Chi-Mei Ku and Jin-Yuarn Lin. 1. Introduction

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1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 2015, Article ID , 12 pges Reserch Article Frnesol, Sesquiterpene Alcohol in Herl Plnts, Exerts Anti-Inflmmtory nd Antillergic Effects on Ovlumin-Sensitized nd -Chllenged Asthmtic Mice Chi-Mei Ku nd Jin-Yurn Lin Deprtment of Food Science nd Biotechnology, Ntionl Chung Hsing University, 250 Kuo Kung Rod, Tichung 40227, Tiwn Correspondence should e ddressed to Jin-Yurn Lin; jinlin@nchu.edu.tw Received 15 Mrch 2014; Revised 14 Novemer 2014; Accepted 14 Novemer 2014 Acdemic Editor: Bor-Luen Ching Copyright 2015 C.-M. Ku nd J.-Y. Lin. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. To investigte the effect of frnesol on llergic sthm, three frnesol doses were extr-dded into AIN-76 feed consumed y ovlumin- (OVA-) sensitized nd -chllenged mice continuously for 5 weeks, t pproximtely 5, 25, nd 100 mg frnesol/kg, BW/dy. The results showed tht there were no significnt differences in ody weight, feed intke, nd viscerl orgn weights etween the frnesol supplementtion nd dietry control groups. Frnesol supplementtion decresed interleukin (IL)-6/IL- 10 level rtios in roncholveolr lvge fluid (BALF). Frnesol supplementtion significntly (P < 0.05) restored the cytokine secretion ility of peritonel mcrophges tht ws suppressed s result of OVA sensitiztion nd chllenge nd slightly decresed tumornecrosisfctor(tnf-α)/il-10 cytokine secretion rtios. Frnesol supplementtion slightly (P > 0.05) decresed IL-4 ut significntly (P < 0.05) incresed IL-2 levels secreted y the splenocytes in the presence of OVA, implying tht frnesol might hve systemic ntillergic effect on llergic sthmtic mice. Frnesol supplementtion significntly (P < 0.05) incresed IL-10 levels secreted y the splenocytes in the presence of OVA, suggesting tht frnesol might hve n nti-inflmmtory potentil to llergic sthmtic mice. Overll, our results suggest tht frnesol supplementtion my e eneficil to improve the Th2-skewed llergic sthmtic inflmmtion. 1. Introduction Asthm tht is n llergic disese estimted to ffect t lest 300 million people worldwide hs ttrcted much ttention recently [1, 2]. Allergic sthm is chronic irwy inflmmtory disese ccompnied with incresed inflmmtory cell infiltrtion, lung inflmmtion, nd irwy hyperresponsiveness. Asthm results from irwy inflmmtion involving diversity of ctivted cells including mst cells, eosinophils, T-lymphocytes, neutrophils, mcrophges, nd epithelil cells. These cells re recruited to the site nd relese proinflmmtory cytokine meditors tht ugment nd regulte irwy inflmmtion, resulting in irwy hyperresponsiveness responsile for the chronic symptoms of dyspne, wheezing, nd chest tightness [3]. Airwy inflmmtion results in denudtion of ronchil epithelium nd thickening of suepithelil sement memrne due to deposition of collgen. In ddition, severe sthm hs een chrcterized y occlusion of the ronchil lumen y mucus, hyperplsi nd hypertrophy of the ronchil smooth muscle, nd golet cell hyperplsi [3]. Therefore, the levels of mucus (possily mucin)ndotherproteinsintheronchillumenmye selected s n inflmmtory mrker. There re two distinct helper T lymphocytes, type 1 helper T (Th1) nd Th2 cells tht synthesize differentil cytokines to influence immune responses. Interleukin (IL)- 1, tumour necrosis fctor (TNF), nd IL-6 produced y Th1, Th2 lymphocytes, or other inflmed cells tht highlight the wy nd trigger locl inflmmtion within injured tissues cn e roughly clssified s proinflmmtory cytokines [4]. In contrst, IL-10 produced y Th2 cells, T regultory cells (Th3 cells), mcrophges, nd some B cells to inhiit Th1 synthesis nd other cytokines nd mcrophge functions during the lte inflmmtion phse re recognized s nti-inflmmtory cytokines [5]. An imlnce in Th1/Th2 immune response ptterns nd pro/nti-inflmmtory cytokines produced nd

2 2 Evidence-Bsed Complementry nd Alterntive Medicine () OH Body weight (g) Grouping Bled Bled OVA/Al-sensitiztion Experimentl period (dys) Bled OVA/Al-sensitiztion () OVA/Al-sensitiztion OVA-inhltion Figure 1: Frnesol structure () nd its supplementtion effects with different doses for 5 weeks on ody weight chnges of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice (). Vlues re mens ± SD (n =12 15). Vlues mong groups t the sme experimentl point not shring common smll letter re significntly different (P < 0.05) from ech other nd ssyed y one-wy ANOVA, followed y Duncn s new multiple rnge test., nonsensitized control;, dietry control;, positive control (dexmethsone, 3 mg/kg BW, 0.3 ml/mouse y gvge);, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed). generlly ccompnied with stress hormones my result in differentil diseses, for exmple, persistent infections, severe immunosuppression, utoimmunity, llergy/topy, tumour growth, nd chronic grft-versus-host disese [6, 7]. Allergic sthmischrcterizedsth2-skeweddisese.regultion of the Th1/Th2 imlnce nd nti/proinflmmtory cytokine expression profiles in the host my void immune disorder diseses [8]. Potentil phytochemicls from different food mterilsorhersrecentlyshedlightonimmunomodultion nd my e eneficil for the corresponding humn diseses [9]. Mny drugs re used to tret sthm, such s inhled corticosteroids, leukotriene inhiitors, mst cell stilizers, nd β2-drenergic gonists tht control the inflmmtion responses resulting from nitric oxide (NO), proinflmmtory enzymes, nd cytokines produced y mcrophges [10]. The overccumultion of proinflmmtory meditors my cuse severe dmge to the hert, lung, nd nerve system. At present, inhled glucocorticoid is widely used to tret sthm; however, out 50% of sthm ptients re not improved y the drug, which my induce dverse side effects, suggesting tht lterntive gents need to e developed [11]. Asthm excertions nd erly mnifesttions of the disese must e prevented to stop the diseses evolution to severe sthm [12]. Therefore, nturl trditionl herl medicines, helth foods, nd their possile ctive compounds show potentil in treting sthm. Among the possile ctive compounds ginst sthm, frnesol ws found to hve potentil. Frnesol is sesquiterpene lcohol tht exists widely in fruits such s peches, vegetles such s tomtoes nd corn, hers such s lemon grss nd chmomile, nd in the essentil oils of mrette seeds nd citronell [13, 14]. Frnesol is found to llevite mssive inflmmtion, oxidtive stress, nd lung injury induced y the intrtrchel instilltion of cigrette smoke extrct in rts [15]. Frnesol meliortes 1,2-dimethylhydrzine induced oxidtive stress, inflmmtion, nd poptotic in the colon of Wistr rts [14]. Frnesol hs een shown to e potent in treting ntimetolic disorders, nti-inflmmtion, showing ntioxidnt, nticncer, nd ntiiotic effects [14, 16, 17]. Recently, we found tht frnesol exhiited reltive Th1- inclintion nd nti-inflmmtory property on immune cells tht my e pplied to improve Th2-skewed llergic sthmtic inflmmtion in vivo [18]. We hypothesized tht frnesol hs immunomodultion potentil ginst llergic sthmtic inflmmtion. To vlidte this ssumption, frnesol t different doses ws dministered to ovlumin- (OVA-) sensitized nd chllenged mice for 5 weeks. The nti-inflmmtory effects of frnesol supplementtion on the experimentl mice were determined. 2. Mterils nd Methods 2.1. Chemicls. Frnesol tht is sesquiterpene lcohol (C 15 H 26 O) in mny plnts ws purchsed t the highest ville purity (>95%, mixture of isomers) (Sigm, St. Louis, MO, USA). The chemicl structure is shown in Figure 1() Animl Grouping nd Feeding. The experimentl feed ws prepred ccording to the Americn Institute of

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 Nutrition AIN-76 recommendtion tht stisfies the nutritionl requirement for mouse growth nd vried only in frnesol composition [19]. Three frnesol doses, low dose (0.003%),mediumdose(0.017%),ndhighdose(0.067%), were dded to the AIN-76 feed (Tle 1)[20]. Ech feed ws prepred y thoroughly mixing in frnesol nd storing t 20 C. Approximtely, 3 grms of AIN-76 feed wsconsumedperdyyechindividulmousewith20 grms of ody weight (BW). Frnesol low (), medium (), nd high () doses, respectively, corresponding to 5, 25, nd 100 mg frnesol/kg BW/dy, were designed for the experimentl mice. It could e estimted tht frnesol supplementtion t the indicted doses might not produce significnt energy in vivo. The energy contriution of ech experimentl diet ws 67.5% from crohydrte, 20.8% from protein, nd 11.7% from ft. The clorie density of ech diet ws 3.85 Kcl/g. The niml use protocol listed elow ws reviewed nd pproved y the Institutionl Animl Cre nd Use Committee (IACUC), Ntionl Chung Hsing University, Tiwn. In our preliminry similr experiments, we found tht the results from either mle or femle mice hd sme trend; however, femle mice were more sensitive to tretments thn mle mice. Therefore, we selected femle mice s our experimentl mice in our following studies. The results from femle mice in immunology hve een ccepted in mny pulished ppers. The femle BALB/cByJNrl mice (7 weeks old) were otined from the Ntionl Lortory Animl Center, Ntionl Applied Reserch Lortories, Ntionl Science Council in Tipei, nd mintined in the Deprtment of Food Science nd Biotechnology t Ntionl Chung Hsing University College of Agriculture nd Nturl Resources in Tichung, Tiwn. The niml room ws kept on 12 h light nd 12 h drk cycle. Constnt temperture (25 ± 2 C) nd humidity were mintined. The mice were housed nd kept on chow diet (lortory stndrd diet, Diet MF 18, Orientl Yest Co., Ltd., Osk, Jpn) to cclimtize for 1 week efore feeding the experimentl diet. After this equilirium period, the mice were divided rndomly into six groups (n = 15) vried y frnesol doses nd sensitized tretments. The tretments were nonsensitized control (treted with phosphteuffered sline (PBS) nd lum (Al(OH) 3 ), nmely, PBS/AL, coded s ), dietry control (treted with OVA nd lum, nmely, OVA/AL, coded s ), frnesol low dose (treted with OVA/AL, supplemented with low dose frnesol out 5 mg/kg BW/dy, coded s ), frnesol medium dose (treted with OVA/AL, supplemented with medium dose frnesol out 25 mg/kg BW/dy, coded s ), frnesol high dose (treted with OVA/AL, supplemented with high dose frnesol out 100 mg/kg BW/dy, coded s ), nd positive control (treted with OVA/AL, treted with dexmethsone, codeds).groupisnonsensitizedcontrolgrouptht is intrperitonelly injected with PBS nd lum. group is not suitle for negtive group in this study ecuse it still induced mild inflmmtory effects. Unfortuntely, the experiment neglected to select norml mice tht were not treted with ny gents for negtive group. The initil verge ody weight of ech group showed no significnt differences mong groups. Mice in ech group were fed with Tle 1: AIN-76 nd frnesol feed formul ingredients. Ingredients (g/kg diet) Formuls AIN-76 Csein DL-methionine Corn strch Sucrose Fier Soyen oil Minerl mixture Vitmin mixture Cholineitrtrte(41%choline) Frnesol 0 38 μl 188μL 753 μl Frnesol (%) Suggested mg frnesol/kg BW/dy Clorie density (Kcl/g) 3.85 Nutrients (% of totl clories) Crohydrte 67.5% Protein 20.8% Ft 11.7% Totl 100%, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed). the specified experimentl diet d liitum for 35 consecutive dys. Mouse food intke nd ody weight were mesured twice week during the study period [20] OVA-Sensitized nd -Chllenged Allergic Asthmtic Inflmmtion Mouse Model. The mice (8 weeks old) were sensitized nd chllenged to induce llergic irwy inflmmtion. Mice were sensitized using n intrperitonel injection (i.p.) of 0.2 ml lum-precipitted ntigen contining 8 μg ofovlumin (OVA, lumin chicken egg grde III, Sigm-Aldrich Co.,St.Louis,MO,USA)nd2mgAl(OH) 3 (Sigm-Aldrich Co., St. Louis, MO, USA) to induce primry immunity nd strted the specified experimentl diets (t dy 0). Two ooster injections of this lum-ova mixture were given 14 nd 28 dys lter, respectively. Nonsensitized control mice received lum-phosphte-uffered sline (PBS, 137 mm NCl (Wko Pure Chemicl Industries, Ltd., Osk, Jpn), 2.7 mm KCl (Sigm-Aldrich Co., St. Louis, MO, USA), 8.1 mm N 2 HPO 4 (Sigm-Aldrich Co., Steinheim, Germny), 1.5 mm KH 2 PO 4 (Sigm-Aldrich Co., St. Louis, MO, USA), ph 7.4, 0.2 μm filtered) only. At dys 31 nd 34, the OVAsensitized mice were chllenged using erosolized OVA t concentrtion of 5 mg OVA per milliliter PBS for 60 min (nmely, 9:00 m for 30 min nd 4:00 pm for 30 min). The erosolized OVA ws produced using n ultrsonic neulizer (sw918, Shinmed, Tipei, Tiwn). Nonsensitized control mice received only PBS [21]. Two hours efore erosolized OVA ws dministered, the group ws treted with dexmethsone (DEX, 3 mg/kg BW, 0.3 ml/mouse, Sigm, St. Louis, MO, USA) y gvge to reduce llergic sthmtic inflmmtion [22]. Two dys lter (t dy 36), ll of

4 4 Evidence-Bsed Complementry nd Alterntive Medicine the nimls were nesthetized, exsnguinted using retrooritl venous plexus puncture, nd immeditely euthnized y CO 2 inhltion (Figure 1()). The roncholveolr lvge fluid (BALF), peritonel mcrophges, spleen, nd viscerl orgns were collected nd ssyed for cytokines nd other inflmmtory meditors [20] BALF Collection. After the mice were euthnized, the irwys nd the lungs were immeditely lvged septiclly using cnnul through the trche with 5 liquots of 0.6 ml Hnk s lnced slts solution (HBSS), free of ionized clcium nd mgnesium (HyClone Lortories Inc., Logn, UT, USA). The BALF ws centrifuged t 400 gfor10mint4 C. The superntnt (BALF) volume ws determined nd stored t 80 Cforfuturessy[23] Peritonel Mcrophge Isoltion nd Culture. After BALF collection, peritonel mcrophges from the experimentl mice were collected ccording to the method descried y Lin et l. [24]. Peritonel cells were prepred y lvging the peritonel cvity with 2 liquots of 5 ml sterile Hnks lnced slts solution (HBSS) (50 ml of 10x HBSS (HyClone Lortories Inc., Logn, UT), 2.5 ml of ntiiotic-ntimycotic solution (100x PSA) contining 10,000 units/ml of penicillin, 10 mg/ml of streptomycin, 25 μg/ml of mphotericin B in 0.85% sline (Atlnt Biologicls Inc., Norcross, GA), 20 ml of 3% ovine serum lumin (BSA, Sigm-Aldrich Co., St. Louis, MO) in phosphte-uffered sline (PBS, 137 mm NCl, 2.7 mm KCl, 8.1 mm N 2 HPO 4, 1.5mM KH 2 PO 4, ph 7.4, 0.20 μm filtered),2.5mlof7.5%nhco 3 (Wko, Osk, Jpn), nd 425 ml of sterile wter) for totl of 10 ml through peritoneum. The peritonel lvge fluid ws centrifuged t 400 g for10mint4 C. The cell pellets were collected nd resuspended in tissue culture medium (TCM, serum replcement; Celox Lortories Inc., Lke Zurich, IL), suspended in medium consisting of 10 ml TCM, 500 ml RPMI 1640 medium (Atlnt Biologicls Inc.), nd 2.5 ml ntiiotic-ntimycotic solution (100x PSA) (Atlnt Biologicls Inc.). The peritonel dherent cells (>90% of mcrophges) from ech niml were djusted to cells/ml in TCM medium with hemocytometer using the trypn lue dye exclusion method. The peritonel mcrophges (0.5 ml/well) in the sence or presence of lipopolyscchride stimulus (LPS) (L-2654, Sigm-Aldrich Co., St. Louis, MO; t the finl concentrtion of 2.5 μg/ml, 0.5 ml/well) were cultured in 48-well pltes. LPS, n endotoxin from Grm-negtive cteri, ws selected to ugment mcrophges inflmmtion in vitro [25, 26]. The pltes were incuted t 37 C in humidified incutor with 5% CO 2 nd 95%irfor48h.Theplteswerethencentrifugedt400 g for 10 min to otin the cell culture superntnts. The cell culture superntnts were collected for cytokine ssy using enzyme-linked immunosorent ssy (ELISA) Tissue Collection nd Anlysis. The thorcic nd dominl cvities of the experimentl mice were opened septiclly. The orgns such s hert, lung, liver, spleen, nd kidney were removed nd weighted. To evlute the possile effects of frnesol supplementtion on the ody nd viscerl orgns, the viscerl orgns weights in ech experimentl group were expressed s solute nd reltive viscerl orgn weights, respectively [27]. The reltive orgn weight (%) ws computed using the following eqution: reltive tissue (or orgn) weight (%) = (individul tissue (or orgn) weight (g)/ody weight (g) of the experimentl mouse) Splenocytes Isoltion nd Cultures. The splenocytes were prepred y septiclly removing spleens from the experimentl BALB/c mice. The spleen ws ground with the flt ottom of syringe piston to homogenize the splenocytes. Splenocytes were centrifuged t 400 gfor7mint25 C. The cell pellets were resuspended in 10 ml of red lood cell (RBC) lysis uffer (0.017 M Trizm Bse (Sigm-Aldrich Co., St. Louis, MO, USA) nd M NH 4 Cl (Sigm-Aldrich Co., St. Louis, MO, USA) in deionized wter, ph 7.2, 0.2 μmfiltered) for 3 minutes nd centrifuged t 400 gfor7mint25 C. The cell pellets were wshed with HBSS three times. Splenocytes were resuspended in TCM medium tht contined 20% of TCM Serum Replcement (Protide Phrmceuticls Inc., Illinois, USA) nd 0.5% of Penicillin-Streptomycin Amphotericin B Solution in RPMI 1640 medium (HyClone, Uth, USA). The cells were counted with hemocytometer using the trypn lue dye exclusion method. The cell density ws djusted to cells/ml in TCM medium. The isolted splenocyte suspensions ( cells/ml) were plted into 48-well pltes nd, respectively, incuted in the sence or presence of OVA (30 μg/ml).theplteswereincutedt 37 Cinhumidifiedincutorwith5%CO 2 nd 95% ir for 48 h. The cell culture superntnts were collected nd stored t 80 Cforcytokinessys Inflmmtory Meditors nd Mrkers Assy in BALF Nitric Oxide (NO) Assy. Eighty μl liquotsofbalf smples nd stndrds (0 100 μm sodium nitrite (Sigm- Aldrich Co., St. Louis, MO, USA) dissolved in doule distilled wter) were pipetted into the 96-microplte wells (Nunc, Thermo Fisher Scientific, Rockford, IL, USA). One hundred sixty μl liquots of Griess regent were then dded into ech well to develop the color. The Griess regent ws freshly prepred from Regents A nd B t rtio of 1 : 1 (Regent A: 1% (w/v) sulfnilmide (Sigm-Aldrich Co., St. Louis, MO, USA) dissolved in 5.0% (v/v) phosphoric cid (Riedelde Hen, Seelze, Germny); Regent B: 0.1% (w/v) N-(1- nphthyl)ethylenedimine dihydrochloride (Sigm-Aldrich Co., St. Louis, MO, USA) dissolved in deionized wter). After incutionfor5min,thepltewsredonpltereder (ELISAreder,ASYSHitech,GmH,Austri)t550nm. Using the stndrd curve, the NO concentrtion for ech unknownsmplewsdetermined[20] Protein Level Assy. The BALF protein content ws nlyzed using icinchoninic cid (BCA) protein ssy (Thermo Scientific Inc., Rockford, USA), ccording to the ccompnying instructions using 96-well microtiter plte [23].

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 Tle 2: Effects of frnesol supplementtion with different doses for 5 weeks on initil nd finl ody weights nd food nd energy intke, s well s feed nd energy efficiencies of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice. n Initil ody Finl ody Body weight Energy Feed intke Energy intke Feed efficiency weight weight gin efficiency (g gin/100 g (g gin/100 kcl (g) (g) (g/d) (g/d) (kcl/dy) feed) feed) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.20 Vlues re mens ± SD. There re no significnt differences mong groups within sme column ssyed y one-wy ANOVA, followed y Duncn s new multiple rnge test., nonsensitized control;, dietry control;, positive control;, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed) Eotxin Concentrtion. The BALF eotxin concentrtion ws determined using the mouse eotxin sndwich ELISA kit (Quntikine M murine, R&D Systems, Minnepolis, MN, USA). The eotxin concentrtion ws ssyed ccording to the mnufcturer s instructions. The sensitivity of this ssy ws <3.9 pg/ml [20] Mesurement of Cytokine Levels in BALF nd Peritonel Mcrophge Cultures Using ELISA. Cytokine (IL-4, IL-5, IL-2, IFN-γ, IL-1β, IL-6, TNF-α, nd IL-10) levels in BALF, peritonel mcrophges, or splenocytes cultures were determined using sndwich ELISA kits, respectively. These cytokine concentrtions were ssyed ccording to the cytokine ELISA protocol of mnufcturer s instructions (mouse DuoSet ELISA Development system, R&D Systems, Minnepolis, MN, USA). The sensitivity of these cytokine ssys ws <3.9 pg/ml [28] Sttisticl Anlysis. Dt re expressed s men ± SD. Dt mong groups were nlyzed using nlysis of vrince (ANOVA), followed y Duncn s new multiple rnge test. Differences mong groups were considered sttisticlly significnt if P < Sttisticl tests were performed using SPSS version Results 3.1. Effects of Frnesol Supplementtion on Intke nd Growth of OVA-Sensitized nd -Chllenged Mice. The ody weight nd experimentl procedure during the experimentl period re given in Figure 1(). The men ody weight of ll experimentl mice incresed slightly s the experimentl period ws extended. There were no significnt differences in men ody weight etween the nd other groups t ech sme experimentl point. The feed efficiency nd ody weight chnges in the experimentl mice re shown in Tle 2.There were no significnt differences in the initil nd finl ody weight, gin in ody weight, feed intke, feed efficiency, nd energy efficiency mong groups. In the frnesol-treted mice, their men feed intkes were 4.37 (), 4.81 (), nd 4.70 g feed/dy/mouse (), respectively. Three frnesol doses, low dose (0.003%), medium dose (0.017%), nd high dose (0.067%), were dded to the AIN-76 feed (Tle 1). Therefore, the ctul delivered doses to the frnesol-treted mice were (), (), nd mg frnesol/mouse/dy (). Our results indicted tht the frnesol tretment doses doptedinthisstudyhdnopprenttoxiceffects Effects of Frnesol Supplementtion on Body nd Viscerl Orgn Weights of OVA-Chllenged Mice. The OVAchllenged mice were fed with different doses of frnesol for 5 weeks to evlute frnesol effects on sthmtic inflmmtion. The results showed tht frnesol supplementtion did not significntly ffect the liver, kidney, thymus, nd hert weight mong groups, indicting no toxic effect from frnesol tretments on the viscerl orgns (Tle 3). However, OVA sensitiztion nd chllenge resulted in slight increse (P > 0.05) in the spleen weight of BALB/c mice, indicting tht mildsystemicinflmmtionwsinducedintheovachllenged mice. Importntly, DEX tretment ( group) significntly decresed (P < 0.05) solutendreltive spleen nd thymus weights compred to those in the group, indicting tht DEX tretment effectively decresed systemic inflmmtion in the OVA-chllenged mice. The spleen nd thymus, prticulrly the spleen, re immune orgns in the ody tht reflect systemic inflmmtion sttus. Frnesol tretments (,, nd groups) just slightly, ut not significntly (P > 0.05), decresed solute nd reltive spleen nd thymus weights, implying tht frnesol tretments might llevite systemic inflmmtion little in the OVA-chllenged mice. Interestingly, OVA sensitiztion nd chllenge cused slight decrese (P > 0.05) in the epididyml ft weight of BALB/c mice, indicting decrese in ft deposition resulting from llergic sthm in the OVA-chllenged mice. However, frnesol nd DEX tretment oviously incresed the epididyml ft weight in the OVA-chllenged mice, indicting tht oth frnesol nd DEX tretments might llevite the epididyml ft loss nd improve nutrition sttus in the experimentl mice.

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Tle 3: Effects of frnesol supplementtion with different doses for 5 weeks on solute nd reltive weights of viscerl orgns of OVA/ALsensitized nd -chllenged BALB/c sthmtic mice. Orgn Spleen Liver Kidney Hert Thymus Epididyml ft Group n ATW (g) 0.15 ± ± ± ± ± ± 0.03 RTW (%) 0.76 ± ± ± ± ± ± 0.11 ATW (g) 1.29 ± ± ± ± ± ± 0.21 RTW (%) 6.56 ± ± ± ± ± ± 0.68 ATW (g) 0.30 ± ± ± ± ± ± 0.04 RTW (%) 1.53 ± ± ± ± ± ± 0.18 ATW (g) 0.12 ± ± ± ± ± ± 0.01 RTW (%) 0.61 ± ± ± ± ± ± 0.03 ATW (g) 0.02 ± ± ± ± ± ± 0.01 RTW (%) 0.11 ± ± ± ± ± ± 0.04 ATW (g) 0.34 ± ± ± ± ± ± 0.16 RTW (%) 1.71 ± ± ± ± ± ± 0.78 Vlues re mens ± SD (n = 12 15). Vlues within sme row not shring common smll letter re significntly different (P < 0.05) fromechothernd ssyed y one-wy ANOVA, followed y Duncn s new multiple rnge test., nonsensitized control;, dietry control;, positive control;, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed); ATW, solute tissue weight; RTW, reltive tissue weight. Tle 4: Effects of frnesol supplementtion with different doses for 5 weeks on proinflmmtory nd nti-inflmmtory cytokine levels in BALF of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice. Proinflmmtory nd nti-inflmmtory cytokines secreted in BALF IL-1β (pg/ml) IL-6 (pg/ml) TNF-α (pg/ml) IL-10 (pg/ml) Pro/nti-inflmmtory cytokine rtios TNF-α/IL-10 (pg/pg) IL-6/IL-10 (pg/pg) 35.2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.26 Vlues re mens ± SD (n = 9 13). Vlues within sme column not shring common smll letter re significntly different (P < 0.05) fromechothernd ssyed y one-wy ANOVA,followed y Duncn s new multiple rnge test.the limit of detection (LOD)of these kits used in this study ws <3.9 pg/ml., nonsensitized control;, dietry control;, positive control;, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed) Effects of Frnesol Supplementtion on Cytokine nd Inflmmtory Meditor nd Mrker Levels in BALF of OVA- Chllenged Mice. Tle 4 shows proinflmmtory nd ntiinflmmtory cytokine levels in BALF of OVA-sensitized nd-chllengedsthmticmice.theresultsshowedtht there were no significnt differences in IL-1β, IL-6,TNF- α, nd IL-10 levels, s well s TNF-α/IL-10 (pro/ntiinflmmtory) cytokine level rtios in BALF mong the differentil tretments, suggesting tht there ws just mild inflmmtion induced in the irwys nd lungs in this niml model. Further comprison with proinflmmtory nd ntiinflmmtory cytokine level rtios showed tht oth frnesol nd DEX tretments slightly, ut not significntly (P > 0.05), decresed IL-6/IL-10 (pro/nti-inflmmtory) cytokine level rtios in BALF. These results suggest tht oth frnesol nd DEX tretments just slightly llevite inflmmtion sttus in the lungs nd irwys of llergic sthmtic mice through decresing pro/nti-inflmmtory cytokine level rtios. Tle 5 shows the nitric oxide (NO), protein, nd eotxin levels in BALF. Unfortuntely, there were no significnt differences etween the frnesol tretments nd group. We presumed tht there ws just mild inflmmtion induced in the irwys nd lungs in this niml model. Therefore, there is no significnt difference in the eotxin level in BALF etween nd groups Effects of Frnesol Supplementtion on Proinflmmtory nd Anti-Inflmmtory Cytokine Secretion Levels in Peritonel Mcrophge Cultures from OVA-Chllenged Mice. Tle 6 shows the secretion levels of proinflmmtory cytokines IL-1β, IL-6,nd TNF-α s well s n nti-inflmmtory cytokine IL-10 in peritonel mcrophge cultures from

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 Tle 5: Effects of frnesol supplementtion with different doses for 5 weeks on inflmmtory meditor nd mrker levels in BALF of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice. Inflmmtory meditor levels in BALF n NO (μm) Protein (μg/ml) Eotxin (pg/ml) ± ± 12.4 c 278 ± ± ± ± ± ± ± ± ± 12.7 c 298 ± ± ± 7.6 c 241 ± ± ± 15.6 c 270 ± 96 Vlues re mens ± SD. Vlues within sme column not shring common smll letter re significntly different (P < 0.05) fromechotherndssyedy one-wy ANOVA, followed y Duncn s new multiple rnge test., nonsensitized control;, dietry control;, positive control;, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed). OVA-sensitized nd -chllenged mice through 5 weeks of feeding. In generl, mcrophges tht re typicl inflmmtory cells should secret diverse cytokines when stimulted. However, our results showed tht sensitiztion nd chllenge with OVA significntly inhiited (P < 0.05) proinflmmtory nd nti-inflmmtory cytokine secretion levels y peritonel mcrophges in the sence or presence of LPS, implying tht the decresed immunity might pper in llergic sthmtic mice. Importntly, oth frnesol nd DEX tretments significntly incresed (P < 0.05) proinflmmtorynd nti-inflmmtory cytokine secretion levels y peritonel mcrophges in the sence or presence of LPS s compred to those in the group, implying tht oth frnesol nd DEX tretments incresed immunity tht might e suppressed s result of llergic inflmmtion in llergic sthmtic mice. Further comprison with proinflmmtory nd nti-inflmmtory cytokine secretion level rtios showed tht frnesol tretments oviously decresed TNF-α/IL- 10 (pro/nti-inflmmtory) cytokine secretion level rtios. These results suggest tht frnesol tretments llevite systemic inflmmtion sttus through decresing pro/ntiinflmmtory cytokine secretion level rtios in peritonel mcrophges from llergic sthmtic mice. The frnesol tretment effects were etter thn DEX tretment (Tle 6), implying tht frnesol my e used to improve llergic inflmmtion in sthm ptients in the future Effects of Frnesol Supplementtion on Th1/Th2 Cytokine SecretionLevelsinSplenocyteCulturesfromOVA-Chllenged Mice. To evlute the effects of frnesol supplementtion on systemic immune response in sthmtic sujects, Th1/Th2 cytokine levels in the splenocyte cultures from OVAchllenged mice were determined. The levels of Th1 (IL-2 nd IFN-γ) nd Th2 (IL-4, IL-5, nd IL-10) cytokines in thesplenocyteculturesinthesenceorpresenceofova from OVA-chllenged mice fed different experimentl diets through 5 weeks re shown in Figure 2. Theresultsshowed tht spontneous cytokine secretion levels of Th1 (IL-2 nd IFN-γ) nd Th2 (IL-4 nd IL-5), except IL-10, were too low to e detected. However, oth Th2 (IL-4, IL-5, nd IL-10) nd Th1 (IL-2 nd IFN-γ) cytokine secretion levels in splenocyte cultures in the presence of OVA were significntly (P < 0.05) incresed s compred to those in the sence of OVA. All Th1 nd Th2 except IL-10 cytokine secretion levels in group were significntly(p < 0.05) higher thn those in group. Moreover, spontneous cytokine secretion profiles exhiited tht Th2 (IL-4 + IL-5 + IL-10)/Th1 (IL-2 + IFN-γ) secretion rtios in group were significntly higher thn those in group (Figure 2(f)). These dt indicted tht OVA sensitiztion nd chllenge indeed induced Th2- skewed systemic OVA-specific immune response, reflecting in the spleen. Frnesol supplementtion slightly (P > 0.05) decresed IL-4 ( Th2 cytokine) (Figure 2()) utsignificntly (P < 0.05) incresed IL-2 ( Th1 cytokine) (Figure 2(d))levels secretedythesplenocytesinthepresenceofova,implying tht frnesol supplementtion might hve n ntillergic effect on llergic sthmtic mice. The IL-10 level in group wsslightlyhigherthnthtingroup,implyingtht the decresed immunity might pper in llergic sthmtic mice (Figure 2(c)). Furthermore, frnesol supplementtion significntly (P < 0.05) incresed IL-10 ( Th2 nd ntiinflmmtory cytokine) levels secreted y the splenocytes inthepresenceofova,suggestingthtfrnesolsupplementtion might lso hve n nti-inflmmtory potentil to llergic sthmtic mice (Figure 2(c)). Unfortuntely, high dose frnesol supplementtion ( group) just slightly (P > 0.05) decresed Th2 (IL-4 + IL-5 + IL-10)/Th1 (IL-2 + IFN-γ) secretion rtios y the splenocytes in the sence or presence of OVA s compred to those in group (Figure 2(f)). Consequently, DEX tretments significntly (P < 0.05) decresed IL-4, IL-5 (Th2), nd IFN-γ (Th1) ut incresed IL-10 (nti-inflmmtory) cytokine secretion levels y the splenocytes in the presence of OVA s compred to those in group, implying tht DEX tretments might inhiit immunity ut hve strong nti-inflmmtory potentil in llergic sthmtic mice (Figure 2). However, DEX tretments incresed Th2 (IL-4 + IL-5 + IL-10)/Th1 (IL-2 + IFN-γ) secretion rtios y the splenocytes in the sence or presence of OVA s compred to those in group (Figure 2(f)), suggesting tht DEX tretments might ggrvte the Th2- skewed inclintion in llergic sthmtic mice. 4. Discussion In the present study, there ws no significnt influence of frnesol supplementtions on intke nd growth, indicting

8 8 Evidence-Bsed Complementry nd Alterntive Medicine Tle 6: Effects of frnesol supplementtion with different doses for 5 weeks on proinflmmtory nd nti-inflmmtory cytokine secretions y peritonel mcrophges of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice. Cytokines secreted y splenocytes IL-1β (pg/ml) IL-6 (pg/ml) TNF-α (pg/ml) IL-10 (pg/ml) TNF-α/IL-10 (pg/pg) Tretment Spon. LPS 8.39 ± 3.88 ND 14.0 ± ± ± ± ± ± ± ± 8.0 c 18.1 ± ± ± 95 d 537 ± 156 c 270 ± 124 cd 1047 ± 640 c 943 ± ± ± 278 c 1215 ± ± 292 c 1076 ± 619 c 595 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 35.1 c 91.0 ± ± ± ± ± ± 47.2 c 151 ± ± 46.0 c 159 ± ± 32.0 c 150 ± ± ± ± 0.52 c 2.54 ± ± 1.60 c 2.13 ± ± 0.88 c 2.15 ± ± ± ± 1.22 c 3.52 ± 1.92 Vlues re mens ± SD (n = 12 15). Vlues within sme column not shring common smll letter re significntly different (P < 0.05)from ech other ndssyedyone-wyanova,followedyduncn snewmultiplernge test.thelodofthesekitsusedinthisstudyws<3.9 pg/ml. ND mens not detectle., nonsensitized control;, dietry control;, positive control;, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed). tht the frnesol tretment doses dopted in this study hd no pprent toxic effects (Tle 2). Actul delivered doses to the frnesol-treted mice were (), (), nd mg frnesol/mouse/dy (). Men ody weights of frnesol-treted mice clculted y their initil nd finl ody weights during the experimentl period were 20.7 (), 20.6 (), nd 20.8 g/mouse (), respectively (Tle 2). Thus, the ctul delivered doses to the frnesol-treted mice were mg/20.7 g BW/dy (), mg/20.6 g BW/dy (), nd mg/20.8 g BW/dy (), nmely, 6, 40, nd 151 mg/kg BW/dy, respectively. The ctul supplemented high dose of frnesol (151 mg/kg BW of mouse/dy = 3.02 mg/20 g BW of mouse/dy) to mice is equl to 1171 mg/dy in humns ccording to n pproprite conversion rtio t 1 : for mice (20 g) to humn (70 kg) [20]. Horn et l. indicted tht supplementing rts with 500 mg frnesol/kg BW y gvge for 28 dys did not significntly influence their ody weight, feed intke, nd liver weights [29]. We found tht supplementing experimentl mice with frnesol t the indicted high doses for 5 weeks showed no pprent toxic side effects (Tles 2 nd 3). Frnesol is sesquiterpene lcohol tht widely exists in fruits, vegetles, hers, nd essentil oils [13, 14]. This study further provides frnesol sfety dt for food nd possile phrmcologicl uses for nti-inflmmtory nd ntillergic effects. Our study suggests tht frnesol supplementtion t the indicted high dose is cceptle; however, the iovilility of frnesol supplementtion nd its reltive distriution in the ody remin to e further clrified. Asthm is Th2-skewed llergic disese ccompnied with systemic nd irwy inflmmtion [30]. In this study, we estlished mild sthmtic niml model using OVA sensitiztion nd chllenge nd evluted frnesol ntiinflmmtory nd ntillergic effects in vivo [31]. Both Th2 (IL-4, IL-5, nd IL-10) nd Th1 (IL-2 nd IFN-γ) cytokine secretion levels in splenocyte cultures in the presence of OVA were significntly (P < 0.05) incresed s compred to those in the sence of OVA (Figure 2). In ddition, ll Th1 nd Th2 except IL-10 cytokine secretion levels in group were significntly(p < 0.05) higher thn those in group. OVA sensitiztion nd chllenge mrkedly (P < 0.05) incresed spontneous secretion rtios of Th2 (IL-4 + IL-5 + IL-10)/Th1 (IL-2 + IFN-γ) y the splenocytes of the experimentl mice (Figure 2(f)). The results evidenced tht OVA sensitiztion nd chllenge successfully induced Th2- skewed systemic OVA-specific immune response, reflecting inthespleen.wefoundthtlevelsofno,protein,nd eotxin in BALF showed no significnt differences etween the frnesol tretments nd group (Tle 5), indicting tht the sthmtic niml model used in this study is still mild model tht did not cuse severe injury or inflmmtiontothelungsndirwys.bsedonourresults,ova sensitiztion 3 times with susequent 3 chllenges is recommended to induce more severe sthmtic response in the lungs nd irwys. Eosinophils infiltrtion into the irwys ws detected ut frnesol supplementtion did not ffect eosinophil numers in BALF (dt not shown). However, frnesol supplementtion slightly (P > 0.05) decresed IL- 4(Th2cytokine)(Figure 2()) utsignificntly(p < 0.05) incresed IL-2 ( Th1 cytokine) (Figure 2(d)) levels secreted y the splenocytes in the presence of OVA, implying tht frnesol supplementtion might hve n ntillergic effect on Th2-skewed llergic sthmtic mice. High dose frnesol supplementtion ( group) slightly (P > 0.05), ut not significntly, decresed Th2 (IL-4 + IL-5 + IL-10)/Th1 (IL-2 + IFN-γ) secretion rtios y the splenocytes in the sence or presence of OVA s compred to those in group,

9 Evidence-Bsed Complementry nd Alterntive Medicine 9 IL-4 (pg/ml) c IL-5 (pg/ml) c d c c () () IL-10 (pg/ml) c c c c c IL-2 (pg/ml) c c c c c 0 0 (c) (d) IFN-γ (pg/ml) (IL-4+IL-5+IL-10)/(IL-2 + IFN-γ) (pg/pg) d c cd Spontneous OVA tretment Spontneous OVA tretment (e) (f) Figure 2: Frnesol supplementtion effects with different doses for 5 weeks on IL-4 (), IL-5 (), IL-10 (c), IL-2 (d), IFN-γ (e), nd (IL-4 +IL-5 + IL-10)/(IL-2 + IFN-γ) rtios (f) secreted y splenocytes of OVA/AL-sensitized nd -chllenged BALB/c sthmtic mice. Vlues re mens ± SD (n =12 15). Vlues mong groups within the sme tretment not shring common smll letter re significntly different (P < 0.05) from ech other nd ssyed y one-wy ANOVA, followed y Duncn s new multiple rnge test. The limit of detection(lod) of these kits used in this study ws <3.9 pg/ml., nonsensitized control;, dietry control;, positive control (dexmethsone, 3 mg/kg BW, 0.3 ml/mouse y gvge);, low dose frnesol (0.003% in AIN-76 feed);, medium dose frnesol (0.017% in AIN-76 feed);, high dose frnesol (0.067% in AIN-76 feed).

10 10 Evidence-Bsed Complementry nd Alterntive Medicine further suggesting mild effect of frnesol supplementtion ginst Th2 responses (Figure 2(f)). In ddition, ser from the experimentl mice were collected to mesure ntiody titers. The results showed tht frnesol supplementtion significntly incresed (P < 0.05) OVA-specific IgG2/IgE ntiody titer rtios ut decresed totl IgE levels (dt not shown). Our results evidenced tht frnesol supplementtion meliorted llergic sttus nd reversed Th2-skewed immune responses in the llergic sthmtic mice vi decresing serum OVA-specific IgE titers ut incresing IgG2/IgE titer rtios. Frnesol supplementtion significntly (P < 0.05) reduced IL-4 levels in BALF tht incresed due to OVA sensitiztion nd chllenge, suggesting tht frnesol supplementtion my hve potentil to modulte Th1/Th2 immunelncetowrdth1poleintheirwysndlungs (dt not shown). Unfortuntely, frnesol supplementtion did not hve significnt effects on infiltrtions of totl cells nd eosinophils into the irwys nd lungs (dt not shown). Importntly, frnesol supplementtion significntly (P < 0.05) incresed IL-10 levels secreted y the splenocytes in the presence of OVA, implying tht frnesol supplementtion might lso hve n nti-inflmmtory potentil to llergic sthmtic mice (Figure 2(c)). Th2 cells re the mjor source of IL-10 production. However, Th2 cytokines, prticulrly IL-10, my inhiit the production of Th1 cytokines such s proinflmmtory IL-1β nd TNF-α cytokines. In ddition, IL-10 is produced in lte stge inflmmtion y immune effector cells to inhiit the synthesis of other cytokines. Therefore, IL-10 hs een recognized s Th2 nd ntiinflmmtory cytokine. Unfortuntely, mny cytokines production (IL-2, IL-4, IL-5, nd IL-10) in frnesol-dministered groups did not show dose-response phenomen (Figure 2). It is universl mechnism to induce low- nd high-zone tolernces of immunomodultion. Thus, the optiml dose for frnesol dministrtion is difficult to scertin. Frnesol dministrtion should e crefully considered to chieve the est effect for vrious purposes. Our results suggested tht themosteffectivedoseoffrnesolin vivo might e low dose dministrtion for long term. To compre the frnesol effects, dexmethsone (DEX), potent synthetic memer of the glucocorticoid fmily, wsselectedinthisstudysthepositivecontrolforits nti-inflmmtory nd immunosuppressnt ctivities. We found tht oth frnesol supplementtion nd DEX tretment decresed enlrged spleen weights (Tle 3), IL- 6/IL-10 level rtios in BALF (Tle 4), nd TNF-α/IL-10 (pro/nti-inflmmtory) cytokine secretion rtios y peritonel mcrophges (Tle 6), indicting significnt ntiinflmmtory effects of frnesol nd DEX on the irwys nd systemic inflmmtion. However, the nti-inflmmtory effects of frnesol supplementtion were much etter thn DEX tretment through decresing TNF-α/IL-10 (pro/nti-inflmmtory) cytokine secretion rtios y peritonel mcrophges (Tle 6). To cure sthm, preventing theerlymnifesttionsofthedisesendthuspreventing its evolution into severe sthm re most importnt [12]. Our results suggest tht frnesol my e used s food supplement to prevent nd improve llergic inflmmtion in sthm ptients in the future. We found tht OVA sensitiztion nd chllenge significntly inhiited IL-1β, IL- 6, TNF-α, nd IL-10 productions y peritonel mcrophges (Tle 6); however, frnesol supplementtion significntly restoredthecytokinesecretionlevels,indictingthtfrnesol supplementtion my enhnce the inhiited immunity ut inhiit inflmmtion in sthmtic mice. We ssume tht frnesol might exert its nti-inflmmtory effect through modultingnuclerfctor(nf)-κbpthwy[32]; however, its possile nti-inflmmtory mechnisms remin to e further clrified. In ddition, frnesol is considered to e significnt contct llergen nd it ws recommend tht it should e included in frgrnce ptch-test preprtion nd tht its use should e regulted for consumer sfety resons [33]. The sfety of dietry frnesol should e further studied. 5. Conclusions Our results showed tht ctul frnesol supplementtion t the indicted high dose of 151 mg/kg BW/dy for 5 weeks hd no toxic effect on the experimentl mice. Frnesol supplementtion decresed IL-6/IL-10 level rtios in BALF, suggesting n nti-inflmmtory effect of frnesol on the lungs nd irwys. Frnesol supplementtion significntly restored the secretion ility of peritonel mcrophges nd slightly decresed TNF-α/IL-10 cytokine secretion rtios, indicting frnesol might enhnce systemic immunity ut inhiit inflmmtion in the lungs nd irwys in sthmtic mice. Frnesol supplementtion slightly decresed IL-4 ut significntly incresed IL-2 levels secreted y the splenocytes in the presence of OVA, implying tht frnesol supplementtion might hve systemic ntillergic effect on llergic sthmtic mice. Furthermore, frnesol supplementtion significntly incresed IL-10 levels secreted y the splenocytes in the presence of OVA, suggesting tht frnesol supplementtion might lso hve n nti-inflmmtory potentil to llergic sthmtic mice. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the puliction of this pper. Acknowledgments This study ws kindly supported y reserch Grnt B MY3 from the Ntionl Science Council, Tipei,Tiwn,ndwssupportedinprtytheMinistryof Eduction, Tiwn, under the ATU pln. References [1] J. F. Vsconcelos, M. M. Teixeir, J. M. Bros-Filho et l., The triterpenoid lupeol ttenutes llergic irwy inflmmtion in murine model, Interntionl Immunophrmcology,vol.8,no. 9, pp , [2] L. C. Wu, Immunogloulin E receptor signling nd sthm, TheJournlofBiologiclChemistry,vol.286,no.38,pp , 2011.

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