research article the ethanol extract of osmanthus fragrans...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 304290 10 pageshttpdxdoiorg1011552013304290

Research ArticleThe Ethanol Extract of Osmanthus fragransFlowers Reduces Oxidative Stress and Allergic AirwayInflammation in an Animal Model

Chien-Ya Hung1 Fu-Long Huang2 Li-Shian Shi3 Shuk-Man Ka4 Jing-Yao Wang3

Yu-Cheng Tsai1 Tsung-Jen Hung5 and Yi-Ling Ye3

1 Department of Food Nutrition Chung Hwa University of Medical Technology Tainan 71703 Taiwan2Graduate Institute of Food Science National Chiayi University Chiayi 60004 Taiwan3Department of Biotechnology National Formosa University Yunlin 63201 Taiwan4Graduate Institute of Aerospace and Undersea Medicine National Defense Medical Center Taipei 11490 Taiwan5Department of Graduate Institute of Biomedical Science Chung Hwa University of Medical Technology Tainan 71703 Taiwan

Correspondence should be addressed to Yi-Ling Ye yilingyeyahoocomtw

Received 29 June 2013 Revised 18 November 2013 Accepted 19 November 2013

Academic Editor Y Ohta

Copyright copy 2013 Chien-Ya Hung et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The Osmanthus fragrans flower a popular herb in Eastern countries contains several antioxidant compounds Ben Cao Gang Mutraditional Chinese medical literature describes the usefulness of these flowers for phlegm and stasis reduction arrest of dysenterywith blood in the bowel and stomachache and diarrhea treatment However modern evidence regarding the therapeutic efficacyof these flowers is limited This study was aimed at assessing the antioxidative effects of the ethanol extract of O fragrans flowers(OFE) in vivo and evaluating its antioxidant maintenance and therapeutic effect on an allergic airway inflammation in mice AfterOFErsquos oral administration to mice the values obtained in the oxygen radical absorbance capacity assay as well as the glutathioneconcentration in the lungs and spleens of mice increased while thiobarbituric acid reactive substances decreased significantlyindicating OFErsquos significant in vivo antioxidant activity OFE was also therapeutically efficacious in a mouse model of ovalbumin-induced allergic airway inflammation Orally administered OFE suppressed ovalbumin-specific IgE production and inflammatorycell infiltration in the lung Moreover the antioxidative state of the mice improved Thus our findings confirm the ability of the Ofragrans flowers to reduce phlegm and suggest that OFE may be useful as an antiallergic agent

1 Introduction

Osmanthus fragrans known as sweet olive tea olive andfragrant olive is a species of Oleaceae native to southwesternChina [1] It is widely cultivated as an ornamental plantfor its fragrant flowers in Taiwan southern Japan southernChina Europe North America and elsewhere The flowerof O fragrans called Kwai-fah in China has been used as abeverage and as an additive for tea and foods such as cakepastry paste vinegar and liqueurs It is popular because ofits delicate fruityfloral aroma Various volatile componentsof the flowers are also used primarily for perfumes flavorsand aromatherapy It was recorded in Ben Cao Gang Mu

that the O fragrans flower was used to reduce phlegmand stasis as well as to arrest dysentery with blood inthe bowel Traditional Chinese medicine has also suggestedthe use of O fragrans to treat weakened vision halitosispanting asthma cough toothache stomachache diarrheaand hepatitis However modern evidence for the biomedicaluse of the ethanol extract of O fragrans flowers (OFE) islimited

Overproduction of free radicals can cause oxidativedamage and may eventually lead to chronic diseases Manyplants contain free radical-scavenging molecules such asphenolic acids and flavonoids which show strong antioxidantactivity There are some reports of antioxidant activity of

2 Evidence-Based Complementary and Alternative Medicine

O fragrans Lee et al demonstrated that the dried flowersof O fragrans contain abundant phenols and flavonoids andexhibit antioxidant activity in the ferric reducing ability ofplasma (FRAP) assay 11-diphenyl-2-picryl-hydrazyl (DPPH)radical-scavenging activity and hydroxyl anion scavengingability [2] Our group has also analyzed many antioxidantcomponents of OFE and found that the antioxidant activityofO fragrans is slightly weaker than that of green tea [3] Inthe aforementioned study five phenolic compounds tyrosylacetate (1) (+)-phillygenin (2) (8E)-ligustroside (3) rutin(4) and verbascoside (5) were isolated from the CHCl

3layer

of O fragrans Evaluation of the antioxidative properties ofthe isolated compounds 2 4 and 5 revealed strong DPPHradical-scavenging activity with IC

50values of 191 103 and

62 120583M respectively These isolates also exhibited an H2O2-

scavenging ability with IC50values of 105 234 and 134 120583M

respectivelyAlthough modern evidence for the in vivo therapeutic

activity of O fragrans is yet to be provided a few studieshave examined the in vitro bioactivity of OFE For exampleOFE inhibited lipid peroxidation occurring through ferrouschloride in the mitochondria in rat brain liver heart andkidney [2] OFE may also exert neuroprotective actionsthrough the upregulation of theAKT survival pathway whichattenuates neurotoxicity [2] Several lignans isolated from theflowers of O fragrans var aurantiacus inhibited nitric oxideproduction in lipopolysaccharide (LPS)-stimulated RAW2647 macrophages [4]

Asthma is a chronic respiratory disease characterizedby airway inflammation and airway hyperresponsiveness(AHR) Previous reviews have highlighted the importanceof reactive oxygennitrogen species in airway inflammation[5 6] Reactive oxygen species (ROS) also contribute tolipid peroxidation The pathologic effects of lipid perox-idation including epithelial cell activation disruption ordeath smooth muscle contraction airway hypersensitivityand increased mucus secretion are all observed in asthma[7] Losing control of intracellular oxidants to environmentalcellular stress also may lead to allergic disorders [8] Forexample the polarization of the helper T cells Th1Th2balance is dependent on the intracellular thiol-redox status ofmacrophages [9] In another study Pazmandi et al observedthat H

2O2-exposed plasmacytoid dendritic cells (pDCs)

provided stronger stimuli for Th2 than for Th1 differenti-ation upon autologous activation (cocultivation with naıveautologous T cells) when compared to untreated pDCs [10]Furthermore enhanced oxidative stress may contribute tothe progression or perpetuation of existing airway inflam-mation through enhanced AHR [11 12] via airway smoothmuscle contraction [13]mucus hypersecretion [14] epithelialshedding [15] vascular exudation [16 17] and inductionof various proinflammatory chemical mediators because ofaccumulation of inflammatory cells in the lower respiratorytract [18] All of these actions are believed to be related tosevere asthma [19ndash21]

The increase in ROS during an asthma exacerbationmight overwhelm endogenous antioxidant defenses Glu-tathione (GSH) is a key antioxidant in the lining fluid ofthe respiratory tract Disturbed GSH status is reported in

asthma for total [22] and for oxidized [23] GSH Total GSHconcentration which includes the oxidized form GSSG ishigher in the bronchial and alveolar fluid in patients withmild asthma [22] A recent report suggested that increasingGSH levels and the GSHGSSG ratio with 120574-glutamylcysteineethyl ester (120574-GCE) ameliorated bronchial asthma by alteringthe Th1Th2 cell imbalance through interleukin (IL-) 12production from antigen presenting cells (APC) and bysuppressing chemokine production and eosinophilmigration[24] Enhancing intracellular GSH can also decrease therelease of cytokines from lung cells by decreasing NF-120581Bactivation [25 26] In addition GSH has recently beenreported to attenuate IL-13 induced asthma in mice [27]

Liao et al investigated the total antioxidant capability inserum and the activity of the antioxidant enzymes superoxidedismutase (SOD) and glutathione peroxidase in 46 asthmaticchildren and 52 normal controls [28] The total antioxidantcapability in the serum of asthmatic children is significantlyless than that found in the serum of controls Plasma levelsof lipid peroxides are also significantly increased in patientswith asthma [29 30]

The concentration of ascorbic acid and 120572-tocopherol inlung lining fluid is reportedly low in patients with mildasthma [23 31] Low dietary intake of vitamin C and man-ganese was found to be associated with an increased riskof bronchial activity [30] Dietary supplements of vitaminC and vitamin E to asthmatics decrease the severity ofpollutant-induced bronchial responsiveness [32] Chang andCrapo [33] found that intratracheal treatment of ovalbu-min (OVA-) challenged mice with the catalytic antioxidantmanganese (III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin having high SOD activity drastically reduced theseverity of airway inflammation as evidenced by the reducednumbers of eosinophils neutrophils and lymphocytes foundin bronchoalveolar lavage fluid

The aim of the current study was to examine the mech-anism of the preventive effect of OFE on allergic airwayinflammation The study comprised two parts the first wasto evaluate the antioxidation state following OFE administra-tion and the second was to examine the immunomodulatoryeffects of OFE We evaluated the antioxidative effects of OFEin naıvemice using the followingmethods assessing the totalantioxidant capacity in the lung and spleen by using the valuesobtained in the oxygen radical absorbance capacity (ORAC)assay assessing the free radical-scavenging effects on DPPHmonitoring lipid peroxidation by measuring thiobarbituricacid reactive substances (TBARS) and determining the con-centration of GSHWe used amousemodel of allergic airwayinflammation to investigate the antiallergic and antioxidanteffects of OFE as well as to determine organ weights

2 Materials and Methods

21 Plant Material Dried flowers of O fragrans were col-lected from Nanto Taiwan in 2005-2006 They were exam-ined and authenticated by Professor C S Kuoh Departmentof Biology National Cheng Kung University A voucher

Evidence-Based Complementary and Alternative Medicine 3

specimen (no Hung-0201) was deposited in the Herbariumof Chung Hwa University of Medical Technology

22 Preparation of OFE The dried flowers of O fragranswere ground into a fine powder using a mill (RT-08 RongTsong Taiwan) collected and sealed in a polyethylene plasticbag and then stored at 0ndash4∘C for further use O fragransflowers (200 g) were soaked (72 h) in 75 ethanol (3 L)twice and filtered through Whatman no 1 filter paper Thecombined extracts were concentrated under reduced pressureand freeze-dried to provide a dark syrup that was storedat minus20∘C for further use The extract was endotoxin free(le01 EU) according to the Limulus amebocyte lysate (LAL)assay (Cambrex Walkersville MD)

221 Determination of Total Phenolic Content in OFE Fol-lowing themethod described by Yen andHung [34] the sam-ple solution in methanol (01mL 1mgmL) was well mixedwith 2 Na

2CO3(2mL) After 3min 50 Folin-Ciocalteu

agent (01mL)was addedThemixturewas allowed to stand atroom temperature (RT) for 30min with intermittent mixingThe absorbance at 750 nm was recorded A standard curveusing gallic acid was preparedThe total phenolic content wasexpressed as gallic acid equivalents (mg ofGAEper g extract)

222 Determination of Total Flavonoid Content in OFEFollowing themethods described byWoisky and Salatino [35]and also by Chang et al [36] the sample solution (05mL)was mixed with 95 EtOH (15mL) 10 AlCl

3(01mL) 1M

KOAc (01mL) and distilled water (28mL) The mixturewas allowed to stand at RT for 30min and the absorbancewas measured at 415 nmThe amount of sample solution wassubstituted by the same amount of a quercetin solution (0ndash200120583gmL) as a standard The amount of 10 aluminumchloride was substituted by the same amount of distilledwater to serve as a blank The total flavonoid content wascalculated from the plot of absorbance against quercetinconcentration using linear regression analysis and expressedas quercetin equivalents (120583g of QE per g extract)

23 DPPH Free Radical-Scavenging Assay DPPH is a stablefree radical with a purple color that is reduced by antioxidantsto a colorless compound We employed DPPH in an assaymodified from the method of Shimada et al [37] MeOH(38mL) sample solution in methanol (02mL 1mgmL)and 1mM DPPH solution (10mL) were mixed well and leftto stand in the dark at RT for 30minThe final concentrationof the sample was 40 120583gmL The absorbance at 517 nm wasmeasuredThe sample inmethanol was used as a blank whileDPPH radical inmethanol solutionwas used as a controlTheDPPH radical scavenging activity was calculated according tothe following equation

of DPPH radical scavenging activity

= [1 minus (119860 sample minus 119860blank)

119860control] times 100

(1)

where 119860 is the absorbance at 517 nm

The concentration providing 50 inhibition (IC50) of the

DPPH radical-scavenging activity was calculated from theplot of the percent inhibition against sample concentrationby using a linear regression analysis

24 Animals Female BALBc mice between six and eightweeks of age were purchased from the National Labora-tory Animal Center in Taiwan The animal room was kepton a 12 h light dark cycle and maintained at a constanttemperature (25∘C plusmn 2∘C) and humidity Animal care andhandling conformed to theNIHGuide for the Care and Use ofLaboratory Animals All experiments were performed underprotocols approved by the biotechnology department ofNational Formosa Universityrsquos affidavit of approval of animaluse protocol Pentobarbital (intraperitoneal ip) injectionswere used to anesthetize (10mgmL 60 120583L per mouse) orsacrifice (10mgmL 200 120583L per mouse) the mice

For the results obtained in the antioxidant evaluationassays mice were divided into two groups comprised of fourBALBc mice each Group 1 (control) received only distilledwater Group 2 (OFE) received 1000mgkg body weight OFEdaily for 14 days by oral gavage

25 Organ Collection Preparation and Protein QuantizationAfter sacrifice organs from the mice were collected andweighed The spleens and lungs (1 g) were homogenizedin PBS (pH 72) on ice by using a homogenizer (MotorDrives Glas-Col Inc Terre Haute IN)The homogenate wasthen centrifuged at 2200timesg for 10min and the filtrate wascollected Protein was determined by using the BCA assaykit (Pierce Rockford IL) using bovine serum albumin as astandard

26 Determination of Antioxidant Activity

261 Oxygen Radical Absorbance Capacity Assay (ORAC)The total antioxidant activity of the organ samples wasmeasured by using the oxygen radical absorbance capacity(ORAC) assay according to Chung et al [38] This assaywas carried out in black-walled 96-well plates at 37∘CAll solutions were prepared in 75mM phosphate buffer(Na2HPO4 NaH

2PO4 pH 70) and preincubated at 37∘C for

30min before use Fifteen 120583L of organ homogenate (diluted100 times) and 100 120583L of 01 120583M120573-PE (120573-phycoerythrin) weretransferred directly into the well to incubate for 10min usingthe FLUOstar OPTIMA microplate reader system (GalaxyBMG LABTECH Inc Cary NC) We rapidly added 75mM221015840-azobis (2-amidinopropane) dihydrochloride (AAPH85 120583L) and immediately measured the resulting fluorescenceby using fluorescence filters with an excitation wavelengthof 480 nm and an emission wavelength of 520 nm Thefluorescence was recorded at 5min intervals for 120min untilthe final value was less than 5 of the initial value ORACvalues from samples were calculated by using the followingequation and expressed as Trolox equivalents ORAC value(mM) = 20times119896times(119878sampleminus119878blank)(119878Troloxminus119878blank) where 119896was


the sample dilution factor The area under the curve (119878) wascalculated by the following equation

119878 = (05 +1198915

1198910

+11989110

1198910

+11989115

1198910

+11989120

1198910

+11989125

1198910

+ sdot sdot sdot +119891120

1198910

) times 5

(2)

where 1198910was the initial fluorescence reading at 0min and 119891

119899

represented the measurement at time 119899

262 TBARS Assay Lipid peroxidation was measured bydetermining the formation of malondialdehyde based on thepresence of TBARS in the lung and spleen [39] A standardcurve was prepared using 1133-tetraethoxypropane (SigmaSt Louis Mo) The organ filtrate (1mL) and standard weremixed with 10 trichloroacetic acid (1mL) 04 thiobarbi-turic acid (1mL) and 02 butylated hydroxy toluene (BHT)(01mL) (both reagents from Sigma St Louis Mo) Thereaction mixture was incubated at 95∘C for 1 h cooled underlight-protected conditions and then centrifuged at 2200timesgfor 10min The absorbance of the supernatant was measuredusing a microplate fluorescence reader (F-2500 HitachiJapan) in 96-well format with the excitation and emissionfilters set at 515 nm and 550 nm respectively

263 Measurement of Glutathione The glutathione concen-tration was determined according to the method describedby Sedlak and Lindsay [40] with little modification Theorgan homogenate (150 120583L) wasmixedwith 5TCA solution(450120583L) The resulting solution was centrifuged at 16770timesgfor 10min to remove protein The supernatant (30 120583L) and001M 551015840-dithio-bis-(2-nitrobenzoic acid) (DTNB 10 120583L)were added to 04MTris buffer (140 120583L pH 89)The solutionremained at RT for 5min The absorbance at 412 nm wasmeasured by n ELISA Reader (VersaMax Molecular DevicesInc Sunnyvale CA) A GSH solution (0ndash12 120583M) was usedas standard Tris buffer was used as blank The plasma GSHcontent was calculated from the plot of absorbance againstGSH concentration

27 Establishment of the Animal Model of Allergic AirwayInflammation The schedule for OVA sensitization and chal-lenge as well as the oral administration schedule of OFE aresummarized in Figure 1 Briefly themice were sensitized withip injections of OVA (20 120583g in 1x PBS Sigma) mixed withalum (2mg Pierce Rockford III) as an adjuvant on day 0 Onday 14 the mice were boosted with OVA (50 120583g) mixed withalum (2mg)Themice that were given ip injections of 1x PBSserved as negative controls Either one of two doses of OFE innormal saline (200 120583L) (high OFE 1000mgkg body weightor low OFE 100mgkg body weight) or normal saline (forpositive and negative control groups) was orally administereddaily from day 1 to day 29 Each group consisted of six miceOn days 35ndash38 the mice were exposed to aerosolized 5OVA for a 20min period by placing them in a chamberThe aerosols were generated and conducted into the chamberusing an ultrasonic nebulizer (DeVibiss PA) The output of

the nebulizer was 03mLmin and the particles size producedwas 05ndash5120583m

271 Serum Levels of Anti-OVA Antibodies After all groupsof mice were anesthetized they were bled from the retro-orbital venous plexus Serum was collected after centrifuga-tion at 12000timesg for 10min Sera anti-OVA IgE and IgG2aantibody titers were determined by ELISA Briefly 96-wellmicrotiter plates were coated with OVA (10 120583gmL) Serumsamples were added to each well for an overnight periodThen biotin-conjugated anti-mouse IgE or IgG2a was addedfor 1 h at 37∘C Streptavidin-conjugated alkaline phosphatasewas added for an additional 2 h at RT Finally the reactionwas developed by 221015840-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) and the absorbance was determined at 420 nmin a microplate reader

272 Bronchoalveolar Lavage and Cell Differential CountsAfter all groups of mice were anesthetized they were bledfrom the retroorbital venous plexus and then sacrificedUsinga cannula their lungs were immediately lavaged throughthe trachea three times with 1x HBSS (1mL) minus ionizedcalcium and magnesium The lavaged fluid was centrifugedat 400timesg for 10min at 4∘C After washing the cells wereresuspended in 1x HBSS (1mL) and the total cell countswere determined using a hemocytometer Cytocentrifugedpreparations were stained with Liursquos stain for differential cellcounts Based on standard morphologic criteria a minimumof 200 cells was counted and classified asmonocytes lympho-cytes neutrophils or eosinophils

273 Histopathological Study of Lung Organs To evaluatethe effects of OFE treatment the lungs were immediatelyremoved after lavage and fixed in a solution of 3 vvformalin (in 001M phosphate buffer pH 72) The tissueswere subsequently embedded in paraffin cut into 5-120583m-thicksections stained with hematoxylin-eosin and examined bylight microscopy for histopathological changes

28 Statistical Analysis Data from theORACand the TBARSassay as well the GSH concentration and various treatmentsare presented as mean plusmn SD (standard deviation) TheStudentrsquos t test was used for comparison between treatmentsDifferences between two treatment groups or a treatmentgroup compared with a negative or positive control groupwere considered statistically significant at 119875 values less than005 001 or 0001

3 Results and Discussion

31 The Total Phenolic and Flavonoid Content in OFE Thetotal phenolic content in OFE was 3679 plusmn 134mg GAEgextract while the total flavonoid content was 450 plusmn 20 120583gQEg extract Flavonoids have been reported to induceantiallergic and anti-inflammatory effects [41] For exampleflavonoids showed a strong inhibition of IL-4 and IL-13production histamine release andCD40 ligand expression in


Table 1 Effects of orally administered OFE on oxidative status in the lungs and spleen of mice as determined in three different assays

Tissue ORAC (Trolox equivalents mM) TBARS (nMmg protein) GSH (120583Mmg protein)Control OFE Control OFE Control OFE

Lung 242 plusmn 061 360 plusmn 024lowast 020 plusmn 002 012 plusmn 002lowast 1527 plusmn 314 2436 plusmn 301lowastlowastlowast

Spleen 210 plusmn 068 397 plusmn 079lowastlowastlowast 057 plusmn 001 046 plusmn 005lowastlowastlowast 1597 plusmn 113 2523 plusmn 119lowastlowastlowast

OFE (1000mg OFEkg body weight in 200120583L normal saline) was orally administered daily for 14 daysData are expressed as mean plusmn standard deviation 119899 = 4 lowast119875 lt 005 lowastlowastlowast119875 lt 001 as compared to (control) BALBc mice given normal saline

Bleeding AHR Sacrifice

0 1 14 21 29 35sim38 39 40

Sensitization (ip) Oral feeding Challenge (ih)

LOFE 100mg flower extractkg mise BW per dayHOFE 1000mg flower extractkg mise BW per dayPositivenegative control 09 normal saline per day

Day Day Day Day Day Day Day Day

Figure 1 The schedule for the development of the OVA animal model of allergic airway inflammation and OFE administration ipintraperitoneal injection ih inhalation with OVA

IgE

(OD405

nm)

NC PC LOFE HOFE

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

IgG2

a (O

D405

nm)

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)

Figure 2 The effects of daily oral administration of OFE on the OVA-specific IgE and IgG2a antibody production in the OVA animal modelof allergic airway inflammation PC OVA immunized group NC negative control group HOFE high dose of OFE LOFE low dose of OFESignificant increase lowast119875 lt 005lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 or decrease

119875 lt 005 119875 lt 001 and 119875 lt 0001 as compared to NC or PC

groups

basophils [42] The antiasthmatic effect by phenylpropanoidglycosides such as verbascoside has also been reported [43]

32 AntioxidantActivity and Free Radical ScavengingCapacityof OFE as Determined in the ORAC and DPPH AssaysThe total antioxidant capacity of ethanol extract of OFE asdetermined in the ORAC assay was 04 plusmn 00mM TroloxequivalentsTheDPPH IC

50was 84120583gmL which was better

than that of the methanol extract (128 120583gmL) of O fragransflowers [3] but less thanTrolox (49120583gmL)Green tea whichhas been proven to have a strong scavenging effect againstDPPH radicals has been reported to have antiallergic activity

[44] We found that in addition to the other antioxidantphenolic compounds verbascoside and rutin are two majorcompounds in the ethanol extract ofOFE (unpublished data)The antiallergic effects of verbascoside [43] and rutin [45]have also been reported Together these results support ourdecision to evaluate the antiallergic effects of the ethanolextract of OFE

33 The Effects of Orally Administered OFE on Total Antiox-idant Capacity Lipid Peroxidation and GSH Concentrationin Lung and Spleen of BALBc Mice The oxidation of lipidsnucleic acids or proteins may be involved in the oxidative


NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0

lowastlowastlowastlowastlowast

Mon

ocyt

e(lowast10

4ce

lls)

(d)

Figure 3 The effects of OFE on cell (eosinophils neutrophils monocytes or lymphocytes) infiltration in bronchoalveolar fluid Significantincrease lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 or decrease

119875 lt 005 119875 lt 001 and 119875 lt 0001 as compared to NC or PC groups

damage of biological molecules Additionally oxidation isbelieved to play a role in the development of inflammatorydiseases For this reason the total antioxidant capacity (asmeasured in the ORAC assay) GSH concentration and lipidperoxidation (as measured in the TBARS assay) in mouselung and spleen were investigated There are no previousreports on the in vivo antioxidant effects of OFE

We first evaluated the antioxidant capacity of the OFEextract in mice after oral administration of OFE for 14days Lungs and spleens from the OFE group demonstratedsignificantly higher values in the ORAC assay and higherconcentrations of GSH than those in the control group(normal saline) (Table 1) In contrast values derived from theTBARS assay were significantly decreased in the OFE groupcompared with those in the control group Taken togetherthese results indicate that oral administration of OFE pro-motes antioxidant capacity and reduces lipid peroxidation inthe lung and spleen of mice

The phenolic antioxidants in OFE include tyrosyl acetate(+)-phillygenin (8E)-ligustroside rutin and verbascoside[3] The verbascoside obtained from OFE has been reportedto protect cell lines from free radical-induced oxidative stress

as measured by TBARS [46] and 120573-amyloid-induced cellinjury by attenuating ROS production [46] However thein vivo evaluation of the antioxidant effects of these fivecompounds needs further study Oxidative stress is an impor-tant factor that contributes to the pathologic development ofasthma Given our results for the antioxidant capacity ofOFEwe next evaluated OFE for its preventive effects in an animalmodel of allergic airway inflammation

34 OVA-Specific Serum Antibody Levels in an Animal Modelof Allergic Airway Inflammation Following the OFE preven-tive protocol (Figure 1) OFE oral administration inhibitedOVA-specific IgE production (Figure 2(a)) and enhancedthe production of OVA-specific IgG2a (Figure 2(b)) IgEsuppression can alleviate the development of asthma [47]In contrast IgG2a is the major isotype of the Th1 immuneresponse The effect of antioxidants to regulate the immuneresponse has been an important issue in western society[48] The strong Th2 inhibition by flavonoids [42] andphenolic compounds [49] contributes to the suppression ofTh2 inflammation The polarization effect of antioxidants


NC PC

HOFE LOFE

Figure 4 Lung sections were prepared from mice subjected to the OVA animal model of allergic airway inflammation after daily oral OFEor saline (control) administration Arrows indicate immune cell infiltration by hematoxylin-eosin staining (magnification 200x)

Table 2 Effects of OFE on relative organ weights for mice in an animal model of allergic airway inflammation

Relative organ weight to body weight ()Group

Organ NC PC LOFE HOFELiver 600 plusmn 086 504 plusmn 036 534 plusmn 068 544 plusmn 080Lung 138 plusmn 031 113 plusmn 014 133 plusmn 021 134 plusmn 017Kidney 175 plusmn 020 148 plusmn 005 151 plusmn 014 142 plusmn 010

Evaluation of OFE cytotoxicity in an animal model of allergic airway inflammation Mice (119899 = 4) were injected (ip) with OVA for positive control (PC) a lowdose of OFE (LOFE) or a high dose of HOFE Negative control (NC) mice were injected with normal saline During the establishment of the allergic airwayinflammation model mice were treated for 28 days with saline (NC and PC) 1000mg OFEkg body weight (HOFE in 200120583L saline) or 100mg OFEkg bodyweight (LOFE in 200120583L saline) After sacrifice on Day 40 organs were collected and weighed Data are expressed as mean plusmn standard deviation Significantdecrease

119875 lt 005 compared to NC group mice

on Th1 and Th2 has been discussed [50] In a study of theantiallergic effects of curcumin IFN-120574 was increased andIgE was suppressed in the latex allergy model [51] In ourstudy the increase in anti-OVA IgG2a and decrease in IgEexpression indicate that OFE prevents development of Th2suppression A higher GSH induction also contributes to thedevelopment ofTh1 as suggested by theTh1-promoting effectof GSH [24]

35 Cellular Composition in Bronchoalveolar Fluid in anAnimal Model of Allergic Airway Inflammation and theHistopathologic Effects of OFE on Mouse Lung Tissue Inorder to evaluate the effects of OFE on the recruitment ofinflammatory cells into airway cells in bronchoalveolar(BAL)

fluid were determined by Liursquos stain Compared with thepositive control group oral administration of a high dose(HOFE) but not a low dose (LOFE) of OFE could suppressthe recruitment of eosinophils neutrophils and lymphocytes(Figure 3) In the histopathological study extensive cellularinfiltration of the periairway region in lung sections ofpositive control-group mice was found However lung tissuefrom groups administered OFE demonstrated less severeinflammation (Figure 4) Fewer inflammatory cells infiltratedthe periairway region in lungs of mice in the HOFE groupThis result is consistent with the cellular composition of BALfluid in the HOFE group and with the observed antiallergiceffect of OFE Results from previous studies examiningthe antiallergic effects of verbascoside [43] and rutin [45]


lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5

demonstrated that these compounds inOFE contribute to theinhibition of inflammatory cell infiltration in lung

36 Effects of Oral OFE Administration on Relative OrganWeight in an Animal Model of Allergic Airway InflammationA change in organ weights is a good indicator of chemically-

or biologically-induced damage to organs [52] Oral admin-istration of OFE (100 800 or 1000mgkg) for 28 days didnot alter organ weights (liver spleen lung and kidney)in mice (data not shown) However HOFE (1000mgkg)administration for 28 days to mice subjected to the OVAanimal model of allergic airway inflammation lowered therelative kidney weight to body weight percentage whencomparedwithmice in the negative control group (NCgrouptreated with saline) but not when compared with mice inthe positive control (PC group OVA-immunized and treatedwith saline) (Table 2) In contrast there were no significantdifferences among groups in the relative organweight to bodyweight percentages for liver and lung Recently we evaluateda medium dose of OFE (500mgkg) in the OVA-inducedallergic airway inflammation model and found that mediumdose of OFE also protected against the infiltrated cells inBAL fluid and maintained the antioxidative state in lung(data not shown) Further investigation will be required toclarify the organ cytotoxicity effect by a medium dose of OFEorally administered in this animal model of allergic airwayinflammation

37 OFE Improves Antioxidative Status in Mouse Lung follow-ing OVA Administration We measured the severity of theoxidative damage in lungs of mice from each group In lungstaken from animals subjected to the mouse model of allergicairway inflammation the GSH concentration and the totalantioxidant capacity values obtained from the ORAC assaywere significantly decreased comparedwith those of the lungsfrom mice in the negative control group This suggests thatairway inflammation may lead to oxidative stress in lungsHowever these phenomena were improved following oraladministration of OFE (Figures 5(a) and 5(b)) In contrastthe value obtained in the TBARS assay for mice treated withOFE was significantly lower than that obtained for mice inthe positive control group (119875 lt 001 Figure 5(c)) Takentogether these data indicate that OFE improves the totalantioxidant capacity in lung and that this improvement iscorrelated with a lower severity of signs in the OFE-treatedmice subjected to the OVA-induced animal model of allergicairway inflammation Recent reports have suggested thatincreasing the GSH levels could attenuate Th2 development[24ndash27] which offers a possiblemechanism for the alleviationin the severity of airway inflammation observed in the presentstudy following OFE administration

4 Conclusion

The therapeutic success ofmany antioxidant agents in allergicairway inflammation in the clinic is moderate at best [53 54]Conversely researchers have found that antioxidants suchas fruit juice [55] and plant extracts [56] interfere with theTh1 immune response in human peripheral blood mononu-clear cells Such results reflect the need for determiningthe appropriate antioxidants for treatment in different typesof inflammatory disease The present report was the firstto evaluate the antioxidant and immunomodulatory effectsof OFE applying traditional Chinese medicinal knowledge


regarding the O fragrans flower to modern scientific studyWe found that OFE which contains many antioxidantspromotes a positive antioxidative state in an animal modelof allergic airway inflammation It also has protective effectsincluding decreasing the OVA-specific IgE production andinflammatory cell infiltration in lung

Conflict of Interests

The authors declare that they have no competing interestsand that no financial relationship exists with any companymentioned in this paper

Acknowledgments

This study was supported by the National Science Councilof the Republic of China (NSC 94-2313-B-273-004) Theauthors thank Editage for providing editorial assistanceTheyalso appreciate the help of Dong-Jer Shiou (Department ofBiotechnology National Formosa University) for checkingthe references in this paper

References

[1] K Larsen ldquoFlora of Taiwanrdquo Nordic Journal of Botany vol 15no 6 p 574 1995

[2] H-H Lee C-T Lin and L-L Yang ldquoNeuroprotection and freeradical scavenging effects of Osmanthus fragransrdquo Journal ofBiomedical Science vol 14 no 6 pp 819ndash827 2007

[3] C Y Hung Y C Tsai and K Y Li ldquoPhenolic antioxidantsisolated from the flowers ofOsmanthus fragransrdquoMolecules vol17 no 9 pp 10724ndash10737 2012

[4] D-G Lee S-M Lee M-H Bang et al ldquoLignans from the flow-ers of Osmanthus fragrans var aurantiacus and their inhibitioneffect on NO productionrdquo Archives of Pharmacal Research vol34 no 12 pp 2029ndash2035 2011

[5] Y S Cho and H-B Moon ldquoThe role of oxidative stress inthe pathogenesis of asthmardquo Allergy Asthma and ImmunologyResearch vol 2 no 3 pp 183ndash187 2010

[6] G Folkerts J Kloek R B R Muijsers and F P NijkampldquoReactive nitrogen and oxygen species in airway inflammationrdquoEuropean Journal of Pharmacology vol 429 no 1ndash3 pp 251ndash2622001

[7] L G Wood P G Gibson and M L Garg ldquoBiomarkers oflipid peroxidation airway inflammation and asthmardquo EuropeanRespiratory Journal vol 21 no 1 pp 177ndash186 2003

[8] H Matsue D Edelbaum D Shalhevet et al ldquoGeneration andfunction of reactive oxygen species in dendritic cells duringantigen presentationrdquo Journal of Immunology vol 171 no 6 pp3010ndash3018 2003

[9] Y Murata T Shimamura and J Hamuro ldquoThe polarizationof Th1Th2 balance is dependent on the intracellular thiolredox status of macrophages due to the distinctive cytokineproductionrdquo International Immunology vol 14 no 2 pp 201ndash212 2002

[10] K Pazmandi Z Magyarics I Boldogh A Csillag E Rajnavol-gyi and A Bacsi ldquoModulatory effects of low-dose hydrogenperoxide on the function of human plasmacytoid dendriticcellsrdquo Free Radical Biology and Medicine vol 52 no 3 pp 635ndash645 2012

[11] U Katsumata M Miura M Ichinose et al ldquoOxygen radicalsproduce airway constriction and hyperresponsiveness in anes-thetized catsrdquoThe American Review of Respiratory Disease vol141 no 5 pp 1158ndash1161 1990

[12] E B Weiss and J R Bellino ldquoLeukotriene-associated toxicoxygen metabolites induce airway hyperreactivityrdquo Chest vol89 no 5 pp 709ndash716 1986

[13] K J Rhoden and P J Barnes ldquoEffect of hydrogen peroxideon guinea-pig tracheal smooth muscle in vitro role of cyclo-oxygenase and airway epitheliumrdquo British Journal of Pharma-cology vol 98 no 1 pp 325ndash330 1989

[14] K B Adler W J Holden-Stauffer and J E Repine ldquoOxygenmetabolites stimulate release of high-molecular-weight glyco-conjugates by cell and organ cultures of rodent respiratoryepithelium via an arachidonic acid-dependentmechanismrdquoTheJournal of Clinical Investigation vol 85 no 1 pp 75ndash85 1990

[15] C J A Doelman R Leurs W C Oosterom and A Bast ldquoMin-eral dust exposure and free radical-mediated lung damagerdquoExperimental Lung Research vol 16 no 1 pp 41ndash55 1990

[16] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[17] R M Tate K M Vanbenthuysen and D M Shasby ldquoOxygen-radical-mediated permeability edema and vasoconstriction inisolated perfused rabbit lungsrdquo The American Review of Respi-ratory Disease vol 126 no 5 pp 802ndash806 1982

[18] I Rahman S-R Yang and S K Biswas ldquoCurrent concepts ofredox signaling in the lungsrdquo Antioxidants and Redox Signalingvol 8 no 3-4 pp 681ndash689 2006

[19] S A A Comhair K S Ricci M Arroliga et al ldquoCorrelationof systemic superoxide dismutase deficiency to airflow obstruc-tion in asthmardquo American Journal of Respiratory and CriticalCare Medicine vol 172 no 3 pp 306ndash313 2005

[20] A M FitzpatrickW G Teague F Holguin M Yeh and L A SBrown ldquoAirway glutathione homeostasis is altered in childrenwith severe asthma evidence for oxidant stressrdquo The Journalof Allergy and Clinical Immunology vol 123 no 1 pp 146e8ndash152e8 2009

[21] A M Fitzpatrick L A S Brown F Holguin andW G TeagueldquoLevels of nitric oxide oxidation products are increased in theepithelial lining fluid of children with persistent asthmardquo TheJournal of Allergy and Clinical Immunology vol 124 no 5 pp990e9ndash996e9 2009

[22] L J Smith M Houston and J Anderson ldquoIncreased levels ofglutathione in bronchoalveolar lavage fluid from patients withasthmardquo The American Review of Respiratory Disease vol 147no 6 part 1 pp 1461ndash1464 1993

[23] F J Kelly I Mudway A Blomberg A Frew and T SandstromldquoAltered lung antioxidant status in patients with mild asthmardquoThe Lancet vol 354 no 9177 pp 482ndash483 1999

[24] Y Koike T Hisada M Utsugi et al ldquoGlutathione redoxregulates airway hyperresponsiveness and airway inflammationin micerdquo American Journal of Respiratory Cell and MolecularBiology vol 37 no 3 pp 322ndash329 2007

[25] F Antonicelli M Parmentier E M Drost et al ldquoNacystelyninhibits oxidant-mediated interleukin-8 expression and NF-120581Bnuclear binding in alveolar epithelial cellsrdquo Free Radical Biologyand Medicine vol 32 no 6 pp 492ndash502 2002

[26] T Aoki Y Suzuki K Suzuki et al ldquoModulation of ICAM-1expression by extracellular glutathione in hyperoxia-exposed


human pulmonary artery endothelial cellsrdquo American Journalof Respiratory Cell and Molecular Biology vol 15 no 3 pp 319ndash327 1996

[27] M H Lowry B P McAllister J-C Jean et al ldquoLung liningfluid glutathione attenuates IL-13-induced asthmardquo AmericanJournal of Respiratory Cell and Molecular Biology vol 38 no5 pp 509ndash516 2008

[28] M-F Liao C-C Chen and M-H Hsu ldquoEvaluation of theserum antioxidant status in asthmatic childrenrdquo Acta Paedi-atrica Taiwanica vol 45 no 4 pp 213ndash217 2004

[29] H Tsukagoshi Y Shimizu S Iwamae et al ldquoEvidence ofoxidative stress in asthma andCOPD potential inhibitory effectof theophyllinerdquo Respiratory Medicine vol 94 no 6 pp 584ndash588 2000

[30] A Soutar A Seaton and K Brown ldquoBronchial reactivity anddietary antioxidantsrdquoThorax vol 52 no 2 pp 166ndash170 1997

[31] S O Olusi O O Ojutiku W J E Jessop and M I IbokoldquoPlasma and white blood cell ascorbic acid concentrations inpatients with bronchial asthmardquo Clinica Chimica Acta vol 92no 2 pp 161ndash166 1979

[32] C A Trenga J Q Koenig and P V Williams ldquoDietary antiox-idants and ozone-induced bronchial hyperresponsiveness inadults with asthmardquo Archives of Environmental Health vol 56no 3 pp 242ndash249 2001

[33] L-Y Chang and J D Crapo ldquoInhibition of airway inflammationand hyperreactivity by an antioxidant mimeticrdquo Free RadicalBiology and Medicine vol 33 no 3 pp 379ndash386 2002

[34] G-C Yen and C-Y Hung ldquoEffects of alkaline and heat treat-ment on antioxidative activity and total phenolics of extractsfrom Hsian-tsao (Mesona procumbens Hemsl)rdquo Food ResearchInternational vol 33 no 6 pp 487ndash492 2000

[35] R G Woisky and A Salatino ldquoAnalysis of propolis someparameters and procedures for chemical quality controlrdquo Jour-nal of Apicultural Research vol 37 no 2 pp 99ndash105 1998

[36] C-C Chang M-H Yang H-M Wen and J-C ChernldquoEstimation of total flavonoid content in propolis by twocomplementary colometric methodsrdquo Journal of Food and DrugAnalysis vol 10 no 3 pp 178ndash182 2002

[37] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cycodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 pp 945ndash948 1992

[38] K-Y Chung S-J Lee S-M Chung M-Y Lee O-N Baeand J-H Chung ldquoGeneration of free radical by interaction ofiron with thiols in human plasma and its possible significancerdquoThrombosis Research vol 116 no 2 pp 157ndash164 2005

[39] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[40] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 no 1 pp 192ndash2051968

[41] Y Bellik L Boukraa H A Alzahrani et al ldquoMolecular mech-anism underlying anti-inflammatory and anti-allergic activitiesof phytochemicals an updaterdquoMolecules vol 18 no 1 pp 322ndash353 2012

[42] MKawai THirano S Higa et al ldquoFlavonoids and related com-pounds as anti-allergic substancesrdquo Allergology Internationalvol 56 no 2 pp 113ndash123 2007

[43] J Y Lee J G Lee S S SimW-K Whang and C J Kim ldquoAnti-asthmatic effects of phenylpropanoid glycosides from Clero-dendron trichotomum leaves and Rumex gmelini herbes inconscious guinea-pigs challenged with aerosolized ovalbuminrdquoPhytomedicine vol 18 no 2-3 pp 134ndash142 2011

[44] J-C Heo J R Rho T-H Kim S-Y Kim and S-H Lee ldquoAnaqueous extract of green tea Camellia sinensis increases expres-sion of Th1 cell-specific anti-asthmatic markersrdquo InternationalJournal of Molecular Medicine vol 22 no 6 pp 763ndash767 2008

[45] C H Jung J Y Lee C H Cho and C J Kim ldquoAnti-asthmaticaction of quercetin and rutin in conscious guinea-pigs chal-lenged with aerosolized ovalbuminrdquo Archives of PharmacalResearch vol 30 no 12 pp 1599ndash1607 2007

[46] H Wang Y Xu J Yan et al ldquoActeoside protects humanneuroblastoma SH-SY5Y cells against 120573-amyloid-induced cellinjuryrdquo Brain Research vol 1283 no 4 pp 139ndash147 2009

[47] D A Lebman and R L Coffman ldquoInterleukin 4 causes isotypeswitching to IgE in T cell-stimulated clonal B cell culturesrdquoTheJournal of Experimental Medicine vol 168 no 3 pp 853ndash8621988

[48] G Johanna C Christian B Kathrin F Dietmar and S RobertldquoImmunoregulatoryimpact of food antioxidantsrdquoCurrent Phar-maceutical Design In press

[49] S-Y Chung and E T Champagne ldquoReducing the allergeniccapacity of peanut extracts and liquid peanut butter by phenoliccompoundsrdquo Food Chemistry vol 115 no 4 pp 1345ndash13492009

[50] C Murr K Schroecksnadel C Winkler M Ledochowskiand D Fuchs ldquoAntioxidants may increase the probability ofdeveloping allergic diseases and asthmardquo Medical Hypothesesvol 64 no 5 pp 973ndash977 2005

[51] V P Kurup C S Barrios R Raju B D Johnson M B Levyand J N Fink ldquoImmune response modulation by curcumin ina latex allergy modelrdquo Clinical and Molecular Allergy vol 5article 1 2007

[52] B Michael B Yano R S Sellers et al ldquoEvaluation of organweights for rodent and non-rodent toxicity studies a reviewof regulatory guidelines and a survey of current practicesrdquoToxicologic Pathology vol 35 no 5 pp 742ndash750 2007

[53] E Omenaas Oslash Fluge A S Buist W M Vollmer and AGulsvik ldquoDietary vitamin C intake is inversely related to coughandwheeze in young smokersrdquoRespiratoryMedicine vol 97 no2 pp 134ndash142 2003

[54] L Grievink H A Smit M C Ocke P van rsquoT Veer andD Kromhout ldquoDietary intake of antioxidant (pro)-vitaminsrespiratory symptoms and pulmonary function the MORGENstudyrdquoThorax vol 53 no 3 pp 166ndash171 1998

[55] G Neurauter B Wirleitner K Schroecksnadel H Schennachand D Fuchs ldquoWine and grape juice modulate interferon-120574-induced neopterin production and tryptophan degradation inhuman PBMCrdquo Pteridines vol 15 no 1 pp 1ndash9 2004

[56] C Winkler B Wirleitner K Schroecksnadel H Schennach EMur andD Fuchs ldquoIn vitro effects of two extracts and two purealkaloid preparations ofUncaria tomentosa on peripheral bloodmononuclear cellsrdquo Planta Medica vol 70 no 3 pp 205ndash2102004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


O fragrans Lee et al demonstrated that the dried flowersof O fragrans contain abundant phenols and flavonoids andexhibit antioxidant activity in the ferric reducing ability ofplasma (FRAP) assay 11-diphenyl-2-picryl-hydrazyl (DPPH)radical-scavenging activity and hydroxyl anion scavengingability [2] Our group has also analyzed many antioxidantcomponents of OFE and found that the antioxidant activityofO fragrans is slightly weaker than that of green tea [3] Inthe aforementioned study five phenolic compounds tyrosylacetate (1) (+)-phillygenin (2) (8E)-ligustroside (3) rutin(4) and verbascoside (5) were isolated from the CHCl

3layer

of O fragrans Evaluation of the antioxidative properties ofthe isolated compounds 2 4 and 5 revealed strong DPPHradical-scavenging activity with IC

50values of 191 103 and

62 120583M respectively These isolates also exhibited an H2O2-

scavenging ability with IC50values of 105 234 and 134 120583M

respectivelyAlthough modern evidence for the in vivo therapeutic

activity of O fragrans is yet to be provided a few studieshave examined the in vitro bioactivity of OFE For exampleOFE inhibited lipid peroxidation occurring through ferrouschloride in the mitochondria in rat brain liver heart andkidney [2] OFE may also exert neuroprotective actionsthrough the upregulation of theAKT survival pathway whichattenuates neurotoxicity [2] Several lignans isolated from theflowers of O fragrans var aurantiacus inhibited nitric oxideproduction in lipopolysaccharide (LPS)-stimulated RAW2647 macrophages [4]

Asthma is a chronic respiratory disease characterizedby airway inflammation and airway hyperresponsiveness(AHR) Previous reviews have highlighted the importanceof reactive oxygennitrogen species in airway inflammation[5 6] Reactive oxygen species (ROS) also contribute tolipid peroxidation The pathologic effects of lipid perox-idation including epithelial cell activation disruption ordeath smooth muscle contraction airway hypersensitivityand increased mucus secretion are all observed in asthma[7] Losing control of intracellular oxidants to environmentalcellular stress also may lead to allergic disorders [8] Forexample the polarization of the helper T cells Th1Th2balance is dependent on the intracellular thiol-redox status ofmacrophages [9] In another study Pazmandi et al observedthat H

2O2-exposed plasmacytoid dendritic cells (pDCs)

provided stronger stimuli for Th2 than for Th1 differenti-ation upon autologous activation (cocultivation with naıveautologous T cells) when compared to untreated pDCs [10]Furthermore enhanced oxidative stress may contribute tothe progression or perpetuation of existing airway inflam-mation through enhanced AHR [11 12] via airway smoothmuscle contraction [13]mucus hypersecretion [14] epithelialshedding [15] vascular exudation [16 17] and inductionof various proinflammatory chemical mediators because ofaccumulation of inflammatory cells in the lower respiratorytract [18] All of these actions are believed to be related tosevere asthma [19ndash21]

The increase in ROS during an asthma exacerbationmight overwhelm endogenous antioxidant defenses Glu-tathione (GSH) is a key antioxidant in the lining fluid ofthe respiratory tract Disturbed GSH status is reported in

asthma for total [22] and for oxidized [23] GSH Total GSHconcentration which includes the oxidized form GSSG ishigher in the bronchial and alveolar fluid in patients withmild asthma [22] A recent report suggested that increasingGSH levels and the GSHGSSG ratio with 120574-glutamylcysteineethyl ester (120574-GCE) ameliorated bronchial asthma by alteringthe Th1Th2 cell imbalance through interleukin (IL-) 12production from antigen presenting cells (APC) and bysuppressing chemokine production and eosinophilmigration[24] Enhancing intracellular GSH can also decrease therelease of cytokines from lung cells by decreasing NF-120581Bactivation [25 26] In addition GSH has recently beenreported to attenuate IL-13 induced asthma in mice [27]

Liao et al investigated the total antioxidant capability inserum and the activity of the antioxidant enzymes superoxidedismutase (SOD) and glutathione peroxidase in 46 asthmaticchildren and 52 normal controls [28] The total antioxidantcapability in the serum of asthmatic children is significantlyless than that found in the serum of controls Plasma levelsof lipid peroxides are also significantly increased in patientswith asthma [29 30]

The concentration of ascorbic acid and 120572-tocopherol inlung lining fluid is reportedly low in patients with mildasthma [23 31] Low dietary intake of vitamin C and man-ganese was found to be associated with an increased riskof bronchial activity [30] Dietary supplements of vitaminC and vitamin E to asthmatics decrease the severity ofpollutant-induced bronchial responsiveness [32] Chang andCrapo [33] found that intratracheal treatment of ovalbu-min (OVA-) challenged mice with the catalytic antioxidantmanganese (III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin having high SOD activity drastically reduced theseverity of airway inflammation as evidenced by the reducednumbers of eosinophils neutrophils and lymphocytes foundin bronchoalveolar lavage fluid

The aim of the current study was to examine the mech-anism of the preventive effect of OFE on allergic airwayinflammation The study comprised two parts the first wasto evaluate the antioxidation state following OFE administra-tion and the second was to examine the immunomodulatoryeffects of OFE We evaluated the antioxidative effects of OFEin naıvemice using the followingmethods assessing the totalantioxidant capacity in the lung and spleen by using the valuesobtained in the oxygen radical absorbance capacity (ORAC)assay assessing the free radical-scavenging effects on DPPHmonitoring lipid peroxidation by measuring thiobarbituricacid reactive substances (TBARS) and determining the con-centration of GSHWe used amousemodel of allergic airwayinflammation to investigate the antiallergic and antioxidanteffects of OFE as well as to determine organ weights

2 Materials and Methods

21 Plant Material Dried flowers of O fragrans were col-lected from Nanto Taiwan in 2005-2006 They were exam-ined and authenticated by Professor C S Kuoh Departmentof Biology National Cheng Kung University A voucher








3(01mL) 1M






(1)













119878 = (05 +1198915

1198910

+11989110

1198910

+11989115

1198910

+11989120

1198910

+11989125

1198910


1198910

) times 5

(2)


119899



















0 1 14 21 29 35sim38 39 40





IgE

(OD405

nm)

NC PC LOFE HOFE

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

IgG2

a (O

D405

nm)

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)



groups








NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0


Mon

ocyt

e(lowast10

4ce

lls)

(d)









NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3(01mL) 1M






(1)













119878 = (05 +1198915

1198910

+11989110

1198910

+11989115

1198910

+11989120

1198910

+11989125

1198910


1198910

) times 5

(2)


119899



















0 1 14 21 29 35sim38 39 40





IgE

(OD405

nm)

NC PC LOFE HOFE

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

IgG2

a (O

D405

nm)

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)



groups








NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0


Mon

ocyt

e(lowast10

4ce

lls)

(d)









NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















119878 = (05 +1198915

1198910

+11989110

1198910

+11989115

1198910

+11989120

1198910

+11989125

1198910


1198910

) times 5

(2)


119899



















0 1 14 21 29 35sim38 39 40





IgE

(OD405

nm)

NC PC LOFE HOFE

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

IgG2

a (O

D405

nm)

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)



groups








NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0


Mon

ocyt

e(lowast10

4ce

lls)

(d)









NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























0 1 14 21 29 35sim38 39 40





IgE

(OD405

nm)

NC PC LOFE HOFE

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

IgG2

a (O

D405

nm)

lowastlowastlowast

5

4

3

2

1

0

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)



groups








NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0


Mon

ocyt

e(lowast10

4ce

lls)

(d)









NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC PC LOFE HOFE

40

30

20

10

0

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Eosin

ophi

l (lowast10

4ce

lls)

(a)

lowastlowastlowast

30

25

15

10

5

0

NC PC LOFE HOFE

20

Lym

phoc

yte(

lowast10

4ce

lls)

(b)

NC PC LOFE HOFE

lowastlowast

15

10

5

0

lowastlowastlowast

Neu

troph

il(lowast10

4ce

lls)

(c)

NC PC LOFE HOFE

lowast

120

100

80

60

40

20

0


Mon

ocyt

e(lowast10

4ce

lls)

(d)









NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC PC

HOFE LOFE











lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowastlowast

NC PC LOFE HOFE

GSH

(120583M

mg

prot

ein)

50

40

30

20

10

0

lowastlowastlowast

(a)

NC PC LOFE HOFE

lowastlowast

lowastlowastlowast

ORA

C (m

M T

rolo

x eq

uiva

lent

s)

5

4

3

2

1

0

lowast

(b)

NC PC LOFE HOFE

lowastlowast

TBA

Rs (n

mol

mg

prot

ein)

04

03

02

01

00

(c)

Figure 5





4 Conclusion






Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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