortechnik, Staufen,Germany) at a temperature not exceeding 25∘C. The extractwas concentrated by evaporation under reduced pressure,lyophilized to give 240 g of reddish semisolid residue, andprotected from light at 4∘C to use later.

2.3. 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 GAE/g 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(mgQE/gDE)with a slightmodification [20].The free radicalscavenging activity of the extracts, based on the scavengingactivity of the stable 1,1-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 equation:%inhibition = (𝐴Control − 𝐴Test) × 100/𝐴Control.

2.4. HPLCAnalysis. Quercetin (Q) and quercetin-4-𝛽-O-D-glucoside (QG) were used as standard marker compounds.QGwas 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 degasser,a G1314A variable wavelength UV detector, and 250 ×4.0mm (particle size 5 𝜇m) ODS column (Lichrospher 100,Merck, Darmstadt, Germany). Chromatographic separationwas achieved by applying a linear gradient system usingmobile phase A (5% formic acid in water v/v) 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% B.The flow rate was maintained at 1.0mL/min and UV wasmonitored at 360 nm.

2.5. Experimental Animals. Adolescent male Wistar rats,aged 50–60 days, were obtained from the animal facilityof King Fahd Research Center, King Abdulaziz University,Jeddah, 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 ± 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 between8–10 a.m. 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 (0.5mg/0.1mL/rat) each day for 30 days. Citral,1M diluted in 70% ethanol, was smeared on a differentshaved area of skin, each time, on the back at a final doseof 185mg/kg 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.

2.6. Animal Treatment. Seven days after castration, the ani-mals were randomly divided into six groups (𝑛 = 7). Group Iserved as a control (shamed operation) group and receivedboth corn oil (0.1mL/rat) and 1% CMC-Na (0.3mL/100 gbody weight) daily during the period of the experiment.Groups from II–VI 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 (100mg/kg) suspended in 1%CMC-Na.Groups from IV–VIwere treatedwithROS at dosesof 75, 150, or 300mg/kg/day, respectively, by oral gavage.All 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 −75∘Cuntil use. The remaining tissues were fixed immediately in0.1M phosphate-buffered 10% formalin (pH 7.4) for 48 h andthen embedded in paraffin and were used for histologicalstudies. Serial 4 𝜇m 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-𝛼,IGF-1, clusterin, and TGF-𝛽1 receptors in the prostatic tissueby immunohistochemistry.

2.7. Histopathology and Histoscore. A part of the ventrallobes was separated and fixed overnight in Stieve’s solution.

Thereafter, the tissue was thoroughly rinsed with water andimmersed overnight in ethanol 70%.Then, it was dehydrated,embedded in paraffin, and 5mm thick sections were cutand stained by Harris’ 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 hyperplasia.An additional histological inflammatory score described byde Nunzio et al. [28] was used to evaluate the inflammation.Score 0: no inflammation, score 1: scattered inflammatory cellinfiltrate without nodules, score 2: no confluent lymphoid,and score 3: large inflammatory areas with confluence.

2.8. Immunohistochemistry and Immunhistoscoring. Hema-toxylin and eosin staining was performed to observehistopathology. For analysis of IL-6 and TGF-𝛽1 expression,sections 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 machine,dewaxed by EZ Prep (Xylene substitute), and rehydrated.Thetissue 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-𝛽1. The dilutions used were IL-6 (dilution 1 : 50)and TGF-𝛽1 (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 background.The positive control used for IL-6 and TGF-𝛽1 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-𝛽1 and IL-6 was evaluated accordingto a semiquantitative scale: 0, no detectable staining at all; 1,less than 10% of the cells stained positive; 2, 10−50% 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].

2.9. 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 manufacturer’s instructions. RT was per-formed in a 10 𝜇L reaction mixture. The RT reaction con-tained 1 𝜇g RNA, 10mM Tris-HCl (pH 8.3), 50mM KCl,1.5mM MgCl

2, 2.5mM dithiothreitol, 500𝜇mol/liter each


Table 1: Sequences of oligonucleotides used as primers.

Gene Forward primer Reverse primer𝛽-Actin 5-GTCACCCACACTGTGCCCATCT-3 5-ACAGAGTACTTGCGCTCAGGAG-3

IL-6 5-GAACTCCTTCTCCACAAGCG-3 5-TTTTCTGCCAGTGCCTCTTT-3

IL-8 5-CTGCGCCAACACAGAAATTA-3 5-ATTGCATCTGGCAACCCTAC-3

TNF-𝛼 5-CAGAGGGAAGAGTTCCCCAG-3 5-CCTTGGTCTGGTAGGAGACG-3

TGF-𝛽1 5-GTTCTTCAATACGTCAGACATTCG-3 5-CATTATCTTTGCTGTCACAAGAGC-3

IGF-1 5-CACAGGCTATGGCTCCAGCAT-3 5-TCTCCAGCCTCCTCAGATCACA-3

Clusterin 5-CTGACCCAGCAGTACAACGA-3 5-TGACACGAGAGGGGACTTCT-3

of dATP, dCTP, dGTP, and dTTP (Bioline), 40U RNasin(Bioline), 25 𝜇g/mL oligo dT pd(T)12–18 (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 using𝛽-actin as a quantitative control. Quantitative PCR amplifi-cation was performed with a 25𝜇L final volume consistingof 1 𝜇L RT reaction mixture, 3mM MgCl

2, 10 pmol of each

sense and antisense primer, and 12.5 𝜇L (Roche Diagnostics).PCR conditions were as follows: initial denaturation at 95∘Cfor 10min and 35 cycles of denaturation at 94∘C for 1min,annealing 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-𝛽1, IGF-1, IL-6, IL-8, and TNF-𝛼 expression in the test samples were normalizedto the corresponding 𝛽-actin level and were reported asthe relative band intensity to the 𝛽-actin gene expression.Sequences of oligonucleotides used as primers for𝛽-actin, IL-6, IL-8, TNF𝛼, TGF-𝛽1, IGF-1, and clusterin are summarizedin Table 1.

2.10. Statistical Analysis. Data were expressed as mean ± 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 Yate’s corrections. Differences wereconsidered significant with a 𝑃 value less than 0.05. Statis-tical analyses were performed using the SPSS for Windows(v. 10.0).

3. Results

3.1. Characterization of ROS Extract. Screening of themethanolic onion extract indicated mainly the presence ofsterols and/or terpenoids, polyphenolic compounds suchas flavonoids, tannins, and the absence of alkaloids andsaponins. Assay of total phenolic content of methanolicextract was determined to be 12.9mg GAE/g DE. Assay oftotal flavonoid content was determined to be 119mg QE/gDE. 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 96.21% inhibition at 1mM/mL. 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 𝜇g/mL. 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 5.68 and 11.01 at 360 nmidentified as quercetin and quercetin-4-𝛽-O-D-glucosidethrough spiking with standard quercetin and the isolatedquercetin-4-𝛽-O-D-glucoside. The amount of quercetin inROS extract was found to be 60.1mg/gm of dry extract usingstandard quercetin (1.5mg/mL).

3.2. 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. Moreover,both 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).

3.3. Effect of ROS on Proinflammatory Cytokines, IL-6, IL-8,and TNF-𝛼. Induction of APH in rats significantly increasedthe tissue levels of IL-6, IL-8, and TNF-𝛼. Saw palmetto treat-ment significantly decreased the IL-6 (20.3%), IL-8 (48.1%),and TNF-𝛼 (39.4%). ROS induced significant reductionsin IL-6, IL-8, and TNF-𝛼, which were greater than thatproduced by saw palmetto (Figure 3). These reductions weredose dependant regarding IL-6 (54.7%, 59%, and 65.6%), IL-8 (93%, 95.3%, and 97.9%), and TNF-𝛼 (50.8%, 65.1%, and91.3%).

3.4. Effect of ROS on Gene Expression of TGF-𝛽R1, IGF-1,and 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-𝛽R1 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-4-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 (mg/g)Start End

Normal rats 199.14 ± 5.16 229 ± 8.81 0.106 ± 0.01 0.452 ± 0.05Rats with APH (negative control) 187.6 ± 8.65 241.4b ± 12.55 0.756∗ ± 0.05 3.136∗ ± 0.19Rats with APH treated with

Saw palmetto (100mg/kg) 192.8 ± 4.59 258.3b ± 10.22 0.698∗ ± 0.03 2.705∗ ± 0.21RO scales (75mg/kg) 227.5 ± 7.44 271.1b ± 7.69 0.681∗ ± 0.02 2.512∗a ± 0.12RO scales (150mg/kg) 225.5 ± 7.15 271.6b ± 9.20 0.593∗a ± 0.023 2.183∗a ± 0.14RO scales (300mg/kg) 196.2 ± 7.39 262.7b ± 16.22 0.547∗a ± 0.075 2.082∗a ± 0.21

∗Significantly different from normal rats at 𝑃 < 0.05.aSignificantly different from rats with APH at 𝑃 < 0.05.bSignificantly different from the value before starting the experiment at 𝑃 < 0.05.

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

APH showed significant increase in clusterin and TGF-𝛽R1 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-𝛽R1 expressionwas not significantly changed compared to the APH group(Figure 4).

3.5. Histological Changes. Ventral prostates from the normalgroup showed normal histology with average histoscorecorresponding to 24.3 ± 1.2 (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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Figure 3: The expression of IL-6, IL-8, and TNF-𝛼 target genes in ventral prostate of Wistar rats after treatment with RO scales at differentdose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using 𝛽-actin as a reference. ∗Significantly differentfrom normal shame control rats at 𝑃 < 0.001. #Significantly different from APH control rats received CMC at 𝑃 < 0.001.

increase in the histoscore corresponding to 41.1 ± 1.8.(Figures 5(b)–5(d)).

The ventral prostate in the saw palmetto group showeddiminution regarding the scale of pathological changes andhistoscore corresponding to 32.2 ± 2.1 (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, regular,and 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 (75mg/kg) 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 35.3 ± 1.2.

Sections examined from ventral prostates of APH-induced rats treated with ROS extract (150mg/kg) 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 group.Intraluminal 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 34.0 ± 0.5.

Histological changes in ventral prostates of rats, withAPH treated with ROS extract (300mg/kg) showed prostatic


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Figure 4: The expression of TGF-𝛽R1, 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 ± S.E. of the relative band intensity (R.B.I.) using 𝛽-actin as a reference. ∗Significantlydifferent from normal shame control rats at 𝑃 < 0.001. #Significantly different from APH control rats receiving CMC at 𝑃 < 0.001.

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 32.2 ± 0.8.

3.6. 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 study,APH-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-𝛽1 which may cause the prostateenlargement in APH group. TGF-𝛽1 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 [38–40]. In the present study, administration ofROS extract induced significant and dose-related reduction


(a) (b)

(c) (d)

(e)

Figure 6: (a)-(b) Prostate of rats treatedwith RO scale extract in a dose of 75mg/kg showing. (a) Irregularly dilated acini filled with secretions.Arrow 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 150mg/kg 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 300mg/kg 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 kaempferol,quercetin, 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 TGF𝛽R-1 and IL-6 in prostates of normal and APH rats. (a) Normal rats TGF𝛽R-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 TGF𝛽R-1 and IL-6 in saw palmetto 100 g treated rats. (a) TGF𝛽R-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) TGF𝛽R-1 score 0.Note 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-𝛼 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-𝛼, a proinflammatory cytokine, may induceinflammation by induction of COX-2, superoxide radical,and hydrogen peroxide in both human and rat mesangialcells [45]. TGF-𝛽, an inflammatory cytokine, has been shownto regulate stromal proliferation and differentiation in BPH,and it is a key factor in the androgen control of prostaticgrowth. Recently, Descazeaud et al. [46] investigated theTGF-𝛽 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-𝛼 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 death.It 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 G1/S phase [50] or at theG2/M 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 expression.The prostate enlargementand the increased net weight of the ventral lobe in case ofAPHmay be partly due to the increased IGF-1 and decreasedTGF-𝛽1 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 antiproliferativeand/or 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. Fetaihi,Chair for Research on Prostatic Diseases, King AbdulazizUniversity, Jeddah, Saudi Arabia.The authors would also liketo thankMr. IslamFarouk, Department of Pharmacology andToxicology, Faculty of Pharmacy, King Abdulaziz University,Jeddah, Saudi Arabia, for his great effort and help in theexperimental study.

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