research article auraptene in the peels of citrus...

Research ArticleAuraptene in the Peels of Citrus kawachiensis(Kawachi Bankan) Ameliorates Lipopolysaccharide-InducedInflammation in the Mouse Brain

Satoshi Okuyama1 Kana Yamamoto1 Hirotomo Mori1 Nobuki Toyoda1

Morio Yoshimura2 Yoshiaki Amakura2 Takashi Yoshida2 Kuniaki Sugawara3

Masahiko Sudo3 Mitsunari Nakajima1 and Yoshiko Furukawa1

1 Department of Pharmaceutical Pharmacology College of Pharmaceutical Sciences Matsuyama University4-2 Bunkyo-cho Matsuyama Ehime 790-8578 Japan

2Department of Pharmacognosy College of Pharmaceutical Sciences Matsuyama University 4-2 Bunkyo-choMatsuyama Ehime 790-8578 Japan

3Department of Planning and Development Ehime Beverage Inc 478 Anjyoji Matsuyama Ehime 791-8603 Japan

Correspondence should be addressed to Yoshiko Furukawa furukawaccmatsuyama-uacjp

Received 27 December 2013 Revised 25 January 2014 Accepted 27 January 2014 Published 15 May 2014

Academic Editor Ke Ren

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

Examination of the dried peel powder of Citrus kawachiensis one of the citrus products of Ehime Japan showed that itcontained naringin (NGIN 4402plusmn 0491mgg) narirutin (NRTN 446plusmn 00563mgg) auraptene (AUR 407plusmn 0033mgg) and356783101584041015840-heptamethoxyflavone (HMF 027plusmn 00039mgg) When this dried peel powder was orally preadministered at thedose of 12 or 24 gkgday for 7 days into lipopolysaccharide- (LPS-) injected mice an animal model of systemic inflammation itsuppressed (1) LPS-induced loss of body weight and abnormal behavior in the open field (2) LPS-induced activation of microgliaand astrocytes in the hippocampus and (3) LPS-induced expression of cyclooxygenase (COX)-2 which were coexpressed inastrocytes of these mice When NGIN or AUR was preadministered to LPS-injected mice at an amount similar to that in thepeel powder AUR but not NGIN had the ability to suppress the LPS-induced inflammation in the brain of these model miceThe dried powder of flavedo tissue (the outer colored layer of the mesocarp of a citrus fruit) and juice which contained sufficientamounts of AUR also had anti-inflammatory effect These results suggest that AUR was the main ingredient responsible for theanti-inflammatory property of the dried peels of C kawachiensis

1 Introduction

Inflammation is a pathophysiological phenomenon that isinvolved in numerous diseases of not only peripheral tis-sues but also the central nervous system (CNS) For theexample one of the representative consequences of ischemiais inflammation in the brain [1] After ischemic onsetinflammatory cells such as blood-derived leukocytes andmicroglia are activated and accumulate within the braintissue subsequently leading to inflammatory injury In theprocesses of ischemiastroke themolecular cues generated by

cerebral ischemia activate components of the innate immu-nity system promote inflammatory signaling and contributeto tissue damage [2] We recently reported by using anischemic mouse model that subcutaneously (sc) admin-istered auraptene (AUR Figure 1(a)) a Citrus coumarineffectively inhibits microglia activation cyclooxygenase-2(COX-2) expression by astrocytes and neuronal cell deathin the hippocampus [3] We also observed that the peelsof Citrus kawachiensis (kawachi bankan) which is one ofthe citrus products of Ehime Japan contain AUR [4] inhigher content than found in any otherCitrus peels [5]These

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 408503 9 pageshttpdxdoiorg1011552014408503

2 Evidence-Based Complementary and Alternative Medicine

OO OH C

CH CH33

3

(a) Auraptene (AUR)

O

O

OCH

H CO

3

OCH3

OCH3

OCH3

OCH3

3

H CO3

(b) 356783101584041015840-Heptamethoxyflavone (HMF)

O

O

O

O

O

O OH

OH

OH

OH

HO

HO

HO

HO

H CH3

(c) Naringin (NGIN)

O

O

O

O

O

O

OHOH

OH

OH

HO

HO

HO

HO CH3

(d) Narirutin (NRTN)

Figure 1 Structures of compounds detected in peel powder of C kawachiensis (kawachi bankan)

results suggested that the peels of C kawachiensismight be agood source of AUR for suppressing inflammation-induceddamage in the CNS

Therefore the objective of this study was to ascertainthe effectiveness of orally administered peel powder of Ckawachiensis against inflammation in the brain As a sam-ple the peel powder of C kawachiensis was prepared byadopting the processes of drying and sieving As a modelanimal of systemic inflammation we used mice that hadbeen intraperitoneally (ip) administered lipopolysaccharide(LPS) as peripheral injected LPS has the ability to induceimmune responses in the brain [6]

2 Materials and Methods

21 Preparation of Citrus Materials and Chemicals Fruitsof C kawachiensis were harvested in Yawatahama EhimeJapan For the preparation of dried peels the peels of thefruits (1555 g) after having been squeezed to obtain the juicewere chopped into small pieces and dried in vacuo at 60∘Cfor 1 day The dried peels (ca 300 g) were then milled andground to a fine powder with a mill mixer (Iwatani IFM-660DG Tokyo Japan)The powder after having been passedthrough a 150-mesh sieve was used as the test sample For thepreparation of the dried flavedo and the dried albedo flavedotissue (exterior yellow peel) and albedo tissue (interior whitepeel) were manually separated from fresh fruit with a smallknife and dried in vacuo at 60∘C for 1 day For the preparationof the dried juice the juice was freeze-dried These materials(dried flavedo dried albedo and dried juice) were thenmechanically ground to a fine powderwith themillmixer and

filtered through a 150-mesh sieve as in the case of the driedpeels Naringin (NGIN Figure 1(c)) and AUR were obtainedfrom LKT Laboratories (St Paul MN USA)

22 Determination of Flavonoid Contents in Samples DriedCitrus samples (1 g) were extracted by sonication for 20minin ethanol (30mL) at room temperature The mixture wasthen centrifuged at 10000timesg for 5min and the resultingsupernatant layer was analyzed by reversed-phase HPLCStandard solutions of NGIN narirutin (NRTN Figure 1(d))heptamethoxyflavone (HMF Figure 1(b) and AUR wereprepared in methanol at a concentration of 10mgmL Stocksolutions were diluted to prepare a calibration curve withlinearity in the range of 001sim10mgmL Each value wasreported as themean of 3 analyses HPLC analysis was carriedout by using a Shimadzu Prominence system (ShimadzuKyoto Japan) HPLC conditions were as follows column L-column ODS (5120583m 150 times 21mm id Chemicals Evaluationand Research Institute Tokyo Japan) mobile phase solventA (5 acetic acid) and solvent B (acetonitrile) 0sim30min 0sim50B inA 30sim35min 50sim85B inA 35sim40min 85sim85Bin A injection volume 2 120583L column temperature 40∘C flowrate 03mLmin detection flavonoids at 280 nm and AURat 320 nm NRTN was obtained from ChromaDex (IrvineCA USA) HMF was prepared from commercial orange oil(Wako Osaka Japan) as described previously [4]

23 Animals Nine-week-old male C57BL6 strain mice werepurchased from Japan SLC (Hamamatsu Japan) Mice in allgroups were kept at 23 plusmn 1∘C under a 12 h lightdark cycle

Evidence-Based Complementary and Alternative Medicine 3

(light on 800sim2000) During the experimental period themice were given free access to tap water and food until 0830and then deprived of food until the time of administrationof samples or vehicle (1600) All animal experiments werecarried out in accordance with the guidelines for animalexperimentation specified by the Animal Care and UseCommittee of Matsuyama University

24 Administration of Citrus Samples For experiments miceweighing about 25 g were randomized into 4 groups Thedried test samples were dissolved in distilled water In the 2test-sample groups mice were administered sample solution(075mL) of 2 different doses per os (po) once a day for 7days Mice in the other 2 groups (nontreated group 119899 = 6and LPS-treated group 119899 = 9) were administered vehicle(distilled water) in the same way The dose for each testsample-administered group was as follows 12 g of driedpeelskgday (119899 = 9) 24 g of dried peelskgday (119899 = 9) 12 gof dried flavedokgday (119899 = 8) 24 g of dried flavedokgday(119899 = 8) 100 g of dried juicekgday (119899 = 6) 200 g of driedjuicekgday (119899 = 6)

25 Administration of NGIN or AUR Mice weighing about25 g were randomized into 6 groups NGIN and AUR wereseparately dissolved inDMSOpolyethylene glycol (PEG) 300(1 1) solutions as previously mentioned [3] The animals in4 groups were subcutaneously administered these solutions(125 120583L) to achieve 50mg of NGINkgday (119899 = 5) 100mgof NGINkgday (119899 = 5) 10mg of AURkgday (119899 = 5) or25mg of AURkgday (119899 = 5) once a day for 7 days Mice ofthe other 2 groups (nontreated group 119899 = 5 and LPS-treatedgroup 119899 = 5) were administered vehicle (DMSOPEG) in thesame way

26 LPS Treatment LPS (from Salmonella enterica serotypetyphimurium) was purchased from Sigma-Aldrich (St LouisMO USA) and dissolved with saline On the seventh dayafter the administration of Citrus samples 25120583g of LPS in0125mL of saline was immediately intraperitoneally (ip)administered (1mgkg of mouse) to the LPS-treated groupand test-sample groups For the nontreated group vehicle(0125mL of saline) was ip administered

27 Open-Field Test One day after the LPS injection micewere weighed and submitted to a test of abnormal behaviorAbnormal activity was evaluated by using the open-fieldtest Each mouse was placed in the center of an open-fieldapparatus (W70 timesD70 timesH50 cm center 35 times 35 cm) and itsbehavior was analyzed for 10min with the ANY-maze VideoTracking System (StoeltingWood Dale IL USA) which wasconnected to a USB digital camera

28 Immunohistochemistry After the tests of abnormalbehavior mice were anesthetized and transcardially per-fused with ice-cold PBS Their brains were then removedand processed for optical microscopy or confocal fluores-cence microscopy as previously reported [6] For opticalmicroscopy a rabbit polyclonal antibody against ionized

Table 1 Amounts of AUR HMF NGIN and NRTN in the driedpeel flavedo albedo and juice

AUR(mgg)

HMF(mgg)

NGIN(mgg)

NRTN(mgg)

Dried peel 407 027 4402 446Dried flavedo 962 137 1391 397Dried albedo 010 001 1407 117Dried juice 032 ND 158 068

calcium-binding adaptor molecule 1 (IBA1 Wako OsakaJapan) was used as the primary antibodyThe secondary anti-body was EnVision-plus system HRP-labeled polymer (anti-rabbit Dako Glostrup Denmark) Immunoreactivity wasdeveloped and visualized by use of DAB substrate (SK-4100Vector Laboratories Burlingame CA USA) and quantifiedby using ImageJ software (NIH Bethesda MD USA) Forconfocal fluorescence microscopy the primary antibodiesused were goat anti-COX-2 (Santa Cruz BiotechnologySanta Cruz CA USA) and mouse anti-glial fibrillary acidicprotein (GFAP Sigma-Aldrich St Louis MO USA) and thesecondary antibodies were Alexa Fluor 488-labeled donkeyanti-goat IgG (H + L) (Invitrogen Carlsbad CA USA) andAlexa Fluor 568-labeled goat anti-mouse IgG (H+ L) respec-tively The mounting medium used was VECTASHIELD(Vector Laboratories Burlingame CA USA) Images of thehippocampus were captured with a confocal fluorescencemicroscopy system (LSM510 Zeiss Oberkochen Germany)

29 Statistical Analysis Data for the individual groups wereexpressed as means plusmn SEM Data were analyzed by one-factor ANOVA followed by Bonferronirsquosmultiple comparisontest (Prism 5 GraphPad Software La Jolla CA USA)Significance was defined as 119875 lt 005

3 Results

31 Contents of Main Compounds in the Dried Peels of Ckawachiensis The contents of 4 selected compounds (AURHMF NRTN and NGIN) in the dried peels were quantifiedby an absolute calibration method with reference to the peakareas on the HPLC chromatogram The chromatogram ofpeel powder of C kawachiensis is depicted in Figure 2 Asshown in Table 1 the primary compound was NGIN at4402 plusmn 0491mgg of the sample NRTN AUR and HMFcontents were much lower being 446 plusmn 00563mgg 407 plusmn0033mgg and 027 plusmn 00039mgg respectively

32 Effect of the Dried Peels of C kawachiensis on LPS-Induced Sickness Response Mice were pretreatedwith sample(dried peels) or vehicle (distilled water) once daily for 7 daysfollowed by a single ip injection of LPS or vehicle (saline)In the present study the dose of the dried peel powder wasdesigned to be 12 gkgday or 24 gkgday The amounts ofAUR HMF NRIN and NRTN contained in these samplesolutions are shown in Table 2


Table 2 Doses of AUR HMF NGIN and NRTN of each group

AUR (mgkgday) HMF (mgkgday) NGIN (mgkgday) NRTN (mgkgday)Peel

12 gkgday-treated group 488 0324 528 53524 gkgday-treated group 977 0648 106 107

Flavedo12 gkgday-treated group 115 164 168 47624 gkgday-treated group 231 329 334 953

Juice100 gkgday-treated group 320 ND 158 68200 gkgday-treated group 640 ND 316 136

0 10 20 30 40 50 60 70(min)

(mAU

)

NRTN AURHMF

NGIN

Figure 2 HPLC profile (280 nm) of peel powder of C kawachiensis

We assessed the changes in body weight loss at 20 h afterinjection of LPS as one index to estimate the degree of theLPS-induced sickness response Previous study showed thatip administration of LPS (01mgkg) significantly reducesfood intake and body weight at 8 h and 1 day after injectionbut not at 2sim4 days [7] Figure 3(a) shows that ip adminis-tration of LPS caused a significant reduction in body weight( lowastlowastlowast119875 lt 0001) The pretreatment of the animals with thedried peel powder attenuated dose-dependently this LPS-induced reduction in body weight though this effect was notsignificant for either 12 gkgday-treated group (119875 = 0267)or 24 gkgday-treated group (119875 = 0142)

As another index of the LPS-induced sickness responsewe examined the LPS-induced abnormal behavior observedin an open-field setting We measured the frequency ofcenter entry in the open-field test which is known to be anindicator of behavioral activity [8] Figure 3(b) shows that thefrequency of center entry of the LPS-treated mice (157plusmn18)was significantly ( lowastlowast119875 lt 001) decreased compared with thatof nontreated group (31 plusmn 25) This value for 12 gkgday-treated group (246 plusmn 39) was significantly (119875 lt 005)greater than that for the LPS-treated group The value forthe higher-dose sample (24 gkgday-treated group) was alsogreater (211 plusmn 29) than that for the LPS-treated group butnot significantly so (119875 = 0184)

These data indicate that acute activation of the periph-eral innate immune system with LPS induced the sicknessresponse in mice and that the peels of C kawachiensis hadthe ability to suppress the symptoms of it

33 Effect of the Dried Peels of C kawachiensis on LPS-InducedMicroglial Activation It is well known that microglia aredistributed throughout the brain as a network of immuno-competent cells and that the cells become rapidly activatedin response to injury or in the presence of pathogens[9] Previous studies indicated that peripheral injected LPSinduced microglial activation in the brain [6 10] So we usedantibody against IBA1 a microglial marker to stainmicrogliain the hippocampal region of the various groups of mice

In the nontreated group only a few IBA1-positive cellswere observed as being in the ramified form (an inac-tivated form) in the hippocampus (Figure 4(a)-none) Inthe LPS-treated group the number of IBA1-positive cellshad increased and their shape had changed to that ofldquoameboid microgliardquo (an activated form Figure 4(a)-LPS) aspreviously reported [6] Quantitative analysis of the numberof these cells (Figure 4(b) lowastlowastlowast119875 lt 0001) and the areasof these cells (Figure 4(c) lowastlowastlowast119875 lt 0001) revealed thatboth had significantly increased compared with those forthe nontreated group In the 12 gkgday-treated group and24 gkgday-treated group the number of IBA1-positive cellswas significantly lower than that in the LPS-treated group(Figures 4(a) and 4(b)

119875 lt 005 for 12 gkgday-treatedgroup and

119875 lt 001 for 24 gkgday-treated group)In the 12 gkgday-treated group and 24 gkgday-treatedgroup the shape of the IBA1-positive cells indicated theinactive ramified form (Figure 4(a)) and the area occupiedby these cells was lower than that for the LPS-treated group(Figure 4(c)119875 = 0076 for the 12 gkgday-treated group and119875 lt 0001 for the 24 gkgday-treated group)These results

indicate that the peels of C kawachiensis had the ability tosuppress LPS-inducedmicroglial activation in the brain of thetreated mice

34 Effect of the Dried Peels of C kawachiensis on LPS-InducedAstroglial Activation and COX-2 Expression Recent studieshave suggested the crucial importance of astrocytes as well asmicroglia in inflammatory responses in the brain [11 12] Wethus stained astrocytes in the hippocampus with anti-GFAPantibody an astrocytic marker In hippocampal regions(between CA1 region of Ammonrsquos horn and dentate gyrus)the expression of GFAP was increased in the LPS-treatedgroup (Figure 5(a)-IV) compared with that in the non-treated group (Figure 5(a)-I) The number of GFAP-positive


Alte

ratio

n of

bod

y w

eigh

t (g)

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(a)

0

10

20

30

40

Cen

ter e

ntry

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

Figure 3 Effect of po administration of the dried peels ofC kawachiensis on LPS-induced loss of bodyweight (a) and LPS-induced abnormalbehavior (b) Symbols indicate significant differences versus nontreated group ( lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) and versus LPS-treated group(119875 lt 005)

None LPS

(24 gkgday)LPS + peel powder


(a)

0102030405060708090

IBA

1-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(b)

IBA

1-po

sitiv

e cel

ls

0

1000

2000

3000

4000

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)

Figure 4 Effect of po administration of the dried peels of C kawachiensis on LPS-induced microglial activation (a) Sagittal sections of thehippocampus were stained with anti-IBA1 antibody Scale bar shows 50 120583m Results of quantitative analysis of number (b) and the area (c)of IBA1-positive cells by use of ImageJ software are shown Values are means plusmn SEM (119899 = 8sim10 for each group) Symbols indicate significantdifferences versus nontreated group ( lowastlowastlowast119875 lt 0001) and versus LPS-treated group (119875 lt 005 119875 lt 001 and

119875 lt 0001)


GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII

LPSNone (24 gkgday)LPS + peel powder


(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)

Figure 5 Effect of po administration of the dried peels of C kawachiensis on LPS-induced expression of GFAP and COX-2 in the mousehippocampus (a) Sagittal sections were stained with antibodies specific for GFAP (red I IV VII and X) or COX-2 (green II V VIII and XI)Merged pictures (III VI IX XII) show cells that co-expressed GFAP and COX-2 (yellow cells) Scale bar indicates 100 120583m (b) GFAP-positivecells and (c) COX-2-positive cells were counted by use of ImageJ software Values are means plusmn SEM (119899 = 8sim10 for each group) Symbolsindicate significant differences versus nontreated group ( lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001) and versus LPS-treated group (119875 lt 005 119875 lt 001and 119875 lt 0001)

cells in the 12 gkgday-treated group (Figure 5(a)-VII) and24 gkgday-treated group (Figure 5(a)-X) was significantlyless than that in the LPS-treated group as shown inFigure 5(b)

We next examined the proinflammatory response inthese regions of the 4 groups (nontreated group LPS-treatedgroup 12 gkgday-treated group and 24 gkgday-treatedgroup) by using COX-2 immunohistochemistry as COX-2 iswell known to be an important enzyme that regulates LPS-induced inflammation [13] In the nontreated group only

weak COX-2 immunoreactivity was detected (Figure 5(a)-II) but the immunoreaction was markedly stronger inthe LPS-treated group (Figure 5(a)-V) In the 12 gkgday-treated group and 24 gkgday-treated group the level ofthis immunoreactivity was reduced compared with that inthe LPS-treated group (Figure 5(a)-VIII and XI resp andFigure 5(c)) In all groups the GFAP-positive cells (astro-cytes) were immunopositive for COX-2 (Figure 5(a)-III VIIX and XII) but the microglia were not (data not shown)These results indicate that activated astrocytes contributed


0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++

0 12 24 0 12 24 0 100 200 0 10 25 50 100 Flavedo powder powderJuicePeel powder AUR NGIN

(gkgday) (mgkgday)(gkgday)(gkgday)

(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)

Figure 6 Effect of various samples derived fromC kawachiensis and of authenticNGINAURonLPS-induced loss of LPS-inducedmicroglialactivation (a) and LPS-induced expression of COX-2 (b) Values aremeansplusmn SEM Symbols indicate significant differences versus LPS-treatedgroup (119875 lt 005 119875 lt 001 and

119875 lt 0001)

to COX-2 expression after ip administration of LPS whoseexpression was attenuated by the dried peels ofC kawachien-sis

35 Effect of the Dried Flavedo and Juice on LPS-InducedMicroglial Activation and COX-2 Expression We thenadministered the dried powder derived from flavedo orjuice to LPS-injected mice The amounts of each compoundin these samples are indicated in Table 2 As was shownin Figure 4 ip administration of LPS induced microglialactivation ( lowastlowastlowast119875 lt 0001) and Figure 6(a) shows the effectof various citrus samples on the LPS-induced microglialactivation taken as 100 this activation was reducedsignificantly in the 12 g of peelkgday-treated group(119875 lt 005) 24 g of peelkgday-treated group (119875 lt 001)12 g of flavedokgday-treated group (119875 lt 005) 24 gof flavedokgday-treated group (119875 lt 001) and 100 gof juicekgday-treated group (119875 lt 0001) Regardingtheir effect on COX-2 expression Figure 6 shows the extentof COX-2 expression of sample groups versus COX-2expression in the LPS-treated group taken as 100 asignificant reduction in expression was found in the 12 g of

peelkgday-treated group (119875 lt 001) 24 g of peelkgday-treated group (119875 lt 0001) 12 g of flavedokgday-treated group (119875 lt 001) 24 g of flavedokgday-treatedgroup (119875 lt 0001) 100 g of juicekgday-treated group(119875 lt 0001) and 200 g of juicekgday-treated group(119875 lt 001) These results indicate that not only the peelsof C kawachiensis but also the flavedo tissuejuice had theability to suppress LPS-induced inflammation in the brain

36 Effect of NGIN or AUR on LPS-Induced MicroglialActivation and COX-2 Expression The primary compoundin the dried peels of C kawachiensiswas NGIN (Figure 2 andTable 1) and the dose of NGIN for the 12 g of peelkgday-treated group and 24 g of peelkgday-treated group wasequivalent to 528mgkgday and 106mgkgday respectively(Table 2) These results suggested that the main candidatefor the anti-inflammatory effect of the dried peels of Ckawachiensis was NGIN We thus sc preadministered com-mercially obtained authentic NGIN to LPS-treated mice atthe dose of 50mgkgday and 100mgkgday As a resultNGIN could suppress neither the microglial activation(Figure 6(a)) nor COX-2 expression (Figure 6(b)) suggesting


that the main candidate for the anti-inflammatory effect ofthe dried peels of C kawachiensis was not NGIN

We also examined the effect of sc administered AURin the amount of 10mgkgday or 25mgkgday on LPS-induced inflammation in the brain The dose of AUR in the24 g of peelkgday-treated group was almost equivalent tothat in the 10mg of AURkgday-treated group As shown inFigure 6(a) and 6(b) the mice of the 25mg of AURkgday-treated group showed significantly suppressed inflammation(119875 lt 005 for IBA-1-positive cells

119875 lt 0001 forCOX-2-positive cells) In the 10mg of AURkgday-treatedgroup IBA-1-positive cells tended to be reduced in number(119875 = 0147) and COX-2 expression was still significantly(119875 lt 0001) suppressed even at this lower dose Thereforethese findings suggest that the main candidate for the anti-inflammatory effect of the dried peels of C kawachiensis wasAUR

4 Discussion

Thepresent study showed that orally administered dried peelsof C kawachiensis could ameliorate LPS-induced inflamma-tion in themouse brainThis is the first report to demonstratethat citrus peel can suppress inflammatory responses in thebrain C reticulata or C unshiu peels are a stomachic andare employed as ldquochinpirdquo by practitioners of ChineseJapanesetraditional herbal medicine and a recent paper showed thatldquochinpirdquo could inhibit the expression of inducible nitric oxidesynthase (iNOS) COX-2 nitric oxide (NO) prostaglandin E

2

(PGE2) tumor necrosis factor (TNF)-120572 and interleukin (IL)-

6 in LPS-stimulated RAW 2647 cells a macrophage cell line[14] But the in vivo effect of ldquochinpirdquo on inflammation in thebrain was not investigated

The dried peel powder of C kawachiensis contained ahigh amount of NGIN middle amounts of NRTN and AURand a low amount of HMF (Figure 2 and Table 1) Althoughthemajor compound in the dried peels ofC kawachiensiswasNGIN the results obtained in this study together with ourprevious results suggest that the major functional compoundwithin the dried peels of C kawachiensis for causing theanti-inflammation effect in these model mice was AURThe reasons for this conclusion are as follows (1) an anti-inflammatory effect on the brain was also observed by theadministration of authentic AUR given alone (Figure 6) (2)the administration of authentic NGIN alone had so mucheffect (Figure 6) (3) NRTN being an isomer of NGIN wouldalso likely have no anti-inflammatory property although wedid not investigate the effect of authentic NRTN given alonein this present study and (4) the amount of HMF in the driedpeels ofC kawachiensiswas too low to have exerted its effects

With respect to NGIN accumulating evidence indicatesthat it potently inhibits oxidative stress not only in vitro [15ndash17] but also in vivo For example in the latter case (1) thepreadministration of NGIN (100mgkgday for 7 days ip)into ischemic model mice attenuates behavioral alterationsand neuronal damage [18] (2) the administration of NGIN(80mgkgday for 7 days ip) into model mice with kainicacid-induced epilepsy attenuates behavioral alterations and

cognitive impairment [19] (3) the administration of NGIN(80mgkgday for 2 weeks po) into model rats with 3-nitropropionic acid-induced Huntingtonrsquos disease suppressesneuronal apoptosis [19 20] (4) the administration of NGIN(40 or 80mgkgday for 25 days po) to colchicine-injectedrats attenuates the cognitive impairment in them [21] (5) theadministration of NGIN (40 or 80mgkgday for 6 weekspo) into mice chronically treated with D-galactose lessenstheir cognitive impairment and mitochondrial dysfunction[22] and (6) the administration of NGIN (100mgkgdayfor 16 weeks) to Alzheimerrsquos disease-model mice (APP-swePS1dE9) improves long-term memory [23] As NGINand its metabolites have been shown to have difficulty incrossing the blood-brain barrier (BBB) [24] the BBB mighthave been destroyed in these cases consequently permittingNGIN (and probably NRTN) to exert a potent antioxidativeeffect on the brain

Themost important question to be answeredwaswhether(1) AUR passed through the BBB and acted directly in thebrain as an anti-inflammatory agent or (2) AUR suppressedthe peripheral inflammation which was followed by thesuppression of central inflammation We suggested that apart of AUR at least could penetrate the BBB because (1)AUR is a hydrophobic small molecule (2) our preliminarydata showed that ip administration of AUR was useful tosuppress inflammation induced by central administrationof LPS (unpublished data) Inflammation in the brain isassociated with various neurodegenerative diseases such asParkinsonrsquos disease and Alzheimerrsquos disease [25 26] as wellas with ischemia and trauma [1] Thus the dried peels of Ckawachiensis which contain high amounts of AUR might beuseful for the reduction of neuroinflammation-related braindiseases

5 Conclusions

Upon pretreatment of LPS-injected mice with dried powderof C kawachiensis inflammatory responses in the brain werereducedThe dried powder derived fromflavedo or juice ofCkawachiensis was also effective as an anti-inflammatory Themain component responsible for these effects was concludedto be AUR

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Wang X N Tang and M A Yenari ldquoThe inflammatoryresponse in strokerdquo The Journal of Neuroimmunology vol 184no 1-2 pp 53ndash68 2007

[2] C Iadecola and J Anrather ldquoThe immunology of stroke frommechanisms to translationrdquo Nature Medicine vol 17 no 7 pp796ndash808 2011

[3] S Okuyama S Minami N Shimada N Makihata M Naka-jima and Y Furukawa ldquoAnti-inflammatory and neuroprotec-tive effect of auraptene a Citrus coumarin following cerebral


global ischemia in micerdquo Eurpean Journal of Pharmacology vol699 no 1ndash3 pp 118ndash123 2013

[4] Y Furukawa S Okuyama Y Amakura et al ldquoIsolation andcharacterization of activators of ERKMAPK from Citrusplantsrdquo International Journal of Molecular Sciences vol 13 no2 pp 1832ndash1845 2012

[5] Y Amakura M Yoshimura K Ouchi S Okuyama YFurukawa and T Yoshida ldquoCharacterization of constituentsin peel of Citrus kawachiensis (Kawachibankan)rdquo BioscienceBiotechnology and Biochemistry vol 77 no 9 pp 1977ndash19802013

[6] S Okuyama N Makihata M Yoshimura et al ldquoOenothein Bsuppresses lipopolysaccharide (LPS)-induced inflammation inthemouse brainrdquo International Journal ofMolecular Science vol14 no 5 pp 9767ndash9778 2013

[7] C B Lawrence D Brough and E M Knight ldquoObese miceexhibit an altered behavioural and inflammatory response tolipopolysacchariderdquo Disease Models amp Mechanisms vol 5 no5 pp 649ndash659 2012

[8] R Yirmiya ldquoEndotoxin produces a depressive-like episode inratsrdquo Brain Research vol 711 no 1-2 pp 163ndash174 1996

[9] G W Kreutzberg ldquoMicroglia a sensor for pathological eventsin the CNSrdquo Trends in Neurosciences vol 19 no 8 pp 312ndash3181996

[10] Z Chen W Jalabi K B Shpargel et al ldquoLipopolysaccharide-induced microglial activation and neuroprotection againstexperimental brain injury is independent of hematogenousTLR4rdquo The Journal of Neuroscience vol 32 no 34 pp 11706ndash11715 2012

[11] A M Blanco S L Valles M Pascual and C Guerri ldquoInvolve-ment of TLR4type I IL-1 receptor signaling in the induction ofinflammatory mediators and cell death induced by ethanol incultured astrocytesrdquoThe Journal of Immunology vol 175 no 10pp 6893ndash6899 2005

[12] Y Dong and E N Benveniste ldquoImmune function of astrocytesrdquoGlia vol 36 no 2 pp 180ndash190 2001

[13] D W Chung K-Y Yoo I K Hwang et al ldquoSystemicadministration of lipopolysaccharide induces cyclooxygenase-2 immunoreactivity in endothelium and increases microglia inthe mouse hippocampusrdquo Cellular andMolecular Neurobiologyvol 30 no 4 pp 531ndash541 2010

[14] Y C Oh W K Cho Y H Joeng G Y Im Y H Hwang and JY Ma ldquoAnti inflammatory effect of Citrus unshiu peel in LPS-stimulated RAW2647macrophage cellsrdquoTheAmerican Journalof Clinical Medicine vol 40 no 3 pp 611ndash629 2012

[15] M Cavia-Saiz M D Busto M C Pilar-Izquierdo N OrtegaM Perez-Mateos and PMuniz ldquoAntioxidant properties radicalscavenging activity and biomolecule protection capacity offlavonoid naringenin and its glycoside naringin a comparativestudyrdquo Journal of the Science of Food and Agriculture vol 90 no7 pp 1238ndash1244 2010

[16] Y-H Lu M-Y Su H-Y Huang L-L Li and C-G YuanldquoProtective effects of the Citrus flavanones to PC12 cells againstcytotoxicity induced by hydrogen peroxiderdquo Neuroscience Let-ters vol 484 no 1 pp 6ndash11 2010

[17] S K Ha H Y Park H Eom Y Kim and I Choi ldquoNarirutinfraction from Citrus peel attenuates LPS-stimulated inflam-matory response through inhibition of NF-120581B and MSPKsactivationrdquo Food Chemical and Tixicology vol 50 no 10 pp3498ndash3504 2012

[18] V Gaur A Aggarwal and A Kumar ldquoProtective effect ofnaringin against ischemic reperfusion cerebral injury possible

neurobehavioral biochemical and cellular alterations in ratbrainrdquo European Journal of Pharmacology vol 616 no 1ndash3 pp147ndash154 2009

[19] M Golechha U Chaudhry J Bhatia D Saluja and D S AryaldquoNaringin protects against kainic acid-induced status epilepti-cus in rats evidence for an antioxidant anti-inflammatory andneuroprotective interventionrdquo Biological and PharmaceuticalBulletin vol 34 no 3 pp 360ndash365 2011

[20] K Gopinath and G Sudhandiran ldquoNaringin modulates oxida-tive stress and inflammation in 3-nitropropionic acid-inducedneurodegeneration through the activation of nuclear factor-erythroid 2-related factor-2 signaling pathwayrdquo Neurosciencevol 227 no 27 pp 134ndash143 2012

[21] A Kumar S Dogra and A Prakash ldquoProtective effect ofnaringin a Citrus flavonoid against colchicine-induced cog-nitive dysfunction and oxidative damage in ratsrdquo Journal ofMedicinal Food vol 13 no 4 pp 976ndash984 2010

[22] A Kumar A Prakash and S Dogra ldquoNaringin alleviates cog-nitive impairment mitochondrial dysfunction and oxidativestress induced by D-galactose in micerdquo Food and ChemicalToxicology vol 48 no 2 pp 626ndash632 2010

[23] D-M Wang Y-J Yang L Zhang F-F Guan and L-F ZhangldquoNaringin enhances CaMK II activity and improves long-termmemory in a mouse model of Alzheimerrsquos diseaserdquo Interna-tional Journal of Molecular Science vol 14 no 3 pp 5576ndash55862013

[24] W Zou C Yang and W Su ldquoTissue distribution study ofnaringin in rats by liquid chromatography-tandem mass spec-trometryrdquo Arzneimittelforschuing vol 62 no 4 pp 181ndash1862012

[25] E GMcGeer A Klegeris and P LMcGeer ldquoInflammation thecomplement system and the diseases of agingrdquo Neurobiology ofAging vol 26 supplement 1 pp S94ndashS97 2005

[26] L Qin X Wu M L Block et al ldquoSystemic LPS causes chronicneuroinflammation and progressive neurodegenerationrdquo Gliavol 55 no 5 pp 453ndash462 2007

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


OO OH C

CH CH33

3

(a) Auraptene (AUR)

O

O

OCH

H CO

3

OCH3

OCH3

OCH3

OCH3

3

H CO3

(b) 356783101584041015840-Heptamethoxyflavone (HMF)

O

O

O

O

O

O OH

OH

OH

OH

HO

HO

HO

HO

H CH3

(c) Naringin (NGIN)

O

O

O

O

O

O

OHOH

OH

OH

HO

HO

HO

HO CH3

(d) Narirutin (NRTN)

Figure 1 Structures of compounds detected in peel powder of C kawachiensis (kawachi bankan)

results suggested that the peels of C kawachiensismight be agood source of AUR for suppressing inflammation-induceddamage in the CNS

Therefore the objective of this study was to ascertainthe effectiveness of orally administered peel powder of Ckawachiensis against inflammation in the brain As a sam-ple the peel powder of C kawachiensis was prepared byadopting the processes of drying and sieving As a modelanimal of systemic inflammation we used mice that hadbeen intraperitoneally (ip) administered lipopolysaccharide(LPS) as peripheral injected LPS has the ability to induceimmune responses in the brain [6]

2 Materials and Methods

21 Preparation of Citrus Materials and Chemicals Fruitsof C kawachiensis were harvested in Yawatahama EhimeJapan For the preparation of dried peels the peels of thefruits (1555 g) after having been squeezed to obtain the juicewere chopped into small pieces and dried in vacuo at 60∘Cfor 1 day The dried peels (ca 300 g) were then milled andground to a fine powder with a mill mixer (Iwatani IFM-660DG Tokyo Japan)The powder after having been passedthrough a 150-mesh sieve was used as the test sample For thepreparation of the dried flavedo and the dried albedo flavedotissue (exterior yellow peel) and albedo tissue (interior whitepeel) were manually separated from fresh fruit with a smallknife and dried in vacuo at 60∘C for 1 day For the preparationof the dried juice the juice was freeze-dried These materials(dried flavedo dried albedo and dried juice) were thenmechanically ground to a fine powderwith themillmixer and

filtered through a 150-mesh sieve as in the case of the driedpeels Naringin (NGIN Figure 1(c)) and AUR were obtainedfrom LKT Laboratories (St Paul MN USA)

22 Determination of Flavonoid Contents in Samples DriedCitrus samples (1 g) were extracted by sonication for 20minin ethanol (30mL) at room temperature The mixture wasthen centrifuged at 10000timesg for 5min and the resultingsupernatant layer was analyzed by reversed-phase HPLCStandard solutions of NGIN narirutin (NRTN Figure 1(d))heptamethoxyflavone (HMF Figure 1(b) and AUR wereprepared in methanol at a concentration of 10mgmL Stocksolutions were diluted to prepare a calibration curve withlinearity in the range of 001sim10mgmL Each value wasreported as themean of 3 analyses HPLC analysis was carriedout by using a Shimadzu Prominence system (ShimadzuKyoto Japan) HPLC conditions were as follows column L-column ODS (5120583m 150 times 21mm id Chemicals Evaluationand Research Institute Tokyo Japan) mobile phase solventA (5 acetic acid) and solvent B (acetonitrile) 0sim30min 0sim50B inA 30sim35min 50sim85B inA 35sim40min 85sim85Bin A injection volume 2 120583L column temperature 40∘C flowrate 03mLmin detection flavonoids at 280 nm and AURat 320 nm NRTN was obtained from ChromaDex (IrvineCA USA) HMF was prepared from commercial orange oil(Wako Osaka Japan) as described previously [4]

23 Animals Nine-week-old male C57BL6 strain mice werepurchased from Japan SLC (Hamamatsu Japan) Mice in allgroups were kept at 23 plusmn 1∘C under a 12 h lightdark cycle









AUR(mgg)

HMF(mgg)

NGIN(mgg)

NRTN(mgg)




3 Results









0 10 20 30 40 50 60 70(min)

(mAU

)

NRTN AURHMF

NGIN












Alte

ratio

n of

bod

y w

eigh

t (g)

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(a)

0

10

20

30

40

Cen

ter e

ntry

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)


None LPS



(a)

0102030405060708090

IBA

1-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(b)

IBA

1-po

sitiv

e cel

ls

0

1000

2000

3000

4000

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)


119875 lt 0001)


GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII



(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)






0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























AUR(mgg)

HMF(mgg)

NGIN(mgg)

NRTN(mgg)




3 Results









0 10 20 30 40 50 60 70(min)

(mAU

)

NRTN AURHMF

NGIN












Alte

ratio

n of

bod

y w

eigh

t (g)

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(a)

0

10

20

30

40

Cen

ter e

ntry

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)


None LPS



(a)

0102030405060708090

IBA

1-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(b)

IBA

1-po

sitiv

e cel

ls

0

1000

2000

3000

4000

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)


119875 lt 0001)


GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII



(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)






0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















0 10 20 30 40 50 60 70(min)

(mAU

)

NRTN AURHMF

NGIN












Alte

ratio

n of

bod

y w

eigh

t (g)

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(a)

0

10

20

30

40

Cen

ter e

ntry

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)


None LPS



(a)

0102030405060708090

IBA

1-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(b)

IBA

1-po

sitiv

e cel

ls

0

1000

2000

3000

4000

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)


119875 lt 0001)


GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII



(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)






0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Alte

ratio

n of

bod

y w

eigh

t (g)

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(a)

0

10

20

30

40

Cen

ter e

ntry

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)


None LPS



(a)

0102030405060708090

IBA

1-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(b)

IBA

1-po

sitiv

e cel

ls

0

1000

2000

3000

4000

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)


119875 lt 0001)


GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII



(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)






0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GFAP

COX-2

Merged

I

II

III VI

V

IV VII

VIII

IX

XI

X

XII



(a)

0

20

40

60

80

100

120

GFA

P-po

sitiv

e cel

ls

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowast

(b)

COX-

2-po

sitiv

e cel

ls

0

20

40

60

80

100

120

140

LPSPeel powder 2412

(gkgday)

minus

minus minus

+ + +

lowastlowastlowast

(c)






0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

Activ

atio

n of

mic

rogl

ia (

)

LPS ++++++++++++++



(a)

0

50

100

150

Expr

essio

n of

CO

X-2

()



LPS ++++++++++++++

(b)


119875 lt 0001)









4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4 Discussion


2







5 Conclusions




References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article auraptene in the peels of citrus...

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