Research Article Immunomodulatory Effect of Red Onion (Allium Cepa Linn) Scale Extract on Experimentally Induced Atypical Prostatic Hyperplasia in Wistar Rats

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Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2014, Article ID 640746, 13 pages http://dx.doi.org/10.1155/2014/640746

Research Article Immunomodulatory Effect of Red Onion (Allium cepa Linn) Scale Extract on Experimentally Induced Atypical 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 Arabia 2 Department of Pharmacology, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt 3 Department of Pathology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia 4 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt 5 Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia 6 Department of Urology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia 7 Department 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 Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Red onion scales (ROS) contain large amounts of flavonoids that are responsible for the reported antioxidant activity, immune enhancement, and anticancer property. Atypical prostatic hyperplasia (APH) was induced in adult castrated Wistar rats by both s.c. injection of testosterone (0.5 mg/rat/day) and by smearing citral on shaved skin once every 3 days for 30 days. Saw palmetto (100 mg/kg) as a positive control and ROS suspension at doses of 75, 150, and 300 mg/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 methanolic 󸀠 extract displayed two major peaks identified as quercetin and quercetin-4 -𝛽-O-D-glucoside. Histopathological examination of APH-induced prostatic rats revealed evidence of hyperplasia and inflammation with cellular proliferation and reduced apoptosis Immunohistochemistry showed increased tissue expressions of IL-6, IL-8, TNF-𝛼, IGF-1, and clusterin, while TGF-𝛽1was decreased, which correlates with the presence of inflammation. Both saw palmetto and RO scale treatment have ameliorated these changes. These ameliorative effects were more evident in ROe scal groups and were dose dependent. In conclusion, methanolic extractofROSshowedaprotectiveeffectagainstAPHinducedratsthatmaybeattributedtopotentialanti-inflammatoryand immunomodulatory effects.

1. Introduction several cancer-like features places APH as an intermediate lesion between BPH and the subset of well-differentiated Atypical prostatic hyperplasia (APH) is a pseudoneoplastic cancers in a hypothetical pathway between BPH and LPC lesion that can mimic prostate adenocarcinoma because of [3]. its architectural and cytological features. In this prostatic The role of inflammation in prostate diseases is suggested disease, there is an imbalance between prostate cell growth by the presence of inflammatory cells within the BPH [4]. and apoptosis [1]. The occurrence rate of APH is not Inflammation is usually a self-limited event, with initial knownanditsassociationswithbenignprostatichypertrophy proinflammatory cytokines, growth factor release, and angio- (BPH) and latent carcinoma of the prostate (LPC) have genesis followed by an anti-inflammatory cytokine-mediated not been completely clarified [2]. The fact that APH arises resolution [5]. Chronic inflammation produces free radicals always in prostates with concomitant BPH and exhibits as various reactive oxygen species [6]. 2 Mediators of Inflammation

∘ There is an interest in the potential health benefits of bulbs were peeled and the ROS were stored at 25 Cinadark Allium vegetables, mainly onion (Allium cepa)andgarlic place till extraction. The plant sample was identified by astaff (A. sativum). Intake of Allium vegetables may be inversely member of the Horticulture Department, Faculty of Agricul- related to cancer at various sites, including prostate [7, 8]. ture, Cairo University. ROS powder (1250 g) was extracted Allium vegetables seem to have antiproliferative action on with 80% ethanol (3 × 5 liters) by using Ultra-Turrax T50 human cancers and to prevent diseases associated with aging homogenizer (Janke and Kunkel, IKA-Labortechnik, Staufen, ∘ [9]. Moreover, onion and garlic intake has been inversely Germany) at a temperature not exceeding 25 C. The extract associated with BPH [10]. Onions contain high levels of was concentrated by evaporation under reduced pressure, nonnutrient antioxidant compounds, flavonoids, and the lyophilizedtogive240gofreddishsemisolidresidue,and ∘ alk(en)yl cysteine sulfoxides (ACSOs), which have protective protected from light at 4 Ctouselater. effects against different degenerative pathologies such as cardiovascular disease, cancer, and other dysfunctions based 2.3. Standardization of Red Onion Scale (ROS) Extract. Pre- on oxidative stress [11]. liminary phytochemical tests to identify the main chemical The uses of bulb in food and manufacture introduce constituents were carried out according to the methods tons of its scales (red dried onion scales, ROS) as a waste of Trease and Evans [16]. Chromatographic TLC profile product. Recently, these scales have attracted the atten- of the methanolic extract was performed using thin layer tion of many researchers to maximize the benefits of this chromatography (solvent system : ethyl acetate-formic acid- waste material. ROS contain large amounts of flavonoids, glacial acetic acid-water, 100 : 11 : 11 : 26) as per fingerprinting mainly ferulic, gallic, kaempferol, quercetin, quercetin dimer, using a chromatographic condition reported by Wagner and quercetin trimer, and quercetin glycosides. These flavonoids Bladt [17] against common reference flavonoids available in are responsible for the reported antioxidant activity of ROS the Pharmacognosy Department, Faculty of Pharmacy, Cairo which is comparable to that of 𝛼-tocopherol [12, 13]. They University. Total phenolics were determined as mg gallic acid arealsoreportedtohavehepatoprotectiveeffect,immune equivalent per gram dry extract (mg GAE/g DE) employing enhancement potential, anti-infectious, antistress and anti- the Folin-Ciocalteu method described by Singleton and Rossi cancer properties [14]. Moreover, quercetin was found to [18]. Aluminum chloride colorimetric assay described by ameliorate significantly prostatitis symptoms by decreasing Hertog [19] was applied for determination of total flavonoids prostatic inflammation15 [ ]. Most of the studies were done content as mg quercetin equivalent per gram of dry extract using an onion bulb per se, bulb extracts, or bulb juice (mgQE/gDE)withaslightmodification[20]. The free radical without studying the protective effect of RO scales, the richest scavenging activity of the extracts, based on the scavenging part of the onion with quercetin. The aim of this study activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) was to investigate the protective effect of the ROS extract free radical, was determined by the method described by onenlargedratprostateandthehistopathologicalchanges Braca et al. [21]. The capability of the extracts to scavenge the related to inflammation, proliferation, and/or apoptosis in DPPH radical was calculated using the following equation: 𝐴 −𝐴 × 𝐴 APH induced experimentally in rats. %inhibition = ( Control Test ) 100/ Control.

󸀠 2. Materials and Methods 2.4. HPLC Analysis. Quercetin (Q) and quercetin-4 -𝛽-O-D- glucoside(QG)wereusedasstandardmarkercompounds. 2.1. Chemicals and Reagents. Solvents of HPLC grade used QG was separated from the ROS extract and identified using a for HPLC analysis, in addition to analytical grade solvents for procedurereportedbyAbouZidandElsherbeiny[22]. HPLC extraction and thin layer chromatography (TLC), were pur- analysis was performed on Agilent HP1200 series HPLC sys- chased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies tem equipped with a G1322A quaternary pump and degasser, against clusterin, phospho-Smad2, and 𝛽-actin were obtained a G1314A variable wavelength UV detector, and 250 × from Santa Cruz Biotechnology (Santa Cruz, CA; anticlus- 4.0 mm (particle size 5 𝜇m)ODScolumn(Lichrospher100, terin for Western blot analysis); Upstate Biotechnology (Lake Merck, Darmstadt, Germany). Chromatographic separation Placid, NY; anticlusterin for immunohistochemistry (IHC)); was achieved by applying a linear gradient system using and Cell Signaling Technology Inc. (Danvers, MA). Antibod- mobile phase A (5% formic acid in water v/v) and mobile ies against TGF-𝛽1 ligands were purchased from Dakocy- phase B (methanol). A linear gradient elution was performed tomation (Carpinteria, CA). Citral was obtained from Fluka using 50% mobile phase B for 10 min, rising to 80% B over Chemie AG, Buchs, Switzerland. Testosterone was obtained 10 min, then to 100% B over 11 min, and back to 50% B. from Sigma-Aldrich and Dakocytomation (Carpinteria, CA), The flow rate was maintained at 1.0 mL/min and UVwas while saw palmetto was obtained from Futurebiotics (NY, monitored at 360 nm. USA). 2.5. Experimental Animals. Adolescent male Wistar rats, 2.2. Collection and Extraction of Dried Red Onion Scales. aged 50–60 days, were obtained from the animal facility The red onion bulbs (Allium cepa L., Giza red cultivar) were of King Fahd Research Center, King Abdulaziz University, collected from a single field cultivated in Sohag city, Sohag Jeddah, Saudi Arabia. They were used in the study according governorate, Egypt. It was cultivated in mid-November and to the guidelines of the Biochemical and Research Ethics collectedinthebeginningofMay,2010.Thefullygrown Committee at King Abdulaziz University, in accordance with Mediators of Inflammation 3 the NIH guidelines. Animals were housed in a well- Thereafter, the tissue was thoroughly rinsed with water and ∘ ventilated, temperature-controlled room at 22 ± 3 Cwitha immersed overnight in ethanol 70%. Then, it was dehydrated, 12 h light-dark cycle. They were provided with standard rat embedded in paraffin, and 5 mm thick sections were cut chow pellets obtained from Grain Silos and Flour Mills orga- and stained by Harris’ hematoxylin eosin, according to the nization F-1005, Jeddah, Saudi Arabia, and tap water ad libi- standard procedures of Lillie [27]. tum. All experimental procedures were performed between A score-chart protocol (histoscore) developed by Scolnik 8–10 a.m. and care was taken to avoid stressful conditions. et al. [26] was used to obtain an objective quantitative Orchidectomy was performed aseptically, under ethyl assessment. The examination, description, and scoring of ether anesthesia, by a midscrotal incision. Following ligation the slides were performed in a blinded manner. The scoring of the spermatic cord and vessels, testes and epididymis were system was presented in arbitrary units to make a better removed. The remaining stump was pushed back through evaluation. In a second step, the cumulative score in each theinguinalcanalintotheabdominalcavity,andthescrotal group was correlated to the final histological diagnosis in sac was closed by sutures [23]. After castration, the rats were order to establish a score range for normal and hyperplasia. maintained under standard laboratory conditions for 7 days An additional histological inflammatory score described by in order to allow a definite involution of the prostatic gland de Nunzio et al. [28] was used to evaluate the inflammation. [24]. Score0:noinflammation,score1:scatteredinflammatorycell APH was induced as previously described by Engelstein infiltrate without nodules, score 2: no confluent lymphoid, et al. [25] in castrated rats using citral and testosterone. Rats and score 3: large inflammatory areas with confluence. were subcutaneously injected with testosterone propionate in corn oil (0.5 mg/0.1 mL/rat) each day for 30 days. Citral, 2.8. Immunohistochemistry and Immunhistoscoring. Hema- 1 M diluted in 70% ethanol, was smeared on a different toxylinandeosinstainingwasperformedtoobserve shaved area of skin, each time, on the back at a final dose histopathology. For analysis of IL-6 and TGF-𝛽1 expression, of 185 mg/kg every 4 days for 30 days. Control group rats sectionsfromtheparaffinembeddedtissueblockswere were smeared with the solvent (ethanol) alone at the shaved ∘ mountedonchargedglassslidesandbakedat60Cfor1h skin. Considering the pungent lime fragrance of citral, the in the oven, then mounted on the Ventana staining machine, control groups were kept in a separate location from the dewaxedbyEZPrep(Xylenesubstitute),andrehydrated.The citral-treated animals to avoid possible false results as citral tissuesectionswereheatedinVentanabufferCC1(pH6)to hassomeinfluenceviatheolfactorytract. facilitate antigen retrieval and treated with H2O2 to eliminate endogenous peroxidase. This was followed by incubation for 2.6. Animal Treatment. Seven days after castration, the ani- 60 min at room temperature with primary antibodies IL-6 mals were randomly divided into six groups (𝑛=7). Group I and TGF-𝛽1. The dilutions used were IL-6 (dilution 1 : 50) served as a control (shamed operation) group and received and TGF-𝛽1 (dilution of 1 : 25). Subsequently, the sections both corn oil (0.1 mL/rat) and 1% CMC-Na (0.3 mL/100 g were incubated with biotinylated secondary antibody using body weight) daily during the period of the experiment. theavidin-biotincomplexmethod.Theimmunoreaction GroupsfromII–VIwerecastratedandhadAPH.Group was visualized using diaminobenzidine. All sections were II served as negative control and received CMC-Na 1% as lightlycounterstainedwithhematoxylinasabackground. previously mentioned. Group III served as positive control The positive control used for IL-6 and TGF-𝛽1wasfromthe and received saw palmetto (100 mg/kg) suspended in 1% colon. The negative controls comprised serial sections that CMC-Na. Groups from IV–VI were treated with ROS at doses were stained using equivalent concentrations of nonimmune of75,150,or300mg/kg/day,respectively,byoralgavage. mouse IgG in place of the primary antibodies. The level All the ROS extracts were suspended in 1% CMC-Na and of staining was evaluated independently by three observers weregiventoratsoncedailybyoralgavagealongwith blinded to experimental conditions. testosterone injection and citral smearing for 30 days as Expression of TGF-𝛽1 and IL-6 was evaluated according described before [26]. After euthanization, ventral prostate to a semiquantitative scale: 0, no detectable staining at all; 1, of each rat in each group was removed from the body, 24 h less than 10% of the cells stained positive; 2, 10−50% positive after the last administration, and weighed. The half of the cells; and 3, more than 50% of cells positive [29]. Staining ∘ tissues were frozen in liquid nitrogen and stored at −75 C intensity was scored as 0 (no detectable stain), 1 (weak until use. The remaining tissues were fixed immediately in staining detected at intermediate to high power), 2 (moderate 0.1 M phosphate-buffered 10% formalin (pH 7.4) for 48 h and detected at low to intermediate power), to 3 (strong detected then embedded in paraffin and were used for histological at low power) [30]. studies. Serial 4 𝜇m thick sections from each tissue specimen were prepared and mounted on poly-L-lysine coated glass 2.9. Reverse Transcription Polymerase Chain Reaction (RT- slides. These were used for detection of IL-6, IL-8, TNF-𝛼, PCR). Total RNA was extracted from the snap-frozen tissue IGF-1, clusterin, and TGF-𝛽1 receptors in the prostatic tissue samples using total RNA isolation kit (Macherey-Nagel) by immunohistochemistry. according to the manufacturer’s instructions. RT was performed in a 10 𝜇L reaction mixture. The RT reaction con- 2.7. Histopathology and Histoscore. Apartoftheventral tained 1 𝜇g RNA, 10mM Tris-HCl (pH 8.3), 50mM KCl, lobes was separated and fixed overnight in Stieve’s solution. 1.5 mM MgCl2, 2.5 mM dithiothreitol, 500 𝜇mol/liter each 4 Mediators of Inflammation

Table 1: Sequences of oligonucleotides used as primers.

Gene Forward primer Reverse primer 󸀠 󸀠 󸀠 󸀠 𝛽-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-𝛽15-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), 40 U RNasin corresponding to a reduction of the absorbance of DPPH (Bioline), 25 𝜇g/mL oligo dT pd(T)12–18 (Bioline), and of 50% (IC50). Ascorbic acid, a commonly used reference 100UMoloney murine leukemia virus reverse transcriptase antioxidant, elicited 96.21% inhibition at 1 mM/mL. Onion ∘ (Bioline). The reaction mixture was incubated at 42 Cfor extract was able to reduce the stable radical DPPH to the ∘ 60 min and then heated to 80 Cfor5min.Theresultant yellow colored diphenylpicrylhydrazine. Thus, it exhibited cDNA was used for PCR. For quantitative real-time RT- observable scavenging activity in a dose-related manner with PCR, we prepared appropriate dilutions of each single-strand IC50 values of 368 𝜇g/mL. Figures 1 and 2 showed HPLC and cDNA followed by normalizing of the cDNA content using TLC profile of ROS extract against Q and QG as marker 𝛽-actin as a quantitative control. Quantitative PCR amplifi- compounds. The HPLC profile of ROS methanolic extract cation was performed with a 25 𝜇Lfinalvolumeconsisting displayed two major peaks at t 5.68 and 11.01 at 360 nm R 󸀠 of 1 𝜇L RT reaction mixture, 3 mM MgCl2, 10 pmol of each identified as quercetin and quercetin-4 -𝛽-O-D-glucoside sense and antisense primer, and 12.5 𝜇L (Roche Diagnostics). through spiking with standard quercetin and the isolated ∘ 󸀠 PCR conditions were as follows: initial denaturation at 95 C quercetin-4 -𝛽-O-D-glucoside. The amount of quercetin in ∘ for 10min and 35 cycles of denaturation at 94 Cfor1min, ROS extract was found to be 60.1 mg/gm of dry extract using ∘ ∘ annealing at 55 C for 1 min, and elongation at 72 Cfor standard quercetin (1.5 mg/mL). ∘ 2sec with a final elongation at 72 C for 10 min. Samples were migrated in 1% agarose gel using electrophoresis, UV 3.2. Changes in Body Weight (BW), Absolute Prostatic Weight visualized, and images were analyzed using total lab120 (APW), and Relative Prostatic Weight (RPW). In APH- 𝛽 (Nonlinear Dynamic Ltd). Clusterin, TGF- 1, IGF-1, IL-6, IL- induced rats, BW has increased significantly at the end of 𝛼 8, and TNF- expressioninthetestsampleswerenormalized the experiment compared to the starting weight. Moreover, 𝛽 to the corresponding -actin level and were reported as bothAPWandRPWwereincreasedsignificantlycompared 𝛽 therelativebandintensitytothe -actin gene expression. to the normal control rats. All treatments with saw palmetto 𝛽 Sequences of oligonucleotides used as primers for -actin, IL- or ROS failed to improve the BW at the end of the experiment 𝛼 𝛽 6, IL-8, TNF ,TGF- 1, IGF-1, and clusterin are summarized compared to the starting weight. However, saw palmetto in Table 1. induced small but insignificant reduction in APW and RPW compared to control rats. However, ROS also induced 2.10. Statistical Analysis. Data were expressed as mean ± SE significantanddose-relatedreductionsinbothAPWand and were analyzed by analysis of variance (ANOVA) fol- RPW compared to control rats with APH (Table 2). lowed by Tukey-Kramer multiple comparisons test. Inflam- mation scores and their significance were calculated by 3.3. Effect of ROS on Proinflammatory Cytokines, IL-6, IL-8, Chi-square test with Yate’s corrections. Differences were and TNF-𝛼. Induction of APH in rats significantly increased considered significant with a 𝑃 valuelessthan0.05.Statis- the tissue levels of IL-6, IL-8, and TNF-𝛼.Sawpalmettotreat- tical analyses were performed using the SPSS for Windows ment significantly decreased the IL-6 (20.3%), IL-8 (48.1%), (v. 10.0). and TNF-𝛼 (39.4%). ROS induced significant reductions in IL-6, IL-8, and TNF-𝛼, which were greater than that 3. Results produced by saw palmetto (Figure 3). These reductions were dose dependant regarding IL-6 (54.7%, 59%, and 65.6%), IL- 𝛼 3.1. Characterization of ROS Extract. Screening of the 8 (93%, 95.3%, and 97.9%), and TNF- (50.8%, 65.1%, and methanolic onion extract indicated mainly the presence of 91.3%). sterols and/or terpenoids, polyphenolic compounds such as flavonoids, tannins, and the absence of alkaloids and 3.4. Effect of ROS on Gene Expression of TGF-𝛽R1, IGF-1, saponins. Assay of total phenolic content of methanolic and Clusterin. Induction of APH is accompanied with a extract was determined to be 12.9 mg GAE/g DE. Assay of significant increase of the clusterin and IGF-1 expression in total flavonoid content was determined to be 119 mg QE/g the ventral lobe of rat prostate, while the TGF-𝛽R1 expression DE. The antioxidant is expressed as inhibition percentage was significantly decreased. Saw palmetto treated rats with Mediators of Inflammation 5

VWD1 A, wavelength =360nm (ESSAMROSD) VWD1 A, wavelength =360nm (ESSAM Q4 GLCO) 5.682 5.115 400 200 11.011 300 150 200 100 (mAU) (mAU) 100 50 2.990 19.131 18.209 16.116 2.129 0 0 0 5 10 15 20 0 24681012 14 (min) (min) (a) (b)

󸀠 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 with APH treated for 30 days at different doses.

Body weight (g) Treatment APW(g) RPW(mg/g) Start End Normal rats 199.14 ± 5.16 229 ± 8.81 0.106 ± 0.01 0.452 ± 0.05 ∗ ∗ Rats with APH (negative control) 187.6 ± 8.65 241.4b ± 12.55 0.756 ± 0.05 3.136 ± 0.19 Rats with APH treated with ∗ ∗ Saw palmetto (100 mg/kg) 192.8 ± 4.59 258.3b ± 10.22 0.698 ± 0.03 2.705 ± 0.21 ∗ ∗ RO scales (75 mg/kg) 227.5 ± 7.44 271.1b ± 7.69 0.681 ± 0.02 2.512 a ± 0.12 ∗ ∗ RO scales (150 mg/kg) 225.5 ± 7.15 271.6b ± 9.20 0.593 a ± 0.023 2.183 a ± 0.14 ∗ ∗ RO scales (300 mg/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.

the expression of IGF-1 was significantly decreased in a dosedependent fashion. On the other hand, TGF-𝛽R1 expression was not significantly changed compared to the APH group (Figure 4).

3.5. Histological Changes. Ventral prostates from the normal group showed normal histology with average histoscore corresponding to 24.3 ± 1.2 (Figure 5(a)). Ventral prostates from the APH-induced rats showed increase in the number of acini (hyperplasia) with irregular distribution. The acini were arranged back to back with intraluminal and stromal papillary projections. All prostatic acini were lined by tall columnar cells with epithelial pilling and nuclear stratification seen among six of seven prostatic sections in the group. Prostatic sections from two rats in this group Figure 2: TLC profile of red onion scales, 1: methanol extract; 2: 󸀠 showed 1-2 mitotic figures. The interstitial stroma was scant quercetin-4 -glucoside, 3: rutin; 4: quercetin; UV 366 nm. and showed edema with congested blood vessels and mixed acute and chronic perivascular inflammatory cells. Polymor- phonuclear leukocytes with eosinophilic granules were con- APH showed significant increase in clusterin and TGF- sidered as acute inflammatory cells. Chronic inflammatory 𝛽R1 expression, an effect which was accompanied with the cells seen were lymphocytes which were distinguished by decrease in IGF-1 expression. All the treatment groups with their darkly stained ink-dot nucleus and thin rim basophilic ROS extract showed a significant increase in clusterin, while cytoplasm. These findings were associated with a significant 6 Mediators of Inflammation

0.4 0.10

∗ ∗ 0.08 0.3 * # 0.06 0.2 # -actin (R.B.I) # -actin (R.B.I) 𝛽 𝛽 0.04 / / 6 # 8 # IL- 0.1 IL- 0.02

# # # 0.0 0.00 0.4

∗ 0.3

0.2 # -actin (R.B.I) 𝛽 / # 𝛼 # 0.1 TNF-

# 0.0

Normal RO scales 75 mg/kg APH RO scales 150 mg/kg Saw palmetto RO scales 300 mg/kg

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 different ∗ dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using 𝛽-actin as a reference. Significantly different # from 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. part of the section were still hyperplastic being lined by tall (Figures 5(b)–5(d)). columnar cells showing nuclear stratificationFigure ( 6(b)). The ventral prostate in the saw palmetto group showed Mitotic figures were absent and the surrounding stroma diminution regarding the scale of pathological changes and showed edema and congested blood vessels. Mixed acute and histoscore corresponding to 32.2 ± 2.1 (Figure 5(e)). The chronic inflammatory cells were seen among four of six rats prostatic acini were less crowded. However, hyperplastic in the group along with clustering of stromal mast cells. Kary- changessuchasacinilinedbytallcolumnarcellswith orrhectic debris was seen within the gland lumina among epithelial pilling and nuclear stratification were seen among four of six rats in the group. These findings corresponded to all rats in the group. The nuclei were round to oval, regular, ahistoscoreof35.3 ± 1.2. and basally placed. There was no loss of polarity noted Sections examined from ventral prostates of APH- in the epithelium. Prostatic sections from two rats in this induced rats treated with ROS extract (150 mg/kg) showed group also showed 1-2 mitotic figures. The interstitial stroma prostatic acini lined by tall columnar epithelium exhibiting was considerable, fibromuscular to edematous, and showed nuclear stratification in all five rats. The acinar distension vascular congestion. Prostatic sections from one rat showed was slightly diminished. Few acini showed scalloped luminal small foci of mixed inflammatory cells. secretions among three of five rats. Mitotic figures were Histological changes in the ventral lobe of APH-induced absent and surrounding stroma showed edema and congested rats treated with ROS extract (75 mg/kg) showed irregular blood vessels. Small foci of mixed acute and chronic inflam- and enormous dilatation of prostatic acini with intraluminal matory cells were seen among three of five rats in the group. secretions among five of six rats in the group. These changes Intraluminal sloughing of karyorrhectic debris was also seen were more pronounced in the central regions of the prostatic among three of five rats (Figures 6(c) and 6(d)). Clustering tissue. Dilated acini showed epithelial changes in the form of of stromal mast cells was seen among three of five rats. These flattening or low cuboidal transformationFigure ( 6(a)). Few findings corresponded to a histoscore of 34.0 ± 0.5. acini showed loss of lining epithelium although the basement Histological changes in ventral prostates of rats, with membrane appeared still intact. Acini seen at the peripheral APH treated with ROS extract (300 mg/kg) showed prostatic Mediators of Inflammation 7

0.8 1.5 ∗ # 0.6 1.0

-actin (R.B.I) 0.4 𝛽 -actin (R.B.I) / 𝛽 1 / R 1 # 𝛽 0.5 # # 0.2 IGF- TGF- ∗ # 0.0 0.0

1.5

# 1.0 # # -actin (R.B.I) 𝛽 # 0.5 ∗ Clusterin/

0.0

Normal RO scales 75 mg/kg APH RO scales 150 mg/kg Saw palmetto RO scales 300 mg/kg

Figure 4: The expression of TGF-𝛽R1, IGF-1, and clusterin target genes in ventral prostate of Wistar rats after treatment with RO scales at ∗ different dose levels. All values are expressed as mean ± S.E. of the relative band intensity (R.B.I.) using 𝛽-actin as a reference. Significantly # different 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 nuclear intensity between moderate to strong. No expression was stratification in all six rats. Acinar distension was slightly observed in the basal or stromal cells (Figures 8(a) and 8(b)). diminished. Scattered mixed acute and chronic inflammatory Prostatic sections from RO scale revealed IL-6 expression cells in the stroma along with intraluminal sloughing of kary- corresponding to score 1 with weak intensity. TGFBR-1 orrhecticdebriswereseeninthreeofsixrats(Figure 6(e)). expression was score 0. No expression was observed in the Mitotic figures were absent and surrounding stroma showed basal or stromal cells. The immunohistochemical profile edema and congested blood vessels. Clustering of stromal expressed by the prostatic tissue showed no notable doze mast cells was seen among two rats. These findings corre- related variation (Figures 8(c) and 8(d)). sponded to a histoscore of 32.2 ± 0.8. 4. Discussion 3.6. Immunohistochemistry. Normal control rats showed no expression of both immunohistochemical markers with pos- It was reported that APH usually arises with concomitant itive internal controls (Figures 7(a) and 7(b)). TGFBR-1 BPH and exhibits several cancer-like features, which makes expression corresponded to score 3 with strong intensity APH an intermediate lesion between BPH and the subset of in the secretory epithelial cells of acini with foci of strong well-differentiated prostate cancers31 [ ]. In the present study, expression among basal cells also in the prostatic sections APH-induced rats revealed prostatic enlargement which was from APH-induced rats. IL-6 expression corresponded to evidencedbyanincreaseintheAPWandRPWofthe score1andshowedweakintensityinthesecretoryepithelial ventral prostate lobe as well as acinar hyperplasia that may cells only. No expression was observed in basal or stromal beattributedtotheinfluenceofandrogenonprostategrowth cells (Figures 7(c) and 7(d)). [32]. The development of hyperplasia was associated with Prostatic sections from saw palmetto treated APH- enhanced proliferation and suppressed apoptosis of prostatic induced rats revealed TGFBR-1 expression corresponding to cells [25, 33].Citral,usedinthecurrentstudy,wasreportedto score 3 exhibiting strong intensity in the secretory epithelial have an estrogenic-like effect, as it binds to estrogen receptors cells of acini with foci of strong expression also in the basal that are located in prostatic epithelial cells of both human cells. IL-6 was variable between score 2-3 with a staining [34]andrat[35]. Type 2 estrogen-binding sites in rat ventral 8 Mediators of Inflammation

(a) (b)

(e)

Figure 5: (a) Prostate of normal rats with round acini and intact basement membranes. Arrow points to acini lined by two layers of low cuboidal epithelium. (b) Prostate of testosterone and citral treated castrated rats with atypical prostatic hyperplasia (APH) reveals increase in the number of prostatic acini (hyperplasia) which are placed back to back with no intervening stroma. Thin arrow points to acini lined by tall columnar cells. Thick arrow points to nuclear stratification and stromal projections. (c) Interstitial stroma shows congested blood vessels and inflammatory cells. (d) Thick vertical down arrow points to polymorphonuclear acute inflammatory cells and the thin slanting arrows point to chronic inlammatory cells (lymphocytes). Very thin arrow points to luminal papillary projections, thick long arrow points to 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 nuclear stratification. Thin arrow points to a small focus of polymorphonuclear acute inflammatory cells in stroma. Acinus at the base of thefigure shows intraluminal projection (at 60X).

prostate could be responsible for this proliferative effect35 [ ]. a manner which influences prostate cell growth, survival, or Moreover, the present study revealed an increase in the IGF-1 apoptosis [37]. andadecreaseintheTGF-𝛽1 which may cause the prostate Fruits and vegetables have health benefits and are good enlargement in APH group. TGF-𝛽1 is known to suppress sources of antioxidants; therefore a lot of recent literature tissueproliferationandinducecellapoptosis[36]. Circulating has focused on nutritional and herbal medicine for prostatic testosterone acts locally in the prostate via the production hyperplasia [38–40]. In the present study, administration of of growth factors such as IGF and TGF families that act in ROS extract induced significant and dose-related reduction Mediators of Inflammation 9

(a) (b)

(e)

Figure 6: (a)-(b) Prostate of rats treated with RO scale extract in a dose of 75 mg/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 cells showing nuclear stratification. Arrow points to polymorphonuclear acute inflammatory cell infiltrate in stroma near the acini. (c)-(d) Prostate of rats treated with RO scale extract in a dose of 150 mg/kg showing. (c) Arrow pointing to reduced acinar distension which are lined by tall columnar cells exhibiting nuclear stratification. (d) Arrow pointing to luminal karyorrhectic debris. (e) Prostate of rats treated with RO scales in a dose of 300 mg/kg showing. Thin arrow points to luminal karyorrhectic debris and thick arrow points to hyperplasia as seen by tall columnar cells lining the acini with nuclear stratification (at 60X).

in both the absolute and relative weight of prostate in which was found to be mainly responsible for the protective the APH-induced rats, an effect that was greater than that effects against different degenerative pathological diseases induced by saw palmetto. These effects may be due to the [11, 14]. It was reported that administration of quercetin along fact that ROS are rich in flavonols which represent 60% of with finasteride resulted in reduction in prostate weight in the total active constituents. These mainly are kaempferol, rats, through a cell cycle-related pathway that may function quercetin, quercetin dimer, quercetin trimer, and other independently of androgens [41]. quercetin glycoside. Flavonols were found to be responsible Moreover, the ROS extract has ameliorated the his- for the antioxidant, hepatoprotective, anticancer, antimicro- topathological changes, significantly modulated the expres- bial, antistress, and other biological activities [12, 13]. Among sion of the proinflammatory cytokines, and decreased the in- the major constituents of the ROS extract, 16% are quercetin, flammatory scores in a dosedependent manner. In the current 10 Mediators of Inflammation

(a) (b)

Figure 7: Immunohistochemical expression of TGF𝛽R-1 and IL-6 in prostates of normal and APH rats. (a) Normal rats TGF𝛽R-1 expression score 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)

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-6 expression score 2-3. (c)-(d) Immunohistochemical expression of TGFBR-1 and IL-6 in RO scales 75 mg 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). Mediators of Inflammation 11 study, ROS significantly decreased IL-6, IL-8, and TNF-𝛼 in proliferative prostatic diseases [56]. Moreover, Senthilkumar the ventral lobe of prostatic tissues of rats with APH. These et al. [57] suggested that quercetin can decrease the survival findings are in accordance with results that are reported by of androgen-independent prostate cancer cells by changing Jung et al. [42]. the expression of IGF-1 signaling and inducing apoptosis in In the context of chronic inflammation and expression cancer patients. of proinflammatory cytokines, IL-6 is one of the major The antiproliferative effect of quercetin is probably medi- physiologic mediators of acute phase reaction that influence ated by interaction with the type II estrogen binding sites [58]. immune responses and inflammatory reactions [43]. IL-6 is Quercetinalsoinhibitscellinvasionandinducesapoptosis also secreted by both normal and prostatic epithelial cells through a pathway involving heat shock proteins [59]. The andactsasagrowthfactorfornormalprostaticepithelial ability of RO extract to decrease the prostate weight in rats cells [44]. TNF-𝛼, a proinflammatory cytokine, may induce withAPHmayalsobeduetothatithassomeactivecon- inflammation by induction of COX-2, superoxide radical, stituents with a specific ability to inhibit phosphodiesterase and hydrogen peroxide in both human and rat mesangial subtype 5A (PDE-5A). These findings are in agreement with cells [45]. TGF-𝛽, an inflammatory cytokine, has been shown that which was reported by Lines and Ono [60]whofound to regulate stromal proliferation and differentiation in BPH, that the improvement in sexual function might be due to and it is a key factor in the androgen control of prostatic quercetin 1. Quercetin 1 was reported to have specific PDE-5A growth. Recently, Descazeaud et al. [46]investigatedthe inhibitory activity [55]. Chronic administration of NO donor TGF-𝛽 receptor II protein (TGFBRII) expression in BPH drugs and PDE5-inhibitors may also induce antiproliferative patients. They observed a significant association between and/or apoptotic effects in the prostate61 [ ]. TGFBRII stromal staining and prostatic volume. In conclusion, the results of the present study showed that The ROS extract contains large amounts of antioxidant the methanolic extract of ROS have the ability to decrease flavonoids, mainly, ferulic, gallic, kaempferol, and quercetin the prostate weight in APH-induced rats. This effect may be or their glycosides [12, 13]. Jung et al. [42]foundthat due to the anti-inflammatory and immunomodulatory effects hepatic expressions of TNF-𝛼 andIL-6indiabeticratswere of the extract. All of these effects are dose-dependent and suppressed by quercetin. These results are in agreement may not only be related to the antioxidant activities of the with previous reports, in which quercetin was found to flavonoids or their glycosides but also due to its phenolic have antioxidative and anti-inflammatory activities47 [ ]. content in ROS. Quercetin significantly decreases prostatitis symptoms by decreasing prostatic inflammation [15]. Moreover, quercetin can affect the prostate cancer biology by inhibiting arachi- Conflict of Interests donic acid metabolism through the blocking of phospho- The authors declare that they have no conflict of interests. lipase A2 and 5 as well as 12-lipooxygenase enzymes [48] and inhibiting androgen receptor mutations [49]. Quercetin has also demonstrated the ability to interrupt the spread Acknowledgments of prostate cancer (metastases) and to promote cell death. This work was supported by Sheikh Ahmed H. Fetaihi, It was able to decrease the activity of specific enzymes Chair for Research on Prostatic Diseases, King Abdulaziz knowntobeinvolvedintumorinvasionandmetastases[50]. University, Jeddah, Saudi Arabia. The authors would also like Furthermore, quercetin was found to have antiproliferative to thank Mr. Islam Farouk, Department of Pharmacology and activity in vitro against several cancer cells [51]andarrested Toxicology, Faculty of Pharmacy, King Abdulaziz University, cell cycle progression either at the G1/S phase [50]oratthe Jeddah, Saudi Arabia, for his great effort and help in the G2/M transitional boundary [52]. Quercetin is also a possible experimental study. option to relieve symptoms for men who have prostate problems, and it has been identified as being beneficial in cases of prostatitis [53]. References The ROS extract significantly reduced the absolute and [1] A. Sciarra, G. Mariotti, S. Salciccia et al., “Prostate growth and relative weight of ventral lobe prostate of rats with APH com- inflammation,” Journal of Steroid Biochemistry and Molecular pared with the negative control rats. This effect may be par- Biology, vol. 108, no. 3–5, pp. 254–260, 2008. tiallyduetothatROSextractmayhaveantiproliferativeand [2] K.Stamatiou,A.Alevizos,M.Natzaretal.,“Associationsamong apoptotic action on the ventral lobe of prostatic tissues. The benign prostate hypertrophy, atypical adenomatous hyperplasia ROS extract significantly decreased the expression of IGF-1 and latent carcinoma of the prostate,” Asian Journal of Androl- and increased clusterin expression. The prostate enlargement ogy,vol.9,no.2,pp.229–233,2007. and the increased net weight of the ventral lobe in case of [3] D. G. Bostwick, J. Srigley, D. Grignon et al., “Atypical ade- APH may be partly due to the increased IGF-1 and decreased nomatous hyperplasia of the prostate: morphologic criteria 𝛽 for its distinction from well-differentiated carcinoma,” Human TGF- 1 expression. This modulation in expression of these Pathology,vol.24,no.8,pp.818–832,1993. cytokines may play a crucial role in prostate cell proliferation [4] G. Novara, A. Galfano, R. B. Berto, V. Ficarra, R. V. Navarrete, andapoptosis.Thesefindingsareinagreementwiththatof and W. Artibani, “Inflammation, apoptosis, and BPH: what is Wu et al. [54]. IGF-1, a mitogenic factor, interacts with IGF- the evidence?” European Urology,vol.5,no.4,pp.401–409, 1 receptor stimulating cell proliferation [55]andinducing 2006. 12 Mediators of Inflammation

[5] J. S. Sandhu, “Prostate cancer and chronic prostatitis,” Current [24] L. Sandford, J. W. Searle, and J. F. Kerr, “Successive waves Urology Reports,vol.9,no.4,pp.328–332,2008. of apoptosis in the rat prostate after repeated withdrawal of [6] B. Rigas and Y. Sun, “Induction of oxidative stress as a testosterone stimulation,” Pathology, vol. 16, no. 4, pp. 406–410, mechanism of action of chemopreventive agents against cancer,” 1984. British Journal of Cancer, vol. 98, no. 7, pp. 1157–1160, 2008. [25] D. Engelstein, J. Shmueli, S. Bruhis, C. Servadio, and A. [7] K. Rahman and G. M. Lowe, “Garlic and cardiovascular disease: Abramovici, “Citral and testosterone interactions in inducing a critical review,” Journal of Nutrition,vol.136,3,pp.736S–740S, benign and atypical prostatic hyperplasia in rats,” Comparative 2006. Biochemistry and Physiology,vol.115,no.2,pp.169–177,1996. [8] C. Galeone, C. Pelucchi, F. Levi et al., “Onion and garlic use and [26] M. D. Scolnik, C. Servadio, and A. Abramovici, “Comparative human cancer,” American Journal of Clinical Nutrition,vol.84, study of experimentally induced benign and atypical hyperpla- no. 5, pp. 1027–1032, 2006. sia in the ventral prostate of different rat strains,” Journal of [9] A. de Martino, G. Filomeni, K. Aquilano, M. R. Ciriolo, and G. Andrology,vol.15,no.4,pp.287–297,1994. Rotilio, “Effects of water garlic extracts on cell cycle and viability [27] R. D. Lillie, Histopathologic Techniques and Practical Histochem- of HepG2 hepatoma cells,” Journal of Nutritional Biochemistry, istry,McGraw-Hill,NewYork,NY,USA,1965. vol. 17, no. 11, pp. 742–749, 2006. [28] C. de Nunzio, G. Kramer, M. Marberger et al., “The contro- [10] F. Bravi, C. Bosetti, L. dal Maso et al., “Food groups and risk of versial relationship between benign prostatic hyperplasia and benign prostatic hyperplasia,” Urology,vol.67,no.1,pp.73–79, prostate cancer: the role of inflammation,” European Urology, 2006. vol. 60, no. 1, pp. 106–117, 2011. [11] G. Griffiths, L. Trueman, T. Crowther, B. Thomas, and B. Smith, [29] A. Hobisch, H. Rogatsch, and A. Hittmair, “Immunohistochem- “Onions—a global benefit to health,” Phytotherapy Research,vol. ical localization of interleukin-6 and its receptor in benign, 16,no.7,pp.603–615,2002. premalignant and malignant prostate tissue,” The Journal of [12] N. L. Tram, C. Hazama, M. Shimoyamada, H. Ando, K. Kato, Pathology, vol. 191, pp. 239–244, 2000. and R. Yamauchi, “Antioxidative compounds from the outer [30] S. K. Gounder, V. T. Chang, D. Hoover et al., “Prostate cancer scales of onion,” Journal of Agricultural and Food Chemistry,vol. immunohistochemical (IHC) stains and survival in stage D3 53, no. 21, pp. 8183–8189, 2005. patients,” JClinOncol,vol.26,22187,no.15,2008. [13] A. Guls¨ ¸en, D. P. Makris, and P. Kefalas, “Biomimetic oxidation [31] D. G. Bostwick, W. H. Cooner, L. Denis, G. W. Jones, P. of quercetin: isolation of a naturally occurring quercetin het- T. Scardino, and G. P. Murphy, “The association of benign erodimer and evaluation of its in vitro antioxidant properties,” prostatic hyperplasia and cancer of the prostate,” Cancer,vol. Food Research International,vol.40,no.1,pp.7–14,2007. 70,no.1,pp.291–301,1992. [14] B. N. Singh, B. R. Singh, R. L. Singh et al., “Polyphenolics [32] A. Abramovici, C. Servadio, J. Shmuely, and U. Sandbank, from various extracts/fractions of red onion (Allium cepa) peel “Experimental induction of atypical hyperplasia in rat ventral with potent antioxidant and antimutagenic activities,” Food and prostate,” Progress in clinical and biological research,vol.243,pp. Chemical Toxicology,vol.47,no.6,pp.1161–1167,2009. 559–568, 1987. [15] D. A. Shoskes, S. I. Zeitlin, A. Shahed, and J. Rajfer, “Quercetin [33] A. A. Geldof, C. Engel, and B. R. Rao, “Estrogenic action of com- in men with category III chronic prostatitis: a preliminary monly used fragrant agent citral induces prostatic hyperplasia,” prospective, double-blind, placebo-controlled trial,” Urology, Urological Research, vol. 20, no. 2, pp. 139–144, 1992. vol. 54, no. 6, pp. 960–963, 1999. [34] J. Brolin, L. Skoog, and P. Ekman, “Immunohistochemistry [16] G. E. Trease and W. C. Evans, Pharmacognosy, Bailliere Tindall and biochemistry in detection of androgen, progesterone, and Press, London, UK, 1983. estrogen receptors in benign and malignant human prostatic [17] H. Wagner and S. Bladt, Plant Drug Analysis: A Thin Layer tissue,” Prostate,vol.20,no.4,pp.281–295,1992. Chromatography Atlas, Springer, London, UK, 1996. [35]S.M.Ho,I.Leav,F.B.Merk,M.Yu,P.W.L.Kwan,andJ. [18] V. L. Singleton and J. A. Rossi, “Colorimetry of total phenolics Ziar, “Induction of atypical hyperplasia, apoptosis, and type II with phosphomolybdic-phosphotungstic acid reagents,” Ameri- estrogen- binding sites in the ventral prostates of noble rats can Journal of Enology and Viticulture,vol.16,pp.144–158,1965. treated with testosterone and pharmacologic doses of estradiol- [19] M. G. L. Hertog, “Content of potentially anticarcinogenic 17𝛽,” Laboratory Investigation,vol.73,no.3,pp.356–365,1995. flavonoids of 28 vegetables and 9 fruits commonly consumed [36] N. Soulitzis, I. Karyotis, D. Delakas, and D. A. Spandidos, in the Netherlands,” Journal of Agricultural and Food Chemistry, “Expression analysis of peptide growth factors VEGF, FGF2, vol. 40, no. 12, pp. 2379–2383, 1992. TGFB1, EGF and IGF1 in prostate cancer and benign prostatic [20]E.A.Abdel-Sattar,S.M.Mouneir,G.F.Asaad,andH. hyperplasia,” International Journal of Oncology,vol.29,no.2,pp. M. Abdallah, “Protective effect of Calligonum comosum on 305–314, 2006. haloperidol-induced oxidative stress in rat,” Toxicology and [37] D. Marinese, R. Patel, and P.D. Walden, “Mechanistic investiga- Industrial Health,vol.30,no.2,pp.147–153,2014. tion of the adrenergic induction of ventral prostate hyperplasia [21] A. Braca, D. Nunziatina, D. B. Lorenzo, P.Cosimo, P.Mateo, and in mice,” Prostate,vol.54,no.3,pp.230–237,2003. M. Ivano, “Antioxidant principles from Bauhinia terapotensis,” [38] I. S. Shin, M. Y. Lee, H. K. Ha, C. S. Seo, and H. K. Shin, Journal of Natural Products,vol.64,pp.892–895,2001. “Inhibitory effect of Yukmijihwang-tang, a traditional herbal [22] S. F. Abou Zid and G. M. Elsherbeiny, “Increase in flavonoids formula against testosterone-induced benign prostatic hyper- content in red onion peel bymechanical shredding,” Journal of plasia in rats,” BMC Complementary and Alternative Medicine, Medicinal Plants Research,vol.2,no.9,pp.258–260,2008. vol. 20, pp. 12–48, 2012. [23] E. Golomb, A. Kruglikova, D. Dvir, and A. Abramovici, [39]C.C.Peng,J.H.Liu,C.H.Changetal.,“Actionmechanism “Induction of atypical prostatic hyperplasia in rats by sympath- of Ginkgo biloba leaf extract intervened by exercise therapy omimetic stimulation,” The Prostate,vol.34,pp.214–221,1998. in treatment of benign prostate hyperplasia,” Evidence-Based Mediators of Inflammation 13

Complementary and Alternative Medicine,vol.2013,ArticleID [55]R.W.Furlanetto,J.N.DiCarlo,andC.Wisehart,“ThetypeII 408734, 12 pages, 2013. insulin-like growth factor receptor does not mediate deoxyri- [40] C. H. Ma, W. L. Lin, S. L. Lui et al., “Efficacy and safety of bonucleic acid synthesis in human fibroblasts,” Journal of Chinese herbal medicine for benign prostatic hyperplasia: sys- Clinical Endocrinology and Metabolism,vol.64,no.6,pp.1142– tematic review of randomized controlled trials,” Asian Journal 1149, 1987. of Andrology,vol.15,pp.471–482,2013. [56] Z. Culig, A. Hobisch, M. V. Cronauer et al., “Regulation of [41]Z.Ma,T.H.Nguyen,T.H.Huynh,P.T.Do,andH.Huynh, prostatic growth and function by peptide growth factors,” “Reduction of rat prostate weight by combined quercetin- Prostate, vol. 28, pp. 392–405, 1996. finasteride treatment is associated with cell cycle deregulation,” [57] K. Senthilkumar, P. Elumalai, R. Arunkumar et al., “Quercetin Journal of Endocrinology,vol.181,no.3,pp.493–507,2004. regulates insulin like growth factor signaling and induces [42] J. Y. Jung, Y. Lim, M. S. Moon, J. Y. Kim, and O. Kwon, “Onion intrinsic and extrinsic pathway mediated apoptosis in andro- peel extracts ameliorate hyperglycemia and insulin resistance gen independent prostate cancer cells (PC-3),” Molecular and in high fat diet/streptozotocin-induced diabetic rats,” Nutrition Cellular Biochemistry,vol.344,no.1-2,pp.173–184,2010. and Metabolism,vol.8,article18,2011. [58] F.O.Ranelletti,R.Ricci,L.M.Laroccaetal.,“Growth-inhibitory [43] B. Djavan, E. Eckersberger, G. Espinosa et al., “Complex effect of quercetin and presence of type-II estrogen-binding Mechanisms in Prostatic Inflammatory Response,” European sites in human colon-cancer cell lines and primary colorectal Urology,vol.8,no.13,pp.872–878,2009. tumors,” International Journal of Cancer,vol.50,no.3,pp.486– [44] D.Giri,M.Ozen,andM.Ittmann,“Interleukin-6isanautocrine 492, 1992. growth factor in human prostate cancer,” The American Journal [59] Y. Q. Wei, X. Zhao, Y. Kariya, H. Fukata, K. Teshigawara, and A. of Pathology,vol.159,no.6,pp.2159–2165,2001. Uchida, “Induction of apoptosis by quercetin: involvement of [45] H. H. Radeke, B. Meier, N. Topley, J. Floge, G. G. Habermehl, heat shock protein,” Cancer Research, vol. 54, no. 18, pp. 4952– and K. Resch, “Interleukin 1-𝛼 and tumor necrosis factor-𝛼 4957, 1994. induce oxygen radical production in mesangial cells,” Kidney [60] T. C. Lines and M. Ono, “FRS 1000, an extract of red onion International,vol.37,no.2,pp.767–775,1990. peel, strongly inhibits phosphodiesterase 5A (PDE 5A),” Phy- [46] A. Descazeaud, N. Weinbreck, G. Robert et al., “Transform- tomedicine,vol.13,no.4,pp.236–239,2006. ing growth factor 𝛽-receptor II protein expression in benign [61] C. H. Deng, H. R. Chen, S. P. Qiu, J. Z. Liu, K. L. Zheng, prostatic hyperplasia is associated with prostate volume and and H. Mei, “Effect of nitric oxide donor and alpha1-receptor inflammation,” BJU International,vol.108,no.2,pp.E23–E28, antagonist on proliferation/apoptosis of hyperplastic prostatic 2011. stromal cells in vitro,” Zhonghua Wai Ke Za Zhi,vol.42,no.4, [47] O. Coskun, M. Kanter, A. Korkmaz, and S. Oter, “Quercetin, pp.201–204,2004. a flavonoid antioxidant, prevents and protects streptozotocin- induced oxidative stress and 𝛽-cell damage in rat pancreas,” Pharmacological Research,vol.51,no.2,pp.117–123,2005. [48] J. S. Bland, Clinical Nutrition: A Functional Approach,The Institute for Functional Medicine, Gig Harbor, Wash, USA, 1999. [49] N. Xing, Y. Chen, S. H. Mitchell, and C. Y. F. Young, “Quercetin inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells,” Carcinogenesis,vol.22,no.3, pp.409–414,2001. [50] M. R. Vijayababu, A. Arunkumar, P. Kanagaraj, P. Venkatara- man, G. Krishnamoorthy, and J. Arunakaran, “Quercetin down- regulates matrix metalloproteinases 2 and 9 proteins expression in prostate cancer cells (PC-3),” Molecular and Cellular Bio- chemistry,vol.287,no.1-2,pp.109–116,2006. [51] N. Bhatia, C. Agarwal, and R. Agarwal, “Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells,” Nutrition and Cancer, vol. 39, no. 2, pp. 292–299, 2001. [52]J.A.Choi,J.Y.Kim,J.Y.Leeetal.,“Inductionofcellcycle arrest and apoptosis in human breast cancer cells by quercetin,” International journal of oncology,vol.19,no.4,pp.837–844, 2001. [53] D. A. Shoskes, J. C. Nickel, and M. W. Kattan, “Phenotypically directed multimodal therapy for chronic prostatitis/chronic pelvic pain syndromepp. A prospective study using UPOINT,” Urology,vol.75,no.6,pp.1249–1253,2010. [54] S. Wu, H. Sun, X. Qi, and Z. Tu, “Effect of epristeride on the expression of IGF-1 and TGF-𝛽 receptors in androgen-induced castrated rat prostate,” Experimental Biology and Medicine,vol. 226, no. 10, pp. 954–960, 2001.