Antinociceptive Effect of 3

16 Pages·2016·2.92 MB·English

molecules
Article
Antinociceptive Effect of 3-(2,3-Dimethoxyphenyl)-1-
(5-methylfuran-2-yl)prop-2-en-1-one in Mice Models
of Induced Nociception
NurIzzatiIsmail1,LeeMing-Tatt2,NordinLajis3,MuhammadNadeemAkhtar4,
AhmadAkira1,EnochKumarPerimal1,DaudAhmadIsraf1andMohdRoslanSulaiman1,*
1 DepartmentofBiomedicalSciences,FacultyofMedicineandHealthSciences,UniversitiPutraMalaysia,
43400Serdang,Malaysia;[email protected](N.I.I.);[email protected](A.A.);
[email protected](E.K.P.);[email protected](D.A.I.)
2 FacultyofPharmaceuticalSciences,UCSIUniversity,56000Cheras,Malaysia;[email protected]
3 LaboratoryofNaturalProducts,InstituteofBioscience,UniversitiPutraMalaysia,43400Serdang,Malaysia;
[email protected]
4 FacultyofIndustrialSciences&Technology,UniversityMalaysiaPahang,26300Gambang,Malaysia;
[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:DerekJ.McPhee
Received:28June2016;Accepted:12August2016;Published:22August2016
Abstract: Theantinociceptiveeffectsproducedbyintraperitonealadministrationofanovelsynthetic
chalcone,3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one(DMFP),wereinvestigated
in several mouse models of induced nociception. The administration of DMFP (0.1, 0.5, 1.0 and
5.0mg/kg)producedsignificantattenuationontheaceticacid-inducedabdominal-writhingtest.
Italsoproducedasignificantincreaseinresponselatencytimeinthehot-platetestandamarked
reductionintimespentlickingtheinjectedpawinbothphasesoftheformalin-inducedpaw-licking
test. In addition, it was also demonstrated that DMFP exhibited significant inhibition of the
neurogenic nociceptive response induced by intraplantar injections of capsaicin and glutamate.
Moreover,theantinociceptiveeffectofDMFPintheaceticacid-inducedabdominal-writhingtest
and the hot-plate test was not antagonized by pretreatment with a non-selective opioid receptor
antagonist,naloxone. Finally,DMFPdidnotshowanytoxiceffectsand/ormortalityinastudyof
acutetoxicityanddidnotinterferewithmotorcoordinationduringtheRota-rodtest. Ourpresent
resultsshowthatDMFPexhibitsbothperipheralandcentralantinociceptiveeffects. Itwassuggested
thatitsperipheralantinociceptiveactivityisassociatedwithattenuatedproductionand/orrelease
of NO and various pro-inflammatory mediators, while central antinociceptive activity seems to
beunrelatedtotheopioidergicsystem,butcouldinvolve,atleastinpart,aninteractionwiththe
inhibitionofcapsaicin-sensitivefibersandtheglutamatergicsystem.
Keywords:3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one;chalcone;antinociceptive
activity;opioidergicsystem;TRPV1receptor;glutamatergicsystem
1. Introduction
Pain remains a major issue in a vast array of medical conditions. Although many effective
andpotentpharmacotherapiessuchasopioidsandnon-steroidalanti-inflammatoryanalgesicdrugs
are available, they all have limitations. The continuous use of these analgesic drugs is generally
followedbyundesirableadversesideeffects,suchasgastrointestinaldamage,renaltoxicity,tolerance
and respiratory depression [1–3]. Therefore, the search for new analgesic compounds with good
efficacyandleastundesirablesideeffectstoalleviatethisobstinatepainfulconditionispotentiallyvery
Molecules2016,21,1077;doi:10.3390/molecules21081077 www.mdpi.com/journal/molecules
Molecules2016,21,1077 2of16
importantandisnowaninterestingstrategy. Takingtheseintoconsideration,compoundsshowingan
analgesiceffecthaveappearedasattractivetherapeuticsourcesforthedevelopmentofnewrelevant
drugsforthemanagementofseveralpainfulconditions.
Chalcones (1,3-diaryl-2-propen-1-ones) represent an important group of natural or synthetic
compoundsbelongingtotheflavonoidfamily. Theyhavebeenreportedtoexhibitbroadspectrum
Molecules 2016, 21, 1077 2 of 15
of biological and pharmacological activities, such as antimicrobial [4], anticancer [5], antiulcer [6],
antinociceptiivmepo[7rt–an9t] ,anadn itsi -nionwfl aanm inmtearetsotirnyg [s1tr0at]e,gaym. Toakninggo ththeseer isn,toa cnodnstidheurasticoon,m copmrpisoeunadsc lsahsoswiwngi thimportant
an analgesic effect have appeared as attractive therapeutic sources for the development of new
therapeutic potential. Many chalcones and their synthetic derivative analogues have been found
relevant drugs for the management of several painful conditions.
to inhibit the syCnhtahlceosniess (o1f,3-ndiiatrryilc-2-opxroidpeen-(1N-onOes)) arenpdrespenrto asnt aimgplaonrtadnitn gsro(uPpG o)f ,nwatuhriacl hor asryenthpertioc ducts of the
nitric oxide csoymnptohuansdes b(eNloOngSin)g aton tdhec fylacvloonooixdy fagmeinlya. sTehe(yC hOavXe )bepena trhepworateyds t,o reexshpibeitc btriovaedl ysp.ecTtrhumes e pathways
of biological and pharmacological activities, such as antimicrobial [4], anticancer [5], antiulcer [6],
constitutethemajorproinflammatorypathwaysandremainthemosttargetedforanti-inflammatory
antinociceptive [7–9], anti-inflammatory [10], among others, and thus comprise a class with important
andantinocictheepratpiveuetidc pruotgenstidale. vMealnoyp cmhaelcnonte[s1 a1n–d1 t4h]e.ir synthetic derivative analogues have been found to
Our onginohiibnigt thien styenrtehsetsisi nof tnhiterics oexaidrec h(NfOo) ranndo pvreosltaagnlaanldginess (iPcGa),g wehnicths ahrea psroledducttso oft thhee niintrvice stigation of
oxide synthase (NOS) and cyclooxygenase (COX) pathways, respectively. These pathways constitute
naturally occurring chemical resources [15–18] as well as synthetic analogues based on natural
the major proinflammatory pathways and remain the most targeted for anti-inflammatory and
products [9,1a9nt]i.noRciececpetinvtel dyr,uwgs edehvealvopemiennvt [e1s1t–i1g4]a. ted that chalcones bearing substituted furanyl group,
3-(2,3-dimethoxyOpuhr eonngyolin)-g1 in-(te5r-emst ient thhye lsfeuarrcah nfo-r2 n-oyvle)lp arnoalpge-s2ic- eagne-n1t-s ohnase le(dD toM thFe Pin,vFesigtiguartieon1 o)f snhatouwralelyd remarkable
occurring chemical resources [15–18] as well as synthetic analogues based on natural products [9,19].
anti-inflammatory activity. It was demonstrated that this compound significantly and potently
Recently, we have investigated that chalcones bearing substituted furanyl group, 3-(2,3-dimethoxyphenyl)-
suppressedN1-(O5-minethiynltfuerrafne-2r-oynl)p-rcop(I-2F-Nen--1c-o)-nea (nDdMFliPp, Foipguorely 1s) ashcocwheadr riedmear(kLabPleS a)n-ati-cintiflvamatmeadtoRryA acWtivi2ty6. 4.7cells[20].
In light of thIte wsaisg dnemifiocnastnratteadc tthiavt itthyis ocofmDpoMunFdP siginnifitchanetlay tatnedn puoatetniotlny soupfpNresOse-dr eNlOat iend inetefrffeercotns-,c which have
(IFN-c)- and lipopolysaccharide (LPS)-activated RAW 264.7 cells [20]. In light of the significant activity
alsobeenassociatedwiththedevelopmentofpain,thiscompoundwasfurthersynthesizedinour
of DMFP in the attenuation of NO-related effects, which have also been associated with the
laboratoryanddeveelvopamlueantt eodf pwaini,t hthirse csopmepcotutnod iwtsasa fnurttihneor scyinctehpestiizveed ipnr ooupr elarbtoierast.orWy aenhd eevrealiunatreedp woitrht thefindings
ontheantinorcesipceecpt ttoiv ites aanctitnivociitcyepotifveD pMropFePrtiuess. iWneg hseerevine rreaploirnt thvei vfiondeinxgps eorni tmhee annttianlocmicoepdtievles aoctfivcithy emicals-and
of DMFP using several in vivo experimental models of chemicals- and heat-induced nociception in
heat-inducednociceptioninmiceandelucidatesomeofitspossiblemechanismsofaction.
mice and elucidate some of its possible mechanisms of action.

Figure 1. Chemical structure of 3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMFP).
Figure1.Chemicalstructureof3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one(DMFP).
2. Results
2. Results
2.1. Antinociceptive Studies
2.1. Antinocic2e.1p.1ti. vAeceStitcu Adciieds-Induced Writhing Test
The effect of DMFP on writhing response in mice is shown in Figure 2, DMFP (0.1, 0.5, 1.0, and
2.1.1. AceticAcid-InducedWrithingTest
5.0 mg/kg, i.p.) administered intraperitoneally (i.p.) caused dose-dependent inhibition on the writhing
response induced by acetic acid with 13.89% (p < 0.01), 32.41% (p < 0.001), 59.95% (p < 0.001) and
TheeffectofDMFPonwrithingresponseinmiceisshowninFigure2,DMFP(0.1,0.5,1.0,and
91.14% (p < 0.001) of inhibition as compared to control, respectively. Such effect was also observed in
5.0mg/kg,i.mp.i)cea pdrem-trienatiesdt ebrye AdSAin wtritahp 4e4.r2i5t%o n(pe <a 0l.l0y5)( iin.phi.b)itcioanu. Tsheed cadlcouslaete-dd empeaenn IdDe50n fotri in.ph. aibdmitiinoisnteorend thewrithing
responseinduDcMeFdP biny thaics emtoicdeal cwidas w0.8i5th m1g3/k.g8 9(C%I, 0(p.76< to0 0.0.915) m,3g2/k.g4)1. %(p<0.001),59.95%(p<0.001)and91.14%
(p<0.001)ofinhibitionascomparedtocontrol,respectively. Sucheffectwasalsoobservedinmice
2.1.2. Formalin-Induced Paw-Licking Test
pre-treatedbyASAwith44.25%(p<0.05)inhibition. ThecalculatedmeanID fori.p. administered
As shown in Figure 3, the i.p. treatment with DMFP at the doses of 0.1, 0.5,1.0 a5n0d 5.0 mg/kg
DMFPinthissigmnioficdaenltlyw inahsib0it.e8d5 tmhe gli/ckkingg (tCimIe, i0n. 7of6 btooth0 n.e9u5romgegni/c k(0g–)5. min, panel A), by 18.85% (p < 0.5),
31.41% (p < 0.001), 41.88% (p < 0.001) and 68.41% (p < 0.001), and inflammatory (15-30 min, panel B), by
2.1.2. Formal1i9n.6-0I%n d(pu < c0e.0d1),P 43a.w10%-L (pic <k 0i.n00g1),T 6e5s.0t7% (p < 0.001) and 80.04% (p < 0.001), phases of formalin-induced
paw-licking test, when compared to control group. The treatment with ASA (100 mg/kg, i.p.) only
AsshowsingniifincaFnitglyu irnehib3i,tetdh ethei .plic.kitnrge atitmme einn tinwflaimthmaDtoMry FpPhaaset bthy e18d.9o6%se (sp o< f00.0.11)., O0.n5 c,1on.0traarny,d 5.0mg/kg
morphine (5 mg/kg, i.p.) significantly inhibited both phases of the test by 51.49% and 69.00% (p < 0.001),
significantlyinhibitedthelickingtimeinofbothneurogenic(0–5min,panelA),by18.85%(p<0.5),
respectively.
31.41%(p<0.001),41.88%(p<0.001)and68.41%(p<0.001),andinflammatory(15-30min,panelB),
by 19.60% (p < 0.01), 43.10% (p < 0.001), 65.07% (p < 0.001) and 80.04% (p < 0.001), phases of
formalin-induced paw-licking test, when compared to control group. The treatment with ASA
(100 mg/kg, i.p.) only significantly inhibited the licking time in inflammatory phase by 18.96%
(p<0.01). Oncontrary,morphine(5mg/kg,i.p.) significantlyinhibitedbothphasesofthetestby
51.49%and69.00%(p<0.001),respectively.
Molecules2016,21,1077 3of16
Molecules 2016, 21, 1077 3 of 15
Molecules 2016, 21, 1077 3 of 15
150
150
s
nggs ((1133..8899))
mber of writhiber of writhin1150500000 **** ((33**22**..44**11)) ((55**99**..99**55)) ((44**44**..22**55))
Nuum ((9911..1144))
N ***
***
0
0
C 0.1 0.5 1 5 ASA
C 0.1 0.5 1 5 ASA
DMFP (mg/kg, i.p.)
DMFP (mg/kg, i.p.)

FFFiiiggguuurrreee 222... EEEffffffeeecccttt ooofff DDDMMMFFFPPP ((00(0..11.1,, ,000..55.5,, ,11 1aannaddn d 55 5mmmgg//gkk/ggk,, gii..p,pi..).)p iin.n) aaincceeattiiccce aaticcciidda--ciiinnddd-uiunccdeeudd c aaebbdddaoobmmdiionnmaalli--nwwarrlii-ttwhhiirnnitggh ttienessgtt
tiienns tmminiicceem.. iEEceaa.cchhE acccoohlluucmmolnnu mrreenpprrreeepsseernensttses nttthhseet hmmeeemaanne a±±n SS±..EES..MM.E... M((nn. ==(n 66=)).. 6TT).hhTee h mmeiimcceeic wweweerreeer eppprreertterrteereaaatteeteddd wwwiiittthhh vvveeehhhiiicccllleee
(((CCC,,, 111000 m mmLLL///kkkggg,,, iii.p..pp.)..),),,D DDMMMFFFPPP( 0((.001..,110,, .500,..551,,. 011..a00n aadnn5dd.0 55m..00g mm/kggg//kk,igg.p,, .ii)..ppo..r)) aoocrre taayccleesttayylillcssyaallliiicccyyallciiiccd aa(ccAiiddS A ((AA,1SS0AA0,,m 110g000/ kmmgg,gi//.kkpgg.),,.
Ti.phe.).a Tshteer iassktesrdiseknso dteensoigten isfiigcnainfcicealnecvee llesv*e*lsp *<* p0 <.0 01.,0*1*,* **p* <p <0 .00.0010,1w, whhenenc coommppaarereddw wiitthh tthhee ccoonnttrrooll
i.p.). The asterisks denote significance levels ** p < 0.01, *** p < 0.001, when compared with the control
vveehhiiccllee ggrroouupp.. SSttaattiissttiiccaall ssiiggnniiffiiccaannccee wwaass ddeetteerrmmiinneedd bbyy oonnee--wwaayy AANNOOVVAA ffoolllloowweedd bbyy DDuunnnneetttt’’ss
vehicle group. Statistical significance was determined by one-way ANOVA followed by Dunnett’s
ppoosstt--hhoocc tteesstt.. VVaalluueess iinn ppaarreenntthheesseess aarree ppeerrcceennttaaggeess ooff iinnhhiibbiittiioonn..
post-hoc test. Values in parentheses are percentages of inhibition.
200
200
110000 ((66..4466))
(18.85)
(18*.85) 150
Licking time (s) Licking time (s) 5500 * ((33**11**..4*4*11)) ((44**11**..88**88)) ((66**88**..44**11)) ((55**11**..44**99)) Licking time (s) Licking time (s)11105050000 ((1199**..**6600)) ((44**33**..11**00)) ((66**55**..00**77)) (80.04) ((1188**..**9966)) ((66*99*..00*00))
50 (8*0*.0*4) ***
***
0
0 CC 00..11 00..55 11 55 AASSAA MMOORR 00 C 0.1 0.5 1 5 ASA MOR
C 0.1 0.5 1 5 ASA MOR
DDMMFFPP ((mmgg//kkgg,, ii..pp..)) DDMMFFPP((mmgg//kkgg,, ii..pp..))
(A) (B)
(A) (B)
Figure 3. Effect of DMFP in formalin-induced paw-licking test (early phase, panel (A); and late phase,
Figure 3. Effect of DMFP in formalin-induced paw-licking test (early phase, panel (A); and late phase,
Figure 3. Effect of DMFP in formalin-induced paw-licking test (early phase, panel (A); and late
panel (B) in mice. Each column represents the mean ± S.E.M. (n = 6). The mice were pretreated with
pphanaseel ,(Bp)a inne lm(iBce). iEnamchi cceo.luEmanc hrecporelusemnnts rtehper meseeannts ±t hS.eE.mMe. a(nn =± 6)S.. ET.hMe .m(nice= w6e).re Tphreetrmeaitceed wweirthe
vehicle C, 10 mL/kg, i.p.), DMFP (0.1, 0.5, 1.0 and 5.0 mg/kg, i.p.), acetylsalicylic acid (ASA, 100 mg/kg,
pvreehtircelaet Ced, 1w0 imthLv/ekhgi,c il.ep.C),, D10MmFPL /(0k.g1,, i0..p5.,) ,1D.0 ManFdP 5(.00. 1m,0g./5k,g1,. 0i.pa.n),d a5c.e0tymlsga/likcygl,iic. pa.c)i,da c(AetSyAls,a 1li0c0y lmicga/ckigd,
i.p.) or morphine (MOR, 5 mg/kg, i.p.). The asterisks denote the significance levels * p < 0.5, ** p < 0.01,
(iA.pS.)A o,r1 m00omrpgh/inkeg ,(Mi.pO.)Ro,r 5m mogr/pkhgi,n ie.p(.M). OThRe, 5asmtegri/skkgs ,di.epn.)o.teT htheea ssitgenriisfkicsadnecne oletevethlse *s pig <n 0ifi.5c,a *n*c pe <le 0v.0el1s,
*** p < 0.001, when compared with control vehicle group. Statistical significance was determined by
***p* <p 0<. 50,.0**01p, <w0h.e0n1, c*o**mpp<ar0e.d0 0w1,itwh hceonntcrooml vpeahriecdlew girtohucpo.n Sttraotlisvteichailc lseiggnriofuicpa.nSceta wtisatsic daeltseirgmniifinceadn bcey
one-way ANOVA followed by Dunnett’s post-hoc test. Values in parentheses are percentage of inhibition.
wonaes-wdeatye rAmNinOeVdAb fyoollonwe-ewda byyA DNuOnnVeAtt’sfo plolostw-heodc tbesyt.D Vuanluneest ti’ns ppaorset-nhtohcesteess ta.rVe apleurecsenintapgae roefn itnhheisbeitsioanre.
percentageofinhibition.
2.1.3. Hot-Plate Test
2.1.3. Hot-Plate Test
2.1.3.AAHss o ddt-eePppliiaccttteeeddT eiisnnt FFiigguurree 44,, DDMMFFPP ttrreeaattmmeenntt aatt ddoosseess ooff 11..00 aanndd 55..00 mmgg//kkgg ssiiggnniiffiiccaannttllyy iinnccrreeaasseedd
the response latency to a heat stimulus 30 min after the treatment and persisted until 180 min in
the response latency to a heat stimulus 30 min after the treatment and persisted until 180 min in
AsdepictedinFigure4,DMFPtreatmentatdosesof1.0and5.0mg/kgsignificantlyincreased
comparison to the control group (vehicle 10 mL/kg).
comparison to the control group (vehicle 10 mL/kg).
the response latency to a heat stimulus 30 min after the treatment and persisted until 180 min in
DMFP at doses of 0.1 and 0.5 mg/kg significantly increased response latency time at 150 min after
DMFP at doses of 0.1 and 0.5 mg/kg significantly increased response latency time at 150 min after
comparisontothecontrolgroup(vehicle10mL/kg).
the treatment in comparison to the control. The positive control group, morphine (5 mg/kg, i.p.),
the treatment in comparison to the control. The positive control group, morphine (5 mg/kg, i.p.),
DMFPatdosesof0.1and0.5mg/kgsignificantlyincreasedresponselatencytimeat150min
increased the response latency to heat stimulation 30 min after administration. This effect was
increased the response latency to heat stimulation 30 min after administration. This effect was
afterthetreatmentincomparisontothecontrol. Thepositivecontrolgroup,morphine(5mg/kg,i.p.),
maintained until 180 min after the treatment (p < 0.001).
maintained until 180 min after the treatment (p < 0.001).
increased the response latency to heat stimulation 30 min after administration. This effect was
maintaineduntil180minafterthetreatment(p<0.001).
Molecules2016,21,1077 4of16
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11441411144414 ********************* ***************************************************************
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Response latency (sec)Response latency (sec)Response latency (sec)Response latency (sec)Response latency (sec)Response latency (sec)11068Response latency (sec)06810681110680680681068 ****** * ****** * *************************************************
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DDuDunDDDunDnuunnueuDnenntnnttunenn’tnste’neetse tt’pnt tttspto’t’e ’sso’pssts stt opp p-t’psohso-otohs sos-pstothct-to-c- oh-htsh hceottooe o-csctchscte tt.ot tse te.ec*e tss* s.sttp t t.*ept. . .< s* *p *<*t 0 pp. p< 0p. * 0<<. <0 0<p5 . 05 000 ;0< .;..5*0. 00 0**0;555 * 5.*p;; ;0 *;p * **5 <* *p**<;* 0 p p*p<0p. * 0<. <<0 0<p1 . 01 00 ;00< .;.1*.0. 0 00**0;11 *1*1.*;* ;0; *;p * *1**p* ** <*;* *p *<** * 0 p *p<p0p. * 0<. <0 <0<p0 .00 00 100<.1.0 0..0 c0 0010c0o00. 1o10cm11 mo 0c ccpcm1oopoo ammcpmamrorepapmppedraadaea rprwdrr eewaeed dirwddti e htw wiwdhw tci h iictiwottt hohchnhi no tct ccchrtnoooroo tnnocnnlrto ltotgtr rrnrgloroo otrgollrl lo urgo ggguorplrrr upooog;o up;uuCr uCop;pp opCu;o;;n ; Cp noCCCtr;tnooo rooCtnnonnlrto ltto(tr rrnr(looo ot(lllr l (o ((()l,) (,) ,) ))),,, , ),
DDMDMDDDMFDFPMMMMPFD (PF FFM(FPP P(PF (( (P( ,( , 0 0,.,, ,1 .,0 001 0;0. ..;1.,11 .1 1;0;; ; ; . 1; , , 0, ,,0 ,,. 00 50.00.5.;.,55.. 5 ;550; ;; ;; . 5; 1 1 1 .1.01.1100.;. 0..; 0;001 ;; ;; . 0; 5 5 .5 .0505.550. .m 0..m0 005m m gm.gmm0g//g gkm/ggk/k/g//kgkggkk,g,g /,ggii ,k,.i. ,, ppi. gip.ii..p...,p))pp. .).i.. )....)p)) .. .. . ).
22.1.21..2224.124.....11.1 .412C ...C.44..4 41aC...a ..p C4 CCpaCs.paas aaaCappspipciaassscsiiapaanicaniisii-icccna-cIiiiIninin-nnncId---nid-IInIuInndnun-cdduIdcdenueucududdeccc cdPuee eePdda cddPaew PP waPdP-waaa a-LPwwwLw-iaLci---cw-kLLiLLkciii-inkiicccLnckkigkkingiic iinnTg nknTgg egigTens TT seTtgTt esee estTss stttet st
TThTheTThTeT ahh ehhaTd eeedaeh m damaeaadmdi dnidammnmiidmnsiisimtininirtnsnriaiitisasnritststtiatirroitrstaoaranittaontrti iationo ooiotno fninof o n D ofonDo fMfD f M oo DDMDFffF MMPDMFP DFPMp FFpPM PrPprFo o prFPpdpodrP rurodpuoocdurdcdeopeucududdrecc codues eesdid cdgdisueg in sdsgncsiiieig nfiiggsfidnicninficgaiiiaficfinnfisnacciticnfiat gaad ntcnndn oatdtiot s nfi dosdedtceos- ooa-ddessdns-eeoeedet-p-s-dpededepee-denedpnepopdenedeseepndneennedn-eddtdnnet eei edntnnin pnetitht ne nh iiiinnhbtinnb dhiihihtbinietiiibiobihnbtoiniiititontbti iiono oiootin fninnof o t h ofothno hiftf ebf h oe t tithc efhthc ai eetaoceph pan ccsecpasaa aapscpipociaasscsifiapanicanisii-iccnta- cihi ini-nnc e--i- n -
ininidndciuiindnuancdpudicdenuescududdaecc cdinue ecende cdidneneu nu end-nruieroe enonurugudogerreroguueonongecrgigoneceieec dgninn ncneiio niccnnoc ce n iocnincucioco coernicceoccpioipeicgctccpetieeeioiptcpnopnietontiti pciioan ooatntn nint aoao ta aanlca ltlati t l cadla aladetl ol lldpolal s d osdtledileossoo osde,ssn s,seao eea,ssass ssa, ,et ,c sa sacaoa s,oscl sm al omc ccsdompoo pmocammpaosrprapeemperadsaadep,r rrdte aeaetods dordt e co ct ctdotoo ooocn m tonc ctocontropo ronctnoanrlott roltrgrn erolgoort dlgrrlol oog rtgugooulrrp rougpooc upwru ouwop pnpiwu ti tw thwprwihot iahi witlatt h hhgh aih tri a ahhgaio g h ihhuahghieip igheghsghsethwiht gseae etiahssc tstcatehtt tica saviaattcvcii ctavttihtityicivivyivt tgioyi iitotvhtfy yyof ie to fosyo ftf f o f
77997.9.9a77971.9c9199%t.7.%19.i999v %11(1. i%9(p%t% p1y ( < p % (<( (o pp0 <p 0 f .( < <0.0p<70 0.0 0900<01..1.00 .0)900) 1001o0. )10o1%1 b)0o)b) s 1 obsoeo()ebsprb breovssvsr<ebeeeverdrsrdve0vev d.ea re0eavdt dd0t ae t 1 tathdaa )httte t h eao t ttdh ethbhd eoetsdeoh se dosdeedreosv ooo desesos feoedoef 5 s ofo5eo.a f0.5f ft 0 o .55m 05tf.m.h 0.005mg e g. m/m0mgk/dk g/ggmgokg/ //skg( kgk(Feg gF/g(ik gF oi ((gg(FuifFFgu iri(5igurgFeg.eur0 uiu5 gerr5r)m eue).5e . r )5A g5e5.A) )/ ) .A.5 s. k AsAi) Agmi.s m iA ssmi(siliiFim amlmsiairlgiira imilliu rlaania nirrrihlre n ha iiiinrhbin5nb hii)hihtbi.nitoiibibohbtrAioriiyittbytoro osiye rrterify oymyffe fre f eeeyicefflcfet fafft efceerw eftcwcf citaewttn a cs wwhwsa ta saiaawabl ssslasi sa otl aaoosas llo lsrsasoy ool s o
oobobseobseooefbsrbbfrveosesvsreebeceverdrtsrdveve vfde rewofedv oddrfae ro f cf dsfrocoao arcrpfr ap aoc scclparsaasa apszpcopzaaesssezappaaopezzsizbpnieaenepseizppnee,eii ,nierwnp nwv,ee iehw,ne,h , wi dewcwhi,ch hihhwhc fiaih ocicahcch rhcahhi ch ca iahacehicceac vhiahvhepeiciveeidsehedveav vi6de eze6e9dv ed9d6.ep 4 .96 64d64i.9944n9% .6.%44.e449 %4,4( 4.p(%4%% p (w4< p <%( ( (ph0p <p0 . i <(0 <.0c<p0 0 h.0 00001<..100.) 000 )a100i 0.nc)i101n 1h)hi0))n h ii1i ienibhin)nbv ihihithbineiitibiioibdhbtoiniiitiontbt iio6oin oiootf9n ni ofn.co 4ofc oano 4afcpf f p% aoc sccpasfaaa apsicppci(aascspisiapnaicaniis-ici<cni-aciniini-innncdi-0nd-i-iuin.nidun0nc-dudc0iedneucd1uuddec) ccnde uenedo idcdnonce noc nihdnciocoic eoibnceccpciiopiecittcccpiteieoeioiptopcpninetontt.iip oin.o ootn.ni nfo.. . n .
capsaicin-inducednociception.

110100100110000100000
Licking time (s)Licking time (s)Licking time (s)Licking time (s)Licking time (s)Licking time (s)468468000Licking time (s)468000468000468468000000000468000 (1(10*(0.11*.(016(1(*1.6)1010)*0.*6(.*1.11)16606)*).()13(*631**)(1.3*5**.(154(*3*(*3.4)*315**1*)1*.4(*.5*.53*)*5*4414*)).()5*4(*444**)(4.4*5**.(450(*4*(4*.0)*445**4*)4*.0(*.5*.54*)*5*0040*)).()5*7(079)(9.79.(991(7(7.1)7999)9.1(.9.97)91191)).)(93(1*36*)*(6.34***.(640(*3(*3*.0)3*64*6*)*6.*0(.*4.4*3)*4*0*060)*).)4*0)
22020202200020 *********************
0000000
CCCCCCC00.1.01.0010..1.110.100.5.05.0050..5.550.51111111 5555555CCACAPCCACPAAPACPPPAP
DDMDMFDMDFDPMMPFMD(PFmF(FMmPP(PgmFg/((k(m/Pmgkmg/gg(,gkg m/,/gik /.kik,pgg.g pg.i/,,.)k ., pi) ig.i..p.p),p ..i).).)p.)
FFigiFguiFFuFgriiuigregFge uru5iu e5gr.r r e.eu5Ee E .5r f5 5eEff..e f. Ef eE5cEfcf.tef ff tfEcofe eeotfcfc cf fottDe t Dfo oc oMDftfMf Do DMFDFfPM MMPFD aP FFMaFgP PgaPaF a gai aPaniaggngs iaaanstaig tinsicn nactass aiscpttnt pa cs cscpsaataaa ipspccpiasacissinaipacani-iisciicn-caniiin-inindnic-dn-iu-iindniuncn-ducdeidenudcuudedc cpcd euepead dcpadw e wpa pdp-waal- aiwlpwc-iwclak-i-k-wclililniikcinc-cgiklkngki iitc nginetkne gsgtigste n t t tsiteg eniets ns st ittmen t mi isn inimtnci mci emimne.c i .ieEmc icEc.eae eEia.c.c . cEah EeEhca .ac ah cEccoch ohaclhu loc c ucchmlooumo llcnulmuoun mmrl nmrue nenprmnp e rr rprrneeeeespr prpseererensreepneetssrssnteese entn snstet tssns ts
Figure5.EffectofDMFPagainstcapsaicin-inducedpaw-lickingtestinmice.Eachcolumnrepresents
ththeteh ttmt ethhmhh eeeemtee ah mm maenmenaee e±me naaa± an nSn e±nS. a ±E± .±S±nE . .S MS E.SS±M..E...E .EMES ...oM ...MMoEM.f fo..6. M .. 6fo oo om 6f.fmff 6o i6m66ci f cm em mim6e.c i. eiTi mciccTc.eh eeeThi..e. c. Teh TeTTm eh.mhh h Teiemeeci h cm emmimee cwi eiiwmciccc eeeweeei r cw rwewewee rep ee eewprrrr reeepreee e -prr ppt-peerrtrr- rreeeepteea-r--r-tatetttrerertareee-edettaadaera ttwted teeewead ddiwdtti e htww wihdw tv ihi ivtitwte thhhevhhi h evtv ivvhciheeecle ihhelvhche iieli(cc iecC(hclllC eele(i,ec C , 1(( (l(CC1e,C0C 0 1 ,,(,m , 0C11 m11 00L0m,0 L 1m/m mkL/m0kLgL/ LmgLk,// /,g/kik kLk.i,gpg. g/gpi,k.,., ,) p.i gi,)i.i ..,p..D,pp )p D,.i.. )M..))D)p,M,, , D .DFMDD)F,PM MMMPFD (P FFF0M(FP0 P.P(P1.0 F 1(, ((. P(0,0010 0.0 ..,.1.(11 5.100,5,,, , .. 0 ,0051 0...,,5.55 50,,, , . 5,
11.0.10 .a1 10a1n.. 0n.0ad01 d na a.a50dn n5n d mad 5dmn 5g 5m5dg / m m k/gm5kg/g ggmkg,/ /,g/kik gk.i,gp.g /gpi,.k,. ), p.i gi ).io. p..p,o)pr .i.r o)..)c )p rcoa oo .arpc)rrp a soc ccpsaaraaaz pspcpzaesaseszpaappaezizsnzpieeaneepipzenp i(ei eniC(npnC e(eAieC An ( (P(CAeCPC, A (AP,0A C0P,.P 1PA.0,1,7 , .07 P01 0m. .7,1m.1 1 07m7m7.m 1m momm7olm m,lmom ,i lo.io,mpo. lpli,l.,. ),o p.i i ).i3l. p..p,3)p0 .i0.3) ..)m)p 03m 33. 0i)0m0n i 3nm m im0b nbi ei mninebfn of ebi bobnrfeereoe febf rfoio oeenirrn fretieoertn irar itnianeprntp atlitrranplraaaantplpnraptlaaltnalaapartnnr anl(t atir(taa.in apr(.rprti l (.a (.pl(i)ir..i. )pl. pi .p(ni)liln .l.j.i)p.e)jn) eic lijni.cnte)nitj cjoeiijeetoncncicntojt etioi noicooof ntn foni o fo o onfff o f
1.0and5mg/kg,i.p.)orcapsazepine(CAP,0.17mmol,i.p.)30minbeforeintraplantar(i.pl.)injection
ccaacppaocsccpsaafaaapispcccpiasaacsisianipapcani iisscic n(caai1(i nii1ni.n(cc6 .1 i6 i( (n.n(1 μ161μ .. g6(.(6μ6g11 / pμ./.gμ66μpa/gg pgaµwμ//w/pagpgp,wa /a,/a 2wpwp,2w0 a0a,2 , ,w μw0 22μ2 L0,,0 μ0L )2 2 μL.)μ00μ .L) TL L.µTμ) h)).TLh.L e. Teh)T )T. a.ehh aThs Teestaeh het s aeeareatsrise sastitrateskseieskrtsrsrteisik esisr dsskrkidk iessssdeskn kn desdodsonete deetondnene toeo tneohttnt oteheetoe teh tett setehhshit egiehtseg hn ie snsgseiiifsingi gifgiscinignfincaiginiaficnfnfiiniaccificiccnaefacaei cnnacn leceanlcc eevneclev e eec llevleeleslelev evs vlle *veese* lvl es pls*slpe s *l <*p*s < * p p 0<p*p 0 . <0<.0p<<0 5 . 0500,<00 .,.50 . .*0000,**55 5*5*.,*,0* ,, * *p5****p* *, *** <*p*** < p*pp 0<p* 0 . <<0<.0p<0 0 . 000001<0..1.0,0 .00 0,010 000.,1110 1,,,0 , 1,
wwhwhewwwehwnnheh hhwc neeecoen nhnocnm em occ cncopmoopo mammcpmaroprepapmpedaraadaer rprewdr eewaedd dirdwti ew htw wdihw tici htitiwchott hhochni cn otc tcohcrtnoonroo tnnoctnlrto rltotrg ornrlgoorl otrlgolrg lo urg ogrguorlopr rupooug o(up urpuo(op opnp((u no oe( (pen(o-noow- enenw(n-eo-aewew-an-y-wwyawe aA y-ayaAaw yNyAyAN a A OANNyAON VNOONAVAOOVVONA VAVAfV OofAA oAfVlfol lo of lAflolfolowloo wollfllwlewooloeodwlewdelw dobd eebewyd bdydb ey D yb bdDb yDuyD yu b nuD DunyDnnunn ueunDenntnenttuenn’ttnstt’neets’ ets’ntp ttstpto’ep ’s’ospstos stt o sp’p-tpstsho-o-to hsohs-pstohctoto-c- o-chsth hcettoot o-escetchcs tes ot )tttste.)ce) et.s .Vs )sttV.tVet )a)V).sa.a l. tuV llVa)Vuu.ela aueeasVllss uleuu aseielen suss e s
inin ip nppiia ninpaanr rrepia pnpeenraann aetrprhttnreehhaeetnnrenehstetssetehhnehesseesst eesahs ssaaereee rsreasssee rea pa aeppsrre re eepeare r rcpre pcpceereeeen cerpnrnertccteactneeaaregtnngcgnaeteetegta anoa egoogfgt ef afeoei gi nfio onn oehfifhhnf iio iibihninbfbniih hitiibhtitniiioiibibothobniiinntiiot.btii ..noio iot.nn in.o. . n.
22.1.21..225.125....1.1 .512G ..G.5..5 51lG..ul .. Gu5 GlGt.utal laGlumtuumattltmaauaaamtmtmteaae-taam-IaetIntt-eneaeId--ndt-IIueIndnun-cduIdcdenuecududdecc cdPue eePd acddPaew P waPdP-waa a-LPwLww-iaLci--cw-kLiLLkcii-ikniiccLnckigkkingic iinTg nknTg egiTgens T seTtgTt see etTss sttet st
2.1.5. Glutamate-InducedPaw-LickingTest
TThTheThTeT rh ehhreT eeerseh s eur rureseele utlssrtssulues utp lsslplttu rtssprsel ep trsppsseerr ersenpeenestsrsneteeeeendtnsndeet ttdie enneid ndtdi e Fn i Fidini nngFi g iF uiFnFguiri igurgeFgeur uiu6 ger6r r eues 6es h r 6 6hs6eo hos sw6showhh o swoto hwhtww hota ha wt tttht ahht tathata hhtttet h eat ttih eth.hi p .eetipeh .. i p .iiae. .p.a.p dp ida...m . pda maad.mid dniammnmiidnsiiismtinintrnsriaiitisasnrtstttiatirroirstaoaanittontrti iaioon oootnf ninof o D o fDono fMDf fM oD DMDFfF MMPDMFP FPaM FFatP PPt aFd ta daPa otdto t s ad osdedteosso osde ssos seoeoef sssof s 0 e o f0o.os 1f.0f f1 ,o.0 0 ,100f ..0,1 .1. 105.0,,5 ,. ,.01 0 ,50 .,.,5 .5 50,, ,. 5,
TheresultspresentedinFigure6showthatthei.p. administrationofDMFPatdosesof0.1,0.5,
11.0.10 .11 a01.a.n 0.001na d .nada0a ndn 5 n5ad. dd0.5n 0 .5d5 m05.m. 0.005mg g .m/m0mgk/ kg/ggmgkg/ //kg ckgkcag g/gacuk ua ccgscuasae aeuscududeass sdeu seesid dsdgise g in sdsgnsiii ignfiggsfininciinfcgaiiiifacfnnfiinaicictcfnat aai dntcndn oattdot s n dosdedteos- oo-ddessds-eeoeede-p-s-dpededepee-enedpneppdenedeeepndnennedneddtnne tee idntnnin nettiht n nh iiiinhtbnin bhiihihtbinitiiibiobihbtoiniiiitontbti iion oiooton fninof o g ofnogo lffgu lf uo gltggufatll alumtugumattltmaauaaamtmtmteaae-taam-iaetnitt-eneaeid-n-dt-iuieindnun-cdudicdenuecududdecc cdue neend cdodnoe c nocdnincioc ocoenicceccpioipeicctccpetieeioiptcpopnietontti,piio n,oo tn,nin o,, , n ,
1.0and5.0mg/kgcausedsignificantdose-dependentinhibitionofglutamate-inducednociception,
wwiwtiwthwwiht ihi2 witt2th1 hh12i. t7. 122h724.114 71%.2.%47.771 %44(4. %7(p%% p4 ( < p % (<( ( pp0 <p 0 .( < <0.0p<0 0.0 000<01..100 .0)00),100 0.,)140 11,4)00 )),04,.1 , 6. 044)647,.007 60%.4.%76.606 %77(.7 %6(p%% p7 ( < p % (<( ( pp0 <p 0 .( < <0.p<0 0 0 .0000<01..10 0.0)00),100 0,.)150 11,5)90 )),95,.1 , 4. 955)455,.995 49%.5.%54.449 %555(. %4(p%% p5 ( < p % (<( ( pp0 <p 0 .( < <0.0p<0 0.0 000<01..100 .0)00) 100a0. )10a1n1 )0na)) d 1 nadaa )ndn8 n8ad1 dd18n. 5. 18d8581.1115 1%.8.%15.551 %11(1. %5(p%% p1 ( < p % (<( ( pp0 <p 0 .( < <0.p<0 0 0 .000<001..1 00.0)00) 100o.0 )10o11 f)0o)f ) i 1 ofnoio )nfifh f n ho iiiinhbfinnb hiihihtbinitiiibioibhbtoiniiiitontbti iiona oioatns ninsa o saana ss s a s
ccoocmomcccompoopmcammpaorprapemperadaadepr rrdte eaetod dordt ec o t ctdoto oocon tonc ctocontroo ronctnonrlott,oltr rn,rrol oort,el rlerls,,o ,s epr rlprseee,ep esscrspcepetpticesevietpcvciectvetetliiycilvevvyt.leei ye.vl lly.yey ..l . y .
Molecules2016,21,1077 5of16
Molecules 2016, 21, 1077 5 of 15
with21.74%(p<0.001),40.67%(p<0.001),59.45%(p<0.001)and81.51%(p<0.001)ofinhibitionas
Molecules 2016, 21, 1077 5 of 15
comparedtocontrol,respectively.
150
150
(21.74)
s) 100 (2*1*.*74)
e (s) 100 *** (40.67)
Licking timLicking time ( 5500 (4**0**.*6*7) ((55**99*.*.4*4*55)) ((8*811*..5*511))
***
0
0 C 0.1 0.5 1 5
C 0.1 0.5 1 5
DMFP(mg/kg, i.p.)

DMFP(mg/kg, i.p.)

Figure 6. Effect of DMFP against glutamate-induced paw-licking test in mice. Each column represents
FFiigguurree 66.. EEffffeecctt ooff DDMMFFPP aaggaaiinnsstt gglluuttaammaattee--iinndduucceedd ppaaww--lliicckkiinngg tteesstt iinn mmiiccee.. EEaacchh ccoolluummnn rreepprreesseennttss
the mean ± S.E.M. of 6 mice. The mice were pre-treated with vehicle (C), or DMFP (0.1, 0.5, 1 and
tthhee mmeeaann ±± SS.E.E.M.M. .ooff 66 mmiiccee.. TThhee mmiiccee wweerree pprree--ttrreeaatteedd wwiitthh vveehhiiccllee ((CC)),, oorr DDMMFFPP ((00..11,, 00..55,, 11 aanndd
5 mg/kg, i.p.) 30 min before i.pl. injection of 20 μL of glutamate (10 μmol/paw). The asterisks denote
55 mmgg//kkgg, ,ii.p.p..)) 3300 mmiinn bbeeffoorree ii..ppll.. iinnjjeeccttiioonn ooff 2200 μµLL ooff gglluuttaammaattee ((1100 µμmmooll//ppaaww)).. TThhee aasstteerriisskkss ddeennoottee
the significance level *** p < 0.001, when compared with control group (one-way ANOVA followed
tthhee ssiiggnniifificcaannccee lleevveell ******p p< <0 0.0.00011,,w whheennc ocommpparaerdedw withithc ocnotnrotrlogl rgoruopu(po n(oen-we-awyaAy NAONVOAVfAol lfoowlloewdebdy
by Dunnett’s post-hoc test). Values in parentheses are percentage of inhibition.
bDyu Dnnuentnt’estpt’oss tp-ohsotc-htoecs tt)e.sVt)a. lVueasluiensp inar penarthenesthesesaerse apreer cpeenrtcaegnetaogfei nohf iibnihtiiobnit.ion.
2.1.6. Involvement of Opioid Receptor
22..11..66.. IInnvvoollvveemmeenntt ooff OOppiiooiidd RReecceeppttoorr
The pre-administration of non-selective opioid receptor antagonist naloxone (5 mg/kg, i.p.)
TThhee pprree--aaddmmiinniissttrraattiioonn ooff nnoonn--sseelleeccttiivvee ooppiiooiidd rreecceeppttoorr aannttaaggoonniisstt nnaallooxxoonnee ((55 mmgg//kkgg,, ii..pp..))
significantly antagonized the antinociceptive effect of morphine (5 mg/kg, i.p.) but was not able to
ssiiggnniiffiiccaannttllyy aannttaaggoonniizzeedd tthhee aannttiinnoocciciceepptitvivee eefffefecct toof fmmoorprphhininee (5(5 mmgg/k/gk, gi,.pi..)p .b)ubt uwtaws ansotn oabtlaeb tloe
antagonized the antinociceptive effect of DMFP (1 mg/kg, i.p.) in both the acetic acid-induced
atontaangtoangioznediz etdhet haenatinntoinciocceipcetipvtei veeffeefcfet cotfo fDDMMFPF P(1(1 mmgg/k/gk,g ,i.ip.p.). )inin bboothth tthhee aacceettiicc aacciidd--iinndduucceedd
abdominal-writhing test (Figure 7) and the hot-plate test (Figure 8). Administration of naloxone per
saaebb dddioodmm niionntaa lal--fwwferrciittth hbiionntggh t teteesssttt (s(F.F iigguurree7 7))a annddt htheeh hoot-tp-plaltaetete tsets(tF (iFgiugruer8e )8.)A. Admdminiinstirsatrtiaotnioonf onfa nloaxlooxnoenpee rpeser
sdei ddindo tnaoftf aecfftebcot tbhottehs ttes.sts.
ns ###
ns ###
150
150
gs (1.42)
hinngs100 (1.42)
Number of writmber of writhi 1550000 ((5*5*9*9.*9.*9*55)) ((6*65*5*.*8.*8*22)) ((8*84*4.1.*188)) ((4*4*44*.*7.*7*88))
u
N ***
0
0
C + + - - - -
C + + - - - -
DMFP - - + + - -
DMFP - - + + - -
NALX - + - + - +
NALX - + - + - +
MOR - - - - + +

MOR - - - - + +

FFiigguurree 7.7 E. ffeEctf foefc ttreoaftmtreenatt mweitnht owpiiothid oapntioagidonaisntt, angaolonxisotn,en oanlo txhoen aeceotinc atchide-iancdeuticceda caibdd-ionmdiuncaeld-
Figure 7. Effect of treatment with opioid antagonist, naloxone on the acetic acid-induced abdominal-
wabridthomingin taels-wt irnit hminicge.t eMsticien wmeicree. pMreic-terewaeterde pwreit-htr neaatleodxowniet h(NnAalLoxXo, n5e m(Ng/AkLgX, i,.p5.m) 1g5/ mkgin,i .bpe.f)o1r5e mthien
writhing test in mice. Mice were pre-treated with naloxone (NALX, 5 mg/kg, i.p.) 15 min before the
tbreeafotmreetnhte wtrietaht mveehnitcwlei t(hC,v e1h0 icmleL(/Ckg,,1 0i.pm),L D/kMgF,Pi.p ()0,.D1,M 0.F5P, 1(0.0.1, ,50.0.5 ,m1g.0/,k5g.,0 im.pg.)/, kogr ,mi.po.r)p,horinme o(rMpOhiRn,e
5t( rMmeaOgt/mRk,ge5n, itm. pwg.)i/.t hEk agvc,ehih. pcioc.)ll.eu Em(aCnc, h r1e0cpo mrleusLme/nkntgsr, etihp.per) em,s DeenaMtnsF ±tPh S e(.E0m..1Me, a.0 no.5f±, s1ixS.0 .mE, 5.iM.c0e. .m oTfghs/eik xagsm, tiei.rpcies.).k, sTo hdr eemnaoostrteep rshiisignknesi fd(iMceanOnocRtee,
5 mg/kg, i.p.). Each column represents the mean ± S.E.M. of six mice. The asterisks denote significance
lseivgenlsifi *c*a* npc e< l0e.v0e0l1s, *w**hepn< co0m.00p1a,rewdh wenithc ovmehpiacrlee dcownittrhol vgerhoiucple; c#o##n tpr o<l 0g.r0o0u1p, ;w#h#e#n pco<m0p.0a0r1ed, wwhitehn
levels *** p < 0.001, when compared with vehicle control group; ### p < 0.001, when compared with
mcoomrpphairneed (wMiOthRm)-torrepahteinde g(rMouOpR; )n-tsr edaetneodtegsr onuop s;ingsnidfiecnaontcees lneovesli gwnhifiecna cnocmelpeavreeldw wheitnh cDoMmpFPar-etrdeawteitdh
morphine (MOR)-treated group; ns denotes no significance level when compared with DMFP-treated
gDroMuFpP (-otrneea-twedayg rAoNupO(VoAne f-owllaoywAedN ObyV DAufonlnloetwt’esd pobsyt-Dhoucn tnesett.t)’ spost-hoctest.)
group (one-way ANOVA followed by Dunnett’s post-hoc test.)
Molecules 2016, 21, 1077 6 of 15
Molecules2016,21,1077 6of16
MMoolleeccuulleess 22001166,, 2211,, 11007777 Molecules 2016, 21, 1077 66 ooff 1155 4 of 15
20 ###
Molecules 2016, 21, 1077 Molecules 2016, 21, 1077 Molecules 20164, o21f ,1 150 77 4 of 15 4 of 15
2200 ####*##** ### 14
****** *** ### ### ***
Response latency (sec) 11102468 ** ****** ****Response latency (sec) 111**024**68** ******* ******Response latency (sec)Response latency (sec)*Response latency (sec)***111105051155055 ****** ********** ******##******#*#***********#**#**** ******##*******##***********#***#*******Response latency (sec)******##*****111**#*#******024***68***#***#****** ##**Response latency (sec)****###***********#**##*******#* 110268##******###************#***##*******#* ****** ** **** ************ *********** ************** **** ********
0
00 0 30 60 90 120 150 0 180
0 0 00 3300 Interv66a00l followin99g00 treatme11n220t00 (min) 115500 118800 0 30 60 90 120 150 180
0 30 60 90Figu1r2e0 8. Eff1e5c00t of t1r8e03a0tment6 0with o9p0ioidIInn tate1enr2rvv0taaall gffooollllonoww1isi5innt0gg, ttnrreeaaalttomm18xee0onnttn ((mem iinon))n the0 hot pl3a0te test6 i0n mice90. Resu1l2ts0 150 180
Interval following treatment (min)

FFFiaiigggrueuu rerreeex p888.r..e EEEsfsffffeefeedccct ttio n oof mfft rtteerraaeenatamt t±mme Snee.ntnEtwt. M wwit.hi iotthhof prooeippospiiioodoiinddasn aeatn nalagttaatoeggnnooicsnnytii, ss(ntts,,a) nnlooaafx ll6ooo xnxmooeinncoeeen .o oStnhnta e ttthhhiseeot ithchpaoolltt a s ptpiegllaantettieefsi tcttiaeensnsttcm eiinn iwc emma.siiRc cdeeee.s. nuRRoleettssseuuadlrl ttebssy
Interval following treatment (min) Int erval following treatment (min) Interval following treatment (min)
eaaxorrpeen reeee-xxswppsreareedyss ssiAneedNdm iOinneV a mmnAee± aafonnSl l±±.oE SwS.M..EEed...MMo bf.. yoor effD srrpueesosnppnnoosenenttssl’esea tllpeaaonttseectnny-hcc(oyysc F ) ((istsoeg)) fs uoot6r.ff e m*66 4pmmi.c <ieiEcc .e0efSf... e0 tSSac5tttt;aa it*stoii*tsfs*i ttc iDipacc alaM<lls iFss0giiP.gg0n n0niofi1iinff ciic ccatoaanhmnncecce peehaw wowrateaa dspss d lwddaeeetinentnho oot tettceeesodddtn bibtbnryyyo ml ice. Results are expressed in mean ± S.E.M. of
Figure 4. Effect of DMFP on the hot Fpilgauter et e4s.t Einff emcti coeoofo. ngnnRDereeeo-M--wswwuuFpaaalPy;tyy s # oAAA#an#NrNN e tpO OOh ee<VVVx ApA0hAr.o 0 efffto0soo ls1plllel,lolo odawwwwt eiheeen deddt enm bsbb ytceyy oai DnmDDn u uupm±nnna niSnrncee.eeeEFtdt.tt t.it ’M’Rg’swssu e p.pip srtooooeuhsssf tl tt 4t-t--shhh.h ooeoEacc crfg trefttereeee oscessstutptxt.. . ppo o***nr f pre ppseDs e<c<s< Mee l 0a00id.vFt..00 0eiP5i5nn5n ;;;gc o **y*m *n*a* *** (pe ts paphpp) n <ero<<o f 0±hp 006. o.0.rS0 0tim0.0a0 E1p1t1i.e cl cMca ceodoot..me mrmS outptpfpage aatas/rictrrse oeetidddimnc wawpwmloi iistittuhcihhgne cn.cdc ooioR fnnanietctttsr arroutoonhllllcte se waraes edxepnroetsesded b yin o mnee-awna y± AS.NE.OMV. Aof followed by
response latency (s) of 6 mice. Statisticraels psiognnsifei claatnecnec wy a(gggssrs)rr o adoooumuuefp nppe6;o; ; md#t##e#o##id#c#s# ee bppp . yw <S<< toi 0at00nht...0ie00os0-00tuw1i11tc,, a,a wnywwl a hsAhlhioegeeNnxnnnoO i cfnccoiVooecmm.amA nCpp pcfaoaoaernrlr ewleetodrddawor weslwwe sd(idpititeth ohhbnn t yothts) hhte,ee ee mdlg agg rotbroreDryoounpu uucophnppynirn n e(errees-eecwt) cect (eo’eiasviify vv ip6 niioAn n,gsm ggNt5-a i.hacOa0poepp pVcm.pp rStAorrgetoopsa /fpktptro.igri rsl*aiil,t aa otpiietctw. ee<pad le.d0d )rds.,urr0 i uumggb5gng;/yo //i* ccrfc*oipoo cpmmhma <inppnp c0ooeoe. u0uu +w1nnn ;ndd ad*as * a aal*dot ttp e xttt hno<hheo nee0te .e0 d0 1b cyo omnpea-wready w AiNthO cVonAtr fooll lgorwouepd; bCyo ntrol ( ),
Dunnett’s post-hoc test. * p < 0.05; ** p <D 0u.0n1n; *e*t*t’ ps p<o 0s.t0-h0o1c scss atao(aemmmmsteepe. *da,dd 5ropooe. s0<sdse e e+ 0 ww .w5w0i.i50titihtht; hmh* oco*ougo uup/tnkt t<n t grnn a0,o aal.ilo0.ll poog1x.xxr;o) o,oo*n D*unne*epeM .p..;C FC<CCoP oo0on .n(nn0trttt0rror1oool Dlllc,( ((o(5(um.n0 npme)a)))g,,,t,r tm/emm’ksdgo oo p,wrr roipDpp.siphtthhM-h.i)ihin n nFocaeeeconP (ndt ((((et DsrotM.l * ,,,g, F p5505rP .o...<001 0+u ;0 mmmpn.0;aggg 5Cl///o;k kox*k,gg *nog0 ,,tnp . ,r5ii eio.<.;.p p p(l 0 ..(.)).),,0, 1mmm; , oo1*5o)*,r..rr 00*ppp ;p+hh h iii<5nnn. 00eee . m0 +++50 gnn.n10/a aak clmllgoooo,mxxgx ioo./oppknnna.ge)eer., e id.p w.).i th control group; Control ( ),
DMFP ( , 0.1; , 0.5; 1.0; DM 5F.0P m (g/kg ,, 0i..p1.;) ((.( ,,, ,555 0...000.5 +++; 555...000 mmm ggg1//./k0kk;ggg ,,, iii...ppp...)) ),,,5 DDD.0MMM mFFFgPPP/ k(((g, i.,,,p D 555...).M000. mmmFPggg ///(kkkggg,,, iii.. .ppp, ...0))) .aa1ann;n ddd DDDMMM F,FF 0PPP. 5+++; nnnaaallloooxxx ooo1nnn.0eee; ((( ,, , 5555....0000 +++m 555g...000/ k mmmggg,g /i//k.kpkggg.,,) ,.ii .i.pp.p..)).).. .
2.2. Motor Performance Study 2.1.4. Capsaicin-Induced Paw-Licking Test
2.1.4. Capsaicin-Induced Paw-Lick2in.1g.4 T. eCsatp saicin-222I.n..222d... uMMMcoeoottdtooo rrrP PPPaeweerrrff-foooLrrrimmmckaaaninnncccgeee STSSttetuuusdddty yy 2.1.4. Capsaicin-Induced Paw-Licking Test
The administration of DMFP produced significant dose-dependent inhibition of the capsaicin-
2.2.1. Rota-Rod Test
The administration of DMFP prodTuhcee da dsmiginniifisctraantti odno soef- DdeMpeFnPd pernotd iuncheibdi tsiiognn iofifc tahneTt hcdaeop sasedai-nimdcdeiinupn-cei sentddra entnieotu nirn oohgfi ebDnitiMcio FnnPo o cpifc rteohpdetu icocanepd as ats iiacglilnn di-fi ocsaens,t adso csoem-dpepareendd teon tc oinnhtriobli tgioronu opf wthieth c aap hsiagihciens-t activity of
R22.o.22t..a11-..R RRooodttaaT--eRRsootdd TTeesstt
induced neurogenic nociception ati nadllu dcoesde ns,e ausr ocogmenpica rneTodch iteco e acpdotminotnirno ails tgt arralolt uidopon sw eosift, hDa saM choFiimPgnh dp(e5uas rctme eaddgc t/tnikoveg 7icu t9oyir..no p9ogt1.rf)e% o ndl ii(gcdp r no<nou o0cp.ti0 c cw0ea1piu)tt hsiooe ban sa heanirtyg va hesleldi gsd tano atis fcteithcsiaev, n aidttsy o ec soofeffme ocpft a5or.n0e d mt htoge /ckmogno (ttForoirg l ugrreo u5p). wAi tshim ai lhairg hinehsti baictotirvyit eyf foefc t was also
79.91% (p < 0.001) observed at the 7d9o.9s1e% o f( p5 .<0 0m.0g0/1k)cg oo o(bFrTTsTidehghhirueneev ra aeaeatdddi do5 mmma)n.ti ii nAnnothiifi sss setttmi rrrmdaaaiottctiiiliseooaoe nrnn a o isoonof f ffah5 Dsi.DD0bsMe MiMmtsoFsFFgrePPPy/d7k (9 e((g5d5.5f9 f um(1emmFr%cgiitgggn / //wu(gkkkpr gagg et<s h o ii i50eb...app)p. sl.0s. R..e))0)Aor o1vd dd t)seiai iidddod-mr b fonnnisoldooeora tr ttrc vt accceieapnaasdutuush assisawbzeeete i httaapaohennnirnneyyyy e d c,ssse oowiiifgggmsfhenenncpi iiicoftfifah iifcwcrc aeaa5aadncn.n0sht tt itmaeeeeolvfff sgfffetoeee/hdk ccce tttg6 o o9oc(.nnoFn4n i 4tgttt%hhhureeoe r( lepmm m g<5ooor )0t.ott o.ooAu0rrr0p 1s )i minihlaibr iitniohni boift ocaryp seafifceinct- iwndaus caelds on ociception.
observed for capsazepine, which achoibesveerdv e6d9 .f4o4r% ca (ppc scc<oa(oo Fz0oooie.rr0rgpddd0uii1iinnrnn)eea aai ,tn 9ttwiii)hooo.hi nnnbIin icotoo hicfoff o a nmmnmc hotiiriifcccae eceevsa teaap,ad sssds a6iaaaai9scssz.sis4sneeee4p-sss%isansseem ed(dddpu o (c<d4bdde 0usudume.r0r rrniigi0vnnno1/eggkgc)d i g ic tnttf,ehhh ohipee.reipt b icR.RoRia)tn opoioso.ttsit angaaa-- -nzorrreioofofp didcdcian atptteneees,st ssawltttiy chw wwirniechhh-dhieeen unnandcc uechccdcooioeem mmtdvhep ppnedaaa o trrr6cieeem9idcdd.4ee p4t ttoo%ootif o ttpt(nhhphe.e ee <r m c0ccoo.oa0nnnn0ttt1errr)nooo icllln e ghgg orirrbonooiu uuttiphppoen of capsaicin-induced nociception.
(((FFFRiiigoggutuuarrr-eeero 999d))).... IIInnn cccooonnntttrrraaasssttt,,, dddiiiaaazzzeeepppaaammm ((4(44 mmmggg//kk/ggk,,g ii,..ppi...p)) .ss)iiggsnnigiiffniiccifiaanncattllnyyt lrryeedd rueudcceeuddc ettdhheet httiiemmteeim ooeff ppoeefrrpmmeaarnmneeannncceeen oocnne ttohhnee
tRRhooe ttRaa--orrtooadd-r.. od. 100
100 100 100

(10.16)
80
Licking time (s) 468000 (10*.16) (3*1*.5*4) (4*4Licking time (s)*.5*0) 468000(79.91) (3*6*.4*(01)0*.1 6)manence (s)manence (s)permanence (s)111102802811(00030000268*10000*.5*4) (4*4*.5*0) (79.91) (3*6Licking time (s)*.4*0) 468000 Licking time (s) 246000(10*.16) (3*1*.5*4)* (4*4*.5*(03*)1*.5*4)(79.9(14*)4*.5*0)(3*6*.4*(07*)9*.9*1) (3*6*.4*0)
200 C 0.1 0.5 1 200*5** C CAP0.1 Time of perTime of perTime of 24624600000024000.5 1 *5** CAP 200 *****C**** 00.1 C 0.50.1 1 0.5 *5** 1 CAP5 CAP
0
C DMFP diazepam DMFP(mg/kg, i.p.)
00
DMFP(mg/kg, i.p.) DM FP(mgC/Ckg, i.p.) DDMMFFPP ddiiaazzeepp aamm DMFP(mg/kg, i.p.)
FiFgiugruere9 .9E. fEfeffcetcot foDf DMMFPFPa gaagianisntsot nonth tehRe oRtoat-aro-rdodtFe istgetu.strE.e aE c5ah. cEhcfo fcelouclmtu omnf nDre MrperpFePrsee asnegtnasti t nsh steth mcea mepasenaai±nci n±S- .SiEn.E.dM.uM.cie.n din p aw-licking test in mice. Each column represents
Figure 5. Effect of DMFP against capsaFiicginu-rien d5.u Ecfefde cpt aowf D-liMckFinPg a tgeasitn isnt mcaipcsea. iEcainc-hi ncdoulucmedn praepwFr-eilgisceuknrietns g5 . t Eesfftte hicnet mmofie cDaenM. E±Fa SPc. hEa .gcMaoil.n uosmft 6nc a mrpeispcaeri.ec Tsinehn-eitn smd iuccee wd epraew p-rleic-tkrienagte tde swt iitnh m veichei.c Elea (cCh, c1o0l ummLn/k rge,p i.rpe.s)e, nDtMs FP (0.1, 0.5,
mFFmiiiggciueuc.erre.eM M 99i.c.i ceEEeffw wffeeeeccrtrte e oo ppff rrDDeettMMrreeFFaaPtPtee dada ggwwaaiiiitnnthhss Dtt D ooMMnnF tFtPhhP ee(5 (RR.50.oo 0mttaamg--r/rgkoo/gddk, titg.eep,ss.itt)... p a EE.n)aadcac hdhn idcacoozdleluiupammazmennp (rra4eem.pp0 rrm(ee4ssg.e0e/nnkmtgtss,g itt/.hhpkee.)g .mm ,Sitee.apaatn.ni)s . t±±iSc SStaa.l.EE tai..snMMtai.lc. y aiisnnli s
the mean ± S.E.M. of 6 mice. The mice wtheer em peraen-t ±re Sa.tEe.dM w.a mmoinwtfhiia cc6a leev ysm.. e sMdMhiiseciiicteccewl.eee rT ma ww(hsCieeen,drr eeme1ed 0ppti ecrrmbreeeym ttLwr reo/eikenaanrgteteeee,d- d dwip. pbwwrae.yy)ii-t,t t hhoArDe nDDNMaetMMO-eFwdPFVF aP PwA(yt 0 ((h i.55tf1Aeho..0,0 l Nm 0 lvmmo.eOe5wghga, V//ienkckAd lgg±e , ,b S f(ii1yo.C..ppE. l0,lD... )o) M1a uaaw0nn.nn dmeoddnd f e5 Ld d6t /bmtii ka’amyszgzg eei,p/D cpkpoie.sguapa.t m,mn-.T )hin,.h o p((Dece44. t )..Mt0mt0 eo’ ssmmFritc P.pcgeg a o* //(w*kpsk0*tggs. e-1pa,h,r , zii oe<..0epcp p.p0.5.)t)r.i,.e.0en SSs0-etttt1 ar.a( etCtciia*ossAt*ttmei*iPccdpaap, llaw0 a<ar.1iennt7dh0aa .l lm0yvwy0ssemiii1htssho i cl l, ei .(pC.), 3100 mmiLn/ kbgef, oi.rpe. )i,n DtrMapFlaPn (t0a.r1 (, i0.p.5l.,) injection of
1.0 and 5 mg/kg, i.p.) or capsazepine (C1A.0P a, n0.d1 75 mmmg/oklg, ,i .ip.cpww.o)c. a)mao3 sson0 p r dtdm arceeoratitelneep dr(r sbCmmawe)ziif ninegotepehrrddeoinc u iobbenpn yyt(.r Ct aoroAopnnllePea(--,Cnww 0t)a.aa1gyyr7 r ( AoAim.upNNmpl.OO).o iVVln, AAjie.p c 1ff.t.oo)i0 oll3 llano0on w womdfee i5ddn m bbbyegycf /oDakDrpguue,s n niain.nnipctee.rit)tnat to’’p ssr(l 1 appc.nooa6sstp tatμ--srhhag o(oz/iccp.e ptptaeelwi.ss)nt ti,.e. n *2*(j**Ce0**c Atpμpi PoL<<n, ) 0.00 o...T001f00 h711 em ccamoosmmtoelppr, iaais.rrkpees.dd) d3ww0e inimttohhit ne bthefeo rseig innitfriacpalnacnet alre v(ie.plsl .)* inp je<c t0i.o0n5 ,o *f* * p < 0.001,
capsaicin (1.6 μg/paw, 20 μL). The asctaeprissakisc idn e(n1o.6t eμ tgh/ep aswig,n 2if0ic μanLc)e. Tlehvee lass t*e rpi s<k s0 .d05e,n o**t*e pt h<ce a 0sp.i0sga0ni1ci,fi inc a(n1c.w6e hμleegnv/ epclaosw m* ,p p2a 0<r e μ0d.L 0w)5., i tT*h*h *ce o pan s<tt re0or.li0 sg0kr1so, udpen (ootnee -twhea ysi gAnNifOicVanAc ef ollelvoewlse d* bp y< D0u.0n5n, e*t*t*’ sp p <o s0t-.h0o0c1 ,t est). Values
ccoonnttrrooll ((CC)) ggrroouupp..
when compared with control group (ownhee-wn acyo mApNaOreVdA w fiothll ocwonetdr obl yg rDouunpn (eotnt’es- pwoasty- hAocN tOesVt)A. wV fhoalellunoe wcso emd pbayinr eD pdua wnrenintehttht ’cesos epnsot rsaotr-lhe g opcr eotruecspet n)(.to aVngaeel- uwoefas iy n hAiNbiOtioVnA. followed by Dunnett’s post-hoc test). Values
2.3. Toxicity Study
in parentheses are percentage of inhibiinti opna.r enthese22s..33 a..r TTe oopxxeiirccciiettyny tSSatgtuued doyyf inhibition. in parentheses are percentage of inhibition.
2.1.5. Glutamate-Induced Paw-Licking Test
2.3.1. Preliminary Acute Toxicity Study
2.1.5. Glutamate-Induced Paw-Lick2.i1n.g5 .T Gelsut tamate-Induced Paw-Licking Test 2.1.5. Glutamate-Induced Paw-Licking Test
22..33..11.. PPrreelliimmiinnaarryy AAccuuttee TTooxxiicciittyy SSttuuddyy The results presented in Figure 6 show that the i.p. administration of DMFP at doses of 0.1, 0.5,
There were neither behavioral abnormalities nor mortality observed during 7 days period of
The results presented in Figure 6 sThhoew r ethsuatl ttsh per ie.pse. natdemd iinni sFtirgautiroen 6 o sfh DowM FthPa at tt hdeo si.epsT. hoafed 0mr.e11is,n.u 00il s.ta5tsrn, apdtri eo5sn.0e n omtfe Dgd/M kingF FPcia gauuts rdeedo 6s se sisgh onofiw f0i c.t1ah,n a0tt . 5dth,o es ei.-pd.e apdemndineinstt rianthioibni toiof nD Mof FgPl uatta dmoastees- ionfd 0u.1ce, d0. 5n,o ciception,
obseTTrhhveearrteieo nww eeinrree t hnneee iitttrhheeearrt ebbdee hhmaaivvciieoo.r rGaallr oaasbbsnn omorrommrapallhiittoiieelossg nnicooarrl mmoboosrrettaravlliiattyyti ooonbb ssoeefrr vsvteeoddm ddauuchrriisnn aggn 77d ddoaathyyess r pp oeerrrgiiooaddn s oo off f
1.0 and 5.0 mg/kg caused significa1n.0t adnodse 5-d.0e pmegn/dkegn ct aiunsheidb itsiiognn iofifc agnlut tdamosaet-ed-einpdeundc1e.e0dn tan niondch i5cibe.0ipwt itmoiiotnghn / o,k2 fg1 g.c7la4uu%tas em(dpa <tse i0g-.in0ni0df1iuc),ca en4d0t .d6n7oo%scei c-(deppe <pt ie0on.n0d,0 e1n),t 5i9n.h4i5b%it i(opn < o 0f. 0g0lu1)t aamndat e8-1i.n5d1%uc e(pd <n 0o.c0i0c1ep) toifo nin, hibition as
ooabbnssieemrrvvaaaltstii oodnnid ii nnn ottthh ieen ttdrrieecaaattteeedd a mnmyii ccheee.. mGGorroorrsshss ammgioocrr lppehhsiooollnoosgg,ii ccuaallcl eoorbb ossere rravvnaaytti iooontnh oeorff asstbtoonmmoraamcchhassl iatainnedsd. ootthheerr oorrggaannss ooff
with 21.74% (p < 0.001), 40.67% (p w< i0t.h0 0211).,7 549%.4 (5p% < (0p. 0<0 01.)0, 0410). 6a7n%d (8p1 <.5 10%.00 (1p) ,< 5 09..04051%)w o(ipft hi<n 20h1.i0b.70i41ti%)oc oan (nm pad ps< a8 0r1.e.05d01 1t%)o, c(4po0 n.<6t 7r0o%.0l, 0 (r1pe) s <op 0fe .ci0nt0ihv1ie)b,l iy5ti.9 o.4n5 a%s (p < 0.001) and 81.51% (p < 0.001) of inhibition as
aanniimmaallss ddiidd nnoott iinnddiiccaattee aannyy hheemmoorrrrhhaaggiicc lleessiioonnss,, uullcceerr oorr aannyy ootthheerr aabbnnoorrmmaalliittiieess..
compared to control, respectively. compared to control, respectively. compared to control, respectively.
Molecules2016,21,1077 7of16
2.3. ToxicityStudy
PreliminaryAcuteToxicityStudy
There were neither behavioral abnormalities nor mortality observed during 7 days period of
observationinthetreatedmice. Grossmorphologicalobservationofstomachsandotherorgansof
animalsdidnotindicateanyhemorrhagiclesions,ulceroranyotherabnormalities.
3. Discussion
The present study established the antinociceptive effects produced by the intraperitoneal
administration of a novel synthetic diarylpropanoid analogue, 3-(2,3-dimethoxyphenyl)-1-
(5-methylfuran-2-yl)prop-2-en-1-one(DMFP)inmousemodelsofinducednociceptionandexplored
potentialmechanismsofaction. ItwasdemonstratedforthefirsttimethattheadministrationofDMFP
producedsignificantperipheralandcentralantinociceptiveactivitywhentestedindifferentinvivo
chemicalsandthermalexperimentalmodelsofinducednociception,namelyaceticacid-,formalin-,
capsaicin-,glutamate-inducednociceptivetestsandthehot-platetest.
Theaceticacid-inducedabdominal-writhingtestisconsideredasaconventionalanimalmodel
ofpainandoneofthemostsensitivemethodsusedforthescreeningofsubstancesthathaveboth
analgesicoriginand/oranti-inflammatoryeffects[21,22]. Itwassuggestedthattheintroductionof
aceticacidintotheperitonealcavityinducesnociceptionbydirectlyactivatingnon-selectivecationic
channelslocatedintheprimaryafferentpathwaysorindirectlybypromotingthereleaseofvarious
endogenousalgesicmediatorssuchasprostaglandins,cytokines,bradykininandothers,aswellas
increasinglipoxygenase(LOX)andcyclooxygenase(COX)productioninperipheraltissues[23–26].
The release of these endogenous substances with subsequent stimulation and sensitization of the
peripheralprimaryafferentC-fibersneuronsintheanimalperitoneumproducedaviscerosomatic
reflexleadingtoabdominalconstrictions[23–25,27]. Thefindingsofthepresentstudyindicatethat
theadministration(i.p.) ofDMFPproducedasignificantdose-dependentreductionintheamountof
abdominalwrithinginducedbyaceticacidascomparedtothecontrolgroup. Thisfindingsuggests
thattheantinociceptiveactionofDMFPintheaceticacid-inducedabdominal-writhingtestcouldbe
theresultofinhibitedreleaseofendogenousalgesicmediatorsordirectinhibitoryactivityatnerve
endingsoftheprimaryafferentneuronsand/orinhibitionofthetransmissionpathwayenteringthe
dorsalhorn.
Althoughtheabdominal-writhingtesthasaverygoodsensitivityhoweveritisnon-specificin
nature, as in some cases its writhing response can be suppressed by muscle relaxants and other
types of drugs, which could lead to misinterpretation of the results. Furthermore, it does not
exactlydiscriminatetheinvolvementofacentralorperipheralmechanism[19,22]. Forthesereasons,
thepossibleinhibitoryeffectofDMFPwasevaluatedfurtherintheformalin-inducedpaw-licking
andhot-platetests,whicharemorespecifictestsfortheinvestigationofperipherallyandcentrally
mediatedantinociceptiveactivity[28,29].
The formalin-induced paw-licking test is a test model that produced two distinct phases of
nociceptiveresponses. Thefirstphase(neurogenic)whichreflectscentrallymediatedpain,isevoked
bydirectformalinstimulationoftheprimaryafferentC-fibers,resultfromthereleaseofsubstanceP,
serotonin,kininsand/CGRP.Incontrast,thesecondphase(inflammatory),whichreflectsperipherally
mediatedpain,ismainlyduetoeitherasubsequentinflammationreactionintheperipheraltissue
mediatedbythereleaseofvariousinflammatorymediatorssuchasprostaglandins,COXandNO,
the effect of sensitizing dorsal horn neurons of the spinal cord or a combination of both [29,30].
Thebiphasicnatureofthepainresponseinthistest,whichreflectsdifferentpathologicalprocesses,
canbeusedtoelucidatethemechanisminvolvedinanalgesia[29]. Inaddition,itiswellappreciated
that centrallyacting drugs, suchas opioids, inhibitboth phases ofpain, while peripherally acting
drugssuchasacetylsalicylicacidanddexamethasonethatinhibitCOXactivityactuallyinhibitonly
the second phase [18,28,30]. The results of the present study show that the i.p. administration of
Molecules2016,21,1077 8of16
DMFPsignificantlyanddose-dependentlyattenuatedthenociceptiveresponseinbothneurogenicand
inflammatoryphasesoftheformalin-inducedpaw-lickingtestinmice. Correspondingly,bothphases
ofnociceptiveresponseinducedbyformalininthepresentstudywasalsoinhibitedbythecentrally
actingdrug,morphine,whiletheperipherallyactingdrugs,ASA,onlyinhibitedthesecondphase
ofthetest.
Similarly, the central antinociceptive effect of DMFP strongly supports the results obtained
in the hot-plate test, which is a preferential method to screen centrally acting analgesic drugs.
Thiseffectisthoughttodemonstratetheinvolvementofcentralmechanisms(supraspinally)[22,31,32].
Itwasdemonstratedthati.p. administrationofDMFPexertssignificantprolongationinthelatency
response time to heat stimuli of the hot-plate test. Taking into account the inhibitory property of
theDMFPintheaceticacid-inducedabdominal-writhingtest,bothphasesoftheformalin-induced
paw-lickingtestandthehot-platetest,itisstronglysuggestedthatDMFPactingbothcentrallyand
peripherally,whichalsoimpliesthatitpossessesnotonlyantinociceptivebutalsoanti-inflammatory
activity. AsfarascentralanalgesiceffectofDMFPisconcerned,however,otherstudiesbymeansof
intracerebroventricularand/orintrathecalroutesofadministrationofDMFParenecessarytofurther
substantiatetheinvolvementofcentralnervoussysteminthecentralanalgesiceffectofDMFP.Another
limitationofthepresentstudyisthelackofinformationonbloodbrainbarrier(BBB)permeability
ofDMFP.SeveralstudieshavereportedtheabilityofchalconesanditsanaloguestocrosstheBBB
andhaveobservedthatitispossibleforsomechalconestodosoduetotheirlipophilicnatureand
theirspecificinteractionswiththeeffluxtransportersexpressedintheepithelialcellsthatmakeupthe
BBB[33,34]butforDMFP,itsabilitytocrosstheBBBisyettodetermine. Thisinformationiscrucial
inordertodeterminethepotentialdirectdrug-proteininteractionofDMFPwithdifferenttypesof
receptorsatthespinalorsupraspinallevelscord,thusascertaintheexactmechanismofitscentral
analgesiceffect.
Numerous reports have indicated the involvement of various endogenous systems in pain
control[25,35,36]. Therefore,inthepresentstudyweconductedpreliminaryexperimentstoexplore
the involvement of opioids, TRVP1 and glutamate receptors in DMFP-induced antinociception.
ThepossibleinvolvementofopioidreceptorsintheDMFP-inducedantinociceptionwasexploredina
separategroupofmiceusingtheaceticacid-inducedabdominal-writhingtestandthehot-platetest,
sincetheopioidreceptorsonperipheralterminalsofprimaryafferentfiberscanbethesitesofthe
intrinsicmodulationofnociception[37]. Inaddition,itwasreportedthatdrugswithactiononthese
receptorsmayexertedanalgesiceffectswithoutthepresenceofthecentraladverseeffectscausedby
opioids[38,39]. Numerousstudieshaveshownthatnaloxoneiscapableofantagonizingtheopioid
receptorsinanon-selectivemanner[27,40]. OurfindingsshowedthattheinhibitoryeffectofDMFPin
bothtestmodels,incontrasttothatofmorphine,wasnotantagonizedbypre-administrationwith
thenon-selectiveopioidreceptorantagonist, naloxone. Thelacksofresponsetonaloxoneexclude
theinvolvementopioidreceptorsinantinociceptiveofDMFP.Thus,theseresultsimplythatboththe
peripheralandcentralmechanismsofDMFP-inducedantinociceptionwereneithermediatedthrough
theactivationofopioidreceptorsnorthemodulationoftheeffectofendogenousopioidpeptides.
Notably,DMFPsignificantlyinhibitedthenociceptiveeffectevokedbycapsaicinandglutamate.
Capsaicinisapungentingredientofredchilipeppersthatactsdirectlytoactivatevanilloidreceptor
type-1(transientreceptorpotentialcationchannelV1orTRPV-1)localizedonnociceptiveafferent
C-fibers, in the dorsal root ganglion of the spinal cord, trigeminal ganglia and CNS [36,41,42].
The stimulation of TRPV1 receptors promotes vascular leakage and vasodilatation, culminating
in the production and release of various neurotransmitters, such as glutamate and substance P,
NOandpro-inflammatorymediatorsfromtheperipheralandcentralterminalsofprimaryafferent
neuronsthatcontributetonociceptivesignaltransmissionandprocessing[41,42]. Theresultsofthe
present study show that DMFP attenuates the neurogenic pain perception caused by capsaicin in
a dose-dependent fashion. Also, it was observed that capsazepine, a selective capsaicin receptor
antagonist, significantly antagonized the nociception induced by capsaicin. Thus, these findings
Molecules2016,21,1077 9of16
suggestthatDMFPpossiblyexertsitsanalgesiceffectviainhibitionofthenociceptivetransmission
initiatedbyTRPV1receptorsactivation.
Itiswellappreciatedthatglutamate,amajorexcitatoryaminoacidneurotransmitter,participates
intheprocessesinvolvedinnociceptivetransmission,developmentandmaintenanceofthenociceptive
responsethroughtheactivationofbothionotropic(iGluRs)andmetabotropicglutamatereceptors
(mGluRs),leadingtotheexcitationandsensitizationofperipheral,centralspinaland/orsupraspinal
nociceptors [43–45]. The iGluRs are nonselective ligand gated cation channels responsible for
fast synaptic transmission eliciting currents of Ca2+, Na+ or K+, whereas the mGluRs belong
to the large family of G-protein coupled receptors and are responsible for the neuromodulatory
activityofglutamatethroughtheformationofsecondmessengers,mainlyinositol1,4,5-triphosphate,
diacylglycerol and cyclic nucleotides. These receptors in different states and on different scales,
participate in induction, modulation or maintenance of pain [46,47]. Indeed, drugs capable of
inhibitingeitheriGluRsor/andmGluRsexhibitantinociceptiveeffects[45,48]. Itwasdemonstrated
that i.pl. injection of glutamate into the mouse hind paw results in an increased influx of calcium
withtheactivationofneuronalNOsynthaseandNOformation[49]. Theincreaseintheamountof
NO subsequently increases the release of pro-inflammatory mediators such as cytokines, reactive
oxygenspecies(ROS)andprostanoidsandactivatestheformationofcyclicGMP,whichenhancesthe
inflammatoryreactionandproducingnociceptivebehaviorsofrapidonsetandshortduration[49–51].
Consequently,inhibitionofthesereceptorsordecreasedglutamatereleaseatthelevelofperipheral,
spinal and/or supraspinal would lead to a decrease in pain perception. It was demonstrated in
thepresentstudythattheadministrationofDMFPproducessignificantdose-dependentinhibition
of thenociceptive response exertedby the i.pl. injection ofglutamate in mice. Hence, the present
resultsstronglysuggestthat,atleastinpart,theantinociceptiveactivityinducedbyDMFPduring
the glutamate test could be due to its interaction with the glutamatergic system and/or its ability
toinhibitNOproduction. However,thishypothesisshouldbeconfirmedbyotherexperimentson
themechanismofactionofDMFP,whicharecurrentlyinprogressinourlaboratory. Thisresultalso
substantiatestheresultsobservedinformalin-andcapsaicin-inducedpaw-lickingtests,asbothstudies
showthatthereisanincreasedlevelofglutamateaswellasNOduetothestimulationofafferent
C-fibersfollowinginjectionofformalinorcapsaicinintothepaw[22,30,42].
It is a matter of great concern in the investigation of analgesic action of compounds if
pharmacological administration of compounds causes other behavioral alterations, such as motor
incoordinationandsedation,whichmightbemisinterpretedasanalgesicactivity. Thereforeinorderto
avoidsuchmisinterpretation,abehavioralevaluationwasconductedtodetermineifDMFP-induced
antinociceptionwereinfluencedbyanydisturbancesonthecentralnervoussystemusingtheRota-rod
test. ItwasobservedthattreatmentswithDMFPdidnotaffectthemotoractivityofmice,thusexclude
possible false-positive results in analgesia or the influence of possible non-specific effects, such as
sedationormotordysfunction. Thisresultindicatedthatthebehavioralresponsesobservedinall
nociceptioninducedexperimentalmodelsinthecurrentstudywerenotduetobehavioralalterations,
butwasratherreflectedaparticularanalgesicpotentialofDMFP.
Finally,toconfirmthesafetyprofilesofDMFP,ourpreliminaryacutetoxicologicalanalysiswas
performedinanimalsthatreceivedsingleoraldosetreatmentofDMFP.Itwasdemonstratedafter
7daysofobservation,theanimalseventreatedwiththehighestdoseofDMFPupto1g/kg,didnot
presentanyalterationinoverallbehaviorandtherewasnooccurrenceofmortality. Therewerealsono
changesinthegrossmacroscopicobservationsofthestomachandothervitalorgans,suchtheheart,
lung,liver,kidneyandspleen. TheseresultsindicatethattreatmentwithDMFPdoesnotimpairorgan
functionasassessedinthisstudy,thuspresentinggoodsafetyconditionsandsuggestingtheefficacy
ofthetestedcompound.
Molecules2016,21,1077 10of16
4. MaterialsandMethods
4.1. GeneralInformation
AllthereagentsandsolventsusedwerepurchasedfromSigma-AldrichCo. (St. Louis,MO,USA)
andwereusedwithoutfurtherpurification. Themeltingpoints(mp.) weredeterminedonahot-stage
meltingpointapparatus,XSP-12500X(BibbyScientificLimited,Staffordshire,UK),andareuncorrected.
IR spectra were recorded on a Perkin–Elmer RXI Fourier transform (FT) IR spectrometer (Perkin
Elmer,Waltham,MA,USA)asKBrdisks. MassspectraweremeasuredonaFinniganMatSSQ710
spectrometer(FinniganMAT,SanJose,CA,USA)withionizationinducedbyelectronimpactat70eV.
Then1H-NMRspectrawererecordedinCDCl usingaVarian500-MHzNMRspectrometer(Varian
3
Associates,PaloAlto,CA,USA).Columnchromatographywasperformedonsilicagel60Merck9385
(230–400mesh,ASTM).Thinlayerchromatography(TLC)wasperformedonpre-coatedsilicaplates
(MerckKiesegel60F254,0.2mmthickness)sheets.
4.2. Synthesisof3-(2,3-Dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one(DMFP)
Thesyntheticdiarylpropanoidanalogue, DMPF(Figure1)waschemicallysynthesizedatthe
Institute of Bioscience, Universiti Putra Malaysia. Briefly, to a mixture of 2-acetyl-5-methylfuran
(1.0mmol)inethanol(15mL)wasaddedwithNaOH(1.5mmol,40%)andstirredfor10minutein
coldwater. Then,addedsubstituted2,5-dimethoxybenzaldehyde(1.0mmol)andstirredthereaction
mixtureatroomtemperaturefor24h. TheprogressofreactionwasmonitoredbyTLCandthereaction
mixturewaspouredovercrushediceandacidifiedwithaceticacid.Thecrudeproductsweredissolved
indistilledwaterandextractedwithethylacetate. TheyellowlayerofEAwaswashedwithwater
anddriedoversodiumsulfateanhydrous. Thecompoundwaspurifiedbycolumnchromatography
usingsilicagelmeshsize(100–200mesh,Merck)andelutionwithpetroleumetherandethylacetate.
Yield:82%;yellowcrystals;m.p. 132–134◦C.IR(CHCl )υ: 2937(C–Hstretch),1652(C=O),1600(C=C),
3
1513(C=C),1269(C–Oaromatic),1074,1001cm−1;1H-NMR(500MHz,CDCl ): δ2.44(s,3H,CH ),
3 3
3.89(s,6H,2×OCH ),6.22(d,J=3.0Hz,1H,H-4furanyl),6.97(d,J=8.0Hz,1H,H-4phenyl),7.11
3
(t,J=8.0Hzeach,1H,H-5phenyl),7.28(d,J=8.0Hz,1H,H-6phenyl),7.46(d,J=16Hz,1H,H-α),
7.24(d,J=3.0Hz,1H,H-3furanyl),8.15(d,J=16Hz,1H,H-β). EIMSm/z(rel. int.) calculatedfor
C H O (M+,%): 272[M+].
16 16 4
4.3. Animals
AdultmaleICRmiceweighing20–30gwereused. Allmicewerehousedandmaintainedinthe
AnimalUnit,FacultyofMedicineandHealthSciences,UniversitiPutraMalaysiaona12hlight/dark
cyclewithstandardcommercializedrodentdietandwateravailableadlibitum. Experimentalgroups
consisted of six randomly selected mice per group (n = 6), acclimatized for at least 1 h prior to
theexperimentandusedonlyoncethroughouttheexperiments. Allanimalcareandexperimental
protocolsconductedinthisstudywereapprovedbytheEthicalCommittee,FacultyofMedicineand
HealthSciences,UniversitiPutraMalaysia(ACUC_UPM⁄FPSK⁄PADS⁄BR-UUH⁄00330). Thenumber
ofanimalsandtheintensitiesofnoxiousstimuliintheexperimentalprotocolsusedweretheminimum,
just the amounts necessary to demonstrate consistent effects of the drug treatments. The animals
usedwereeuthanizedbycervicaldislocationimmediatelyaftercompletionoftheexperiment. Inall
experiments,datawerecollectedinablinded,randomizedandcontrolleddesign.
4.4. DrugsandChemicals
The following drugs and chemicals were used: morphine hydrochloride, acetylsalicylic acid
(ASA), diazepam, Tween 20, absolute ethanol (100%), acetic acid, formalin, capsaicin, capsazepin,
glutamateandnaloxonehydrochloride(Sigma-AldrichCo.). DMPFandASAweredissolvedina
vehiclecontainingabsoluteethanol(5%),Tween20(5%)andnormalsaline(0.9%NaCl,90%). Thefinal
concentrationsofethanolandTween20didnotexceed10%anddidnotcauseanyeffectperse. All