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J Food Biochem. 2019;e12912. wileyonlinelibrary.com/journal/jfbc
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https://doi.org/10.1111/jc.12912
© 2019 Wiley Periodicals, Inc.
Received:13December2018
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Revised:2 0March2019
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Accepted:4May2019
DOI : 10.1111/jfbc .12912
FULL ARTICLE
Effect of ethanol and aqueous extracts of seed pod of
Copaifera salikounda (Heckel) on complete Freund’s adjuvant‐
induced rheumatoid arthritis in rats
Chinyere Aloke1 | Udu Ama Ibiam2 | Nwogo Ajuka Obasi1 | Obasi Uche Orji2 |
Nkiru Nwamaka Ezeani2 | Patrick Maduabuchi Aja2 | Esther Ugo Alum2 |
Joseph Chukwufumnanya Mordi3
1DepartmentofMedicalBiochemistry,
FacultyofBasicMedicalSciences,Alex
EkwuemeFederalUniversity,Abakaliki,
Nigeria
2DepartmentofBiochemistr y,Facultyof
Sciences,EbonyiStateUniversity,Abakaliki,
Nigeria
3DepartmentofBiochemistr y,DeltaSt ate
University,Abraka,Nigeria
Correspondence:
ChinyereAloke,DepartmentofMedical
Biochemistr y,FacultyofBasicMedical
Sciences,AlexEkwuemeFederalUniversity,
Ndufu‐Alike,Ikwo,Abakaliki,EbonyiState,
Nigeria.
Email: alokec2002@yahoo.com
Abstract
Theantirheumatoidarthritispotentialofethanolandaqueousextractsofseedpod
of Copaifera salikounda(SPCS) was evaluatedusing thechickencollagen/complete
Freund'sadjuvant‐inducedarthriticrats model.Adjuvat‐inducedratsweretreated
with varied doses of the ex tracts (400, 600, and 800 mg/kg body weight) and
withreferencedrug,indomethacin for21days. Antiarthritic evaluation wasdone
throughmeasurementofbodyweight,pawsize,inflammatorymakers,hematologi‐
calparameters, cytokines,antioxidantenzymes,reduced glutathione,lipidperoxi‐
dation as wellas histopathological examinations.Treatment withthe ethanol and
aqueousextractsofSPCSmarkedlyinhibitedthepawsizeandcausedweightgain.
Theextractsconsiderablymodulated thehematologicalaswellasthe antioxidant
paramete rs. Likewise, the ext ract restored th e altered lipid peroxidati on, pro‐in‐
flammatorymediators,andinflammatoryfactorswhichfurtheraccentuatetheim‐
plication inadjuvant‐inducedarthritis. Thus, theethanoland aqueous extracts of
SPCS showedasignificantantiarthriticactivitythat was statistically analogousto
that of indomethacin.
Practical applications
Copaifera salikounda (Heckel) has been used in treatment of different ailments in‐
cluding rheumatoid arthritis in folklore medicine. This is the first reported proof of
theantiarthriticpotentialofthe seedpod. Oxidative stress hasbeen implicated in
rheumatoidarthritis.EthanolextractofSPCShasbeenshowntobepredominantly
richin phenols,terpenoids,alkaloids,andflavonoidswhicharenaturalantioxidant.
ThepresentstudyhasdemonstratedthatethanolandaqueousextractsofSPCScan
exertantioxidativeandantiinflammatoryeffects,thusstrengtheningitsantiarthritic
potentials.
KEYWORDS
antioxidant,Copaifera salikounda,Cytokines,indomethacin,lipidperoxidation
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ALOKE Et AL.
1 | INTRODUCTION
Rheumatoid arthritis is an autoimmune systemic disorder with an
unknown cause which is associated with swelling of membrane
liningthejointsanddamageofthejoints(Dhaouadietal.,2007).
The synovial stratum or synovium which is situated within the
joint cavity isthe first joint thatis affected(Smolen, Aletaha,&
Ma c h o ld,2005) .O therti s s uesaro u n dthejo i nt ssucha sskin,b l o o d
vessels,andmusclesareequallyaffectedbytheailment(Asolkar,
Kakkar,&Chakre,1992).Differenttherapeuticapproacheshave
beenemployedinthe treatment of RA. Nevertheless,sufferers
of rheumatoid arthritis still contend with the negative side effects
of synthetic steroidal and nonsteroidal antiinflammatory drugs
(NSAIDS) (M usa et al., 2012). World hea lth organizatio n report
equally assert s that long‐term usage of these drugs is accom‐
panied wi th toxic actio ns or undesir able side effe cts elici ted by
thedrugs(Sarwar,Suryakanta, Hameed,Abul,&Sarfaraz,2011).
Sequel to the negative effects and ineffectiveness of the cur‐
rently us ed orthodox d rugs, atten tion is geared towa rd the use
of herbal drugs which are used in folklore medicine because they
producelessersideeffectsandarecheaper(Dharamsiri,Jayakody,
Galhena,Liyanage,&Ratnasooriya,2003).Oneofsuchplantthat
iscommonlyemployedintreatmentofRAtraditionallyisCopaifera
salikounda but there are no documented evidences to substantial
thisclaim,hencethisstudy.
Copaifera salikounda (C. salikounda) belongs to the family
Caesalpiniaceae (Leguminosae or Fabaceae). Its pulped leaves are
usedinsoretreatment, coldextractoftheseed inmanagementof
vertigo , whereas the dr ied leaves and b ark mixed with b aked and
powdered clay are used in ulcer treatment (Oteng‐Amoako and
Obeng,2012).Theseed podsof Copaifera salikounda with or with‐
out the seeds are underutilized as food material in the cooking of
yam porr idge mostly duri ng famine perio d. In the same vein, t he
grounded seed is used to make sauce for eating of white yam by the
rural po pulace from Ugw ulangwu in Oh aozara Local G overnment
Area of Ebonyi State, Nigeria. In light of the all eged antiarthritic
potential s of the seed pod of t his plant, this s tudy is design ed to
investigatetheeffect ofethanoland aqueousextractsofseedpod
of Copaifera salikounda in chicken collagen/complete Freund's adju‐
vant‐induced rheumatoid arthritis.
2 | MATERIALS AND METHODS
2.1 | Plant material
The seed pods of C. salikounda (Heckel) (SPCS) were sourced
from a far m land located a t Umuigboke villa ge in Ohaozara Lo cal
Governm ent of Ebonyi State, Nige ria in the month of Novem ber.
Theplantwasidentifiedand madeauthentic byMr.AlfredOzioko,
a plant ta xonomist at the Inte rnational Cent re for Ethnomedi cine
and Drug Development Nsukka, Enugu State. A voucher speci‐
men has been deposited with the center (Voucher specimen no.
InterCEDD/16281). The picture of seed pod of C. salikounda is
shown in Figure 1 below.
2.2 | Preparation of extracts
The ethanol and aqueous extracts of SPCS were prepared by the
method describedby Oluduro andAderiye(2009).The driedseed
pods of C. salikounda obtained from the farm were washed with
clean water and further dried in the shade.Thereafter, they were
then pounded in a mortar using pes tle to get them into a fine p article.
The fine particle was passed through a sieve to obtain its powdered
form. Aqueous extract of SPCS was obtainedbysoaking500gof
thepowderedSPCSin200mlofdistilledwateratroomtemperature
for3daysandfilteredthereafter.Theethanolextractwasprepared
bymacerationof500gofpowdered SPCSin1,000mlofabsolute
ethanol for72hr.Then,theextractwasfiltered,concentrated,and
dried in vacuo. The percentage yield for the ethanol and aqueous
extractsofSPCSare6.16and8.35%,respectively.
FIGURE 1 Seedpodsof Copaifera salikounda with some seeds
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ALOKE Et A L.
2.3 | Chemicals and reagents
Chicken t ype 11 collagen, CFA, and ot her reagents use d for this
studywerepurchasedfromSigma‐Aldrich,USA,andwereofstand‐
ard grade.
2.4 | Experimental animals
Albino rats (Wister strain) of eithersex weighing 170–195g were
obtainedfromtheAnimalHouseofFacultyofVeterinaryMedicine,
University of Nigeria, Nsukka. Therats weresafelytransportedto
the Animal House of Department of Biochemistry Ebonyi State
University, Abakaliki and thereafter acclimatized for a period of2
weeks under standard laborator y conditions and fed with growers’
mash and water ad libitum. The Fac ulty of Basic Me dical Science
ResearchandEthicsCommitteeofAlexEkwuemeFederalUniversity,
Ndufu‐AlikeIkwo, Nigeria granted ethical approvalfor this animal
study with ethical code no: FBMS/EC/AE/1812. The procedures
agreewiththeU.S.NationalInstitutesofHealthGuidelinesforCare
andUseofLaboratoryAnimalsinBiomedicalResearch(2001).
2.5 | Quantitative phytochemical screening
Quantitativedeterminationofalkaloid,terpenoids,andglycosidesof
theextractswereconducted accordingtothe methoddescribed by
Harborne(1973).Theflavonoidcontentwasdeterminedusingprevi‐
ouslydescribedmethod(Bohnr&Kocipai,1994).Saponinconcentra‐
tionw asd ete rmin edu sin gear lie rdes crib edm etho d(S ofo wor a,199 3).
TotalphenolicwasdeterminedbymeansofFolin–Ciocalteu'sphenol
reagent (Singleton, Orthofer, & Lamuela‐Reventos, 1999). Tannin
content was determined using gravimetric method as described by
Makkar, Bluemmel,Borowy,and Becker(1993).The composition of
steroidwasdeterminedusingthemethodofAOAC(1999).Reducing
sugar and c arbohydrates were quantified using the method previously
describedbyEkwueme,Nwodo,Joshua,Nkwocha,andEluka(2015).
2.6 | Acute toxicity
WecarriedoutacutetoxicitystudyofSPCSethanolandaqueousex‐
tractsusingmodifiedmethodofLorke(1983).Atotalof68ratswere
used for th e acute toxicity s tudy. The rats were r andomized into
threegroups,1,2,and3,weighedandfastedovernight beforethe
acutetoxicitytesting.Group1(fourrats)servedasthecontrolgroup
andwasgivennormalsaline.Group2wasgiven ethanolextractof
SPCS,while group 3receivedaqueous extractof SPCS. Groups 2
and 3 were further subdivided into eight groups (four rats in each
subgroup). T he subgroups wer e orally given et hanol and aque ous
extractsofSPCSat200,400,800,1,200,1,800,2,000,3,000,and
5,000mg/kgbodyweight,respectively.Theexperimentalratswere
observedfor24hr.Further,theywerecontinuouslyobservedforthe
first2hrformorbidityandupto24hrformortality.
2.7 | Experimental design
2 . 7.1 | Induction of arthritis
RA was i nduced according to t he method previo usly described b y
Pearson (1956) Arthritis was induced intradermally by injection of
0.1‐mlchicken typeIIcollagen inCFA[heat‐killed Mycobacterium tu‐
berculosisandsterileparaffinoil(10mg/ml)]intothelefthindpawsof
rats ingroups 2–9accordingto theirbodyweights. Treatment with
etha nolan daque ousex t rac t sofSBCPsta rte dfromthe10thdayaf ter
arthritis induction and continued for 21 days. The treatment lasted
for3 weeks afterwhichthe animals wereeuthanized humanely via
cervicaldislocation.Thereafter,bloodsampleswerecollectedbycar‐
diac punc ture into sterile bot tles. The blood sample s were centrifuged
(3,000×gfor15min)andserumseparatedforbiochemicalanalyses.
2 . 7. 2 | Experimental groups
Atotal number of 135Wistar albino rats divided intonine groups
of 15 rats ea ch were used. T hey were groupe d as follows: Gro up
1servesas Normalcontroland wasgiven normal saline (5 ml/kg).
Group 2 was induced but not treated (Negative control) and re‐
ceived5ml/kg normalsaline.Group 3 (Positivecontrol) is arthritic
rats treatedwith indomethacin(10mg/kg)standard drug.Group 4
isarthriticrats +400mg/kgethanolextractofseedpodCopaifera
salikounda(EESPCS).Group5isarthriticrats+600mg/kgEESPCS.
Group 6isarthritic rats+800mg/kg EESPCS.Group 7isarthritic
rats+400mg/kgaqueousextractofseedpodCopaifera salikounda
(AESPCS).Group8isarthriticrats+600mg/kgAESPCS).Group9is
arthriticrats+800mg/kgAESPCS).
2.8 | Evaluation of physical parameters
Thechangesinpawsizeandbodyweightweremeasuredbeforein‐
ductionand on10th, 17th, 24th, and31st daysof thestudy using
digital Vernier caliper and weighing balance.
2.9 | Determination of inflammatory parameters
The seru m C‐reactive protein (CR P) level was carried out by the
methoddescribedbyVoilaetal.(1981).Rheumatoidfactor(RF)was
determined according to methoddescribed byJohnson and Faulk
(1976).Theactivityofadenosinedeaminase(ADA) was assayedby
the meth od elucidated by B ergmeyer (1983). Esti mation of ery th‐
rocy te sedimentat ion rate (ESR) wa s carried out ac cording to the
methoddescribedbyWestergreen(1957).S erumlevelsoftumorne‐
crosis factor‐α (TNF‐α),interleukin‐1beta (IL‐1β),andinterleukin‐6
(IL‐6)were quantitated using an ELISA‐based kit for rats following
manufacturer's instructions.
2.10 | Determination of hematological parameters
AnimprovedNeubauer's counting chamber was used for counting
RBC, wh ite blood cell (WB C) count, and plate let as describe d by
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ALOKE Et AL.
Baker and Si lverton (1982). Hemoglobin ( Hb) was determined ac‐
cordingtothemethoddescribedbyICSH(1996),while packedcell
volume(PCV)wasdeterminedbymethoddescribedbyICSH(1980).
2.11 | Assessment of oxidative stress markers and
antioxidant status
The concen tration of malo ndialdehyde (M DA) in serum was sp ec‐
trophotometrically estimated by measuring thiobarbituric acid reac‐
tivesubstance(TBARS)asdescribedbyBuegeandAust(1978),while
nitric oxide(NO) was measured according to method describedby
Tither adge (1998). The activit y of superoxide dism utase (SOD) in
serum was assayedby themethoddescribed byFridovichandMc‐
Cord(1969),whereastheassayfortheactivityofcatalase(CAT)was
donebythemethoddescribedbySinha(1972).
2.12 | Histopathological assessment of joint
The histopathological assessment of the interphalangeal joints was
carriedoutbyfixingtheminformolaceticacidsolutionwhichwere
later removed and cleaned with distilled water twice. The samples
wereimmersedfor2hrinethanolofdifferentgradedseries(30,50,
70,and 95%),and thereafterdehydrated in absolute ethanol over‐
night.Afterdehydration,thesampleswereclearedbyputtingthem
successivelyvia 3:2, 1:1, and1:3 volume/volumeofabsolute etha‐
nol/xyleneseriesandafterwardthroughpurexylenefor3hr.Each
ofthemwaskeptinmoltenwaxat50–60°Cfor48hrforinfiltration
ofthewaxintothe tissues.The specimens were placedinthe em‐
beddingmoldandmoltenwaxpouredonthem,andsetasidetocool.
Plas ticblockwasth enattachedtoth ewa xb loc kcont ain ingthesa m‐
ple and to a rocking microtome for sectioning. The cut sections were
laid on slides daubed with albumin of an egg, cleared with graded
ethanol–xylenesolutions,anddyedwithhematoxylinandeosin.The
slideswereoven‐driedandsubsequently,microscopicexaminations
were carried out using light microscope and photomicrograph.
2.13 | Statistical analysis
AllthedatawereexpressedasMean±Standarddeviation(SD)in
Tables and Figur es. One‐way ANOVA tes t was used for asse ss‐
ment of significant differences between means. A significance
threshold of p<0.05wasadoptedfortheanalyses.Datawere
analyzed using the IBM‐SPSS (version 20) statistical software
(IBM, Co rp., Atlanta , GA). Value of (p <0.05)wasviewedtobe
statistically significant.
3 | RESULTS
3.1 | Quantitative phytochemical screening of
EESPCS
The result of quantitative phytochemical screening of EESPCS is
shown in Table1.The results revealed thatEESPCScontainedthe
followingphytochemicals: tannins,steroids, terpenoids,total phe‐
nolics,alkaloids,flavonoids,glycosides,reducingsugarandcarbohy‐
drateswithtotalphenolics,alkaloids,andterpenoidspredominantly
more abundant.
3.2 | Acute toxicity testing
Theresultoftheacutetoxicitystudydemonstratedthattheextracts
werenottoxic.Therewasnodeathorobservabletoxiceffectinthe
ratsevenatahighdoseof5,000mg/kg.
3.3 | Effect of EESPCS and AESPCS on paw size and
weight of adjuvant‐induced arthritic rats
The data i n Figure 2a showe dt hat administ ration of EESP CS and
AESPCSatdosesof40 0,600,and80 0mg/kgsignificantly( p<0.05)
reduced paw swelling in dose dependent manner in comparison with
thecontrol.Similarobservationwasevidentinindomethacin‐treated
group.Theresultsofalterationsinbodyweightoftheexperimental
rats are shown in Figure 2b. The result revealed significant (p<0.05)
loss in bod y weight of induced a rthritic r ats. However, treatm ent
withEESPCSandAESPCSatdifferentconcentrationsinhibitedloss
in weight in the treated rats in comparison to the negative control.
Thus,thedeclineinweightwasrestoredinthetreatedrats.
3.4 | Effect of EESPCS and AESPCS on
inflammatory parameters of adjuvant‐induced
arthritic rats
The results of inflammatory parameters are depicted in Figures
3(a–d)and4(a–c).AsshowninFigure3(a–d),CRP,ESR,RF,andADA
levels were significantly elevated in the serum of ar thritic control
group relative to the normal control. Treatment with different doses
oftheextractmitigatedtheirconcentrationindose‐dependentman‐
ner.Likewise,theconcentrationsofthepro‐inflammatorymediators
(I L‐1β,IL‐6,TNF‐α)(Figure4a–c)wereattenuatedbytheextractad‐
ministration corollary to the negative control.
TABLE 1 PhytochemicalcompositionofEESPCS
Phytochemical constituents Value (mg/100g)
Tannins 12.57±0.15
Steroids 2.51±0.09
Terpenoids 189.4±10.9
Total phenolics 1,148.99±36.88
Alkaloids 1,04 4.70±28.90
Flavonoids 84.54±5.87
Glycosides 9.61±0.01
Reducing sugar 31.63±1.95
Carbohydrates 165.41±11.67
Note:Resultsaremeanoftriplicatedeterminations±standarddeviation
(SD).EESPCS:ethanolextractofseedpodof Copaifera salikounda.
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ALOKE Et A L.
3.5 | Effect of EESPCS and AESPCS on
hematological parameters of adjuvant‐induced
arthritic rats
Figure 5(a–e) show s the effect of t he extrac ts on RBC, PC V, Hb,
platelet count, and WBC. The resultsshowed that there was con‐
siderablesubsidenceinthelevelsRBC,PCV,andHbwhiletherewas
markedupsurgein plateletandWBCcountintheinducedarthritic
ratsrelativetothenormalcontrol.EESPCSandAESPCStreatments
conferred protection to the rats by helping to restore the altered
hematologicalparametersclosetonormal.Similarly,theeffectofin‐
domethacinwascomparabletotheeffectoftheextracts.
3.6 | Effect of EESPCS and AESPCS on oxidative
stress parameters of adjuvant‐induced arthritic rats
The resul ts are shown in Figur e 6(a–f). As expected, injec tion of
chicken collagen/complete Freund's adjuvant into the hind paw of
the rats markedly increased theserum level of MDA and NO and
decreasedtheactivitiesofCAT,SOD,GPx,andGSHrelativetothe
normal control. However, treatment with indomethacin and var‐
ieddosesoftheextractsat400,600,and800mg/kgbody weight
caused a significant (p<0.05)attenuationintheserumconcentra‐
tionofMDAandNO,whiletheactivitiesofcatalase,superoxidedis‐
mutase,and glutathioneperoxidase and reduced glutathione were
boosted.
3.7 | Effect of EESPCS and AESPCS on
HIstopathology of adjuvant‐induced arthritic rats
The histopathology observations of the normal control and ad‐
juvant‐induced arthritic rats are shown in Figure 7(a–e). A few
numbers of multiple synoviocytes and giant cells (yellow arrow)
without evidence of inflammation was observed in normal control
group(Figure7a).Therewasdestructionof epidermallayer,lossof
thickness,and hyperplasia of synoviocytes (blackarrow)inthe un‐
treated arthriticrats, i.e.,negative controlgroup (Figure7b).Quick
onset of stratum corneum and stratum granulosum regeneration
with intact collagen fibers and dif fuse synoviocytes at the epider‐
malregionwasobservedinindomethacin‐treatedgroup(Figure7c).
Regeneration of stratum corneum with stratum granulosum was
evidentinEESPCS‐treatedgroup(Figure7d).Formationofthethree
stratas(stratumcorneum,Sratumgranulosum, and stratum basale)
inAESPCS‐treatedgroupwasseen(Figure7e).
4 | DISCUSSION
This study has demonstrated that administration of ethanol and
aqueousextracts of seedpod of C. salikounda at different doses
exhibitedpotent antiarthriticpotential against adjuvant‐induced
rheumatoidarthritisinrats.Besides,theextractsreducedthein‐
flamedpawsizeandrestoredthelossinbodyweightofadjuvant‐
inducedar thr it icrats.Theextractsequallyi ncreasedtheactivities
of SOD, CAT, GPx, and G SH as well as decreas ed the levels of
MDA, NO, IL‐1β,IL‐6, and T NF‐α indicating the inhibition of in‐
flammatory reaction by reducing the generation of free radicals. In
thesamevein,treatmentwiththeextractssignificantlyincreased
thelevelsofHb,RBC,andPCV,whiletheWBCandplateletcount
were reduced in the ar thritic rats relative to the negative con‐
trol. The phytochemical analysis of the ethanol extract of C . sa‐
likounda revealed the presence of tannins, steroids, terpenoids,
totalphenolics, alkaloids, flavonoids, glycosides, reducingsugar,
and carbohydrate with total phenolics and alkaloids being the
FIGURE 2 EffectofEESPCSandAESPCSonpawsizeandbodyweightofadjuvant‐inducedarthriticrats.
EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda;Norm.C,normal
control;Neg.C,negativecontrol;Pos.C,positivecontrol;EE,ethanolextract;AE,aqueousextract
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ALOKE Et AL.
most pre dominant chem ical compone nt in the extr act. Many of
these secondar y metabolites from plant s have been discovered
andshowntodisplayantiarthriticpotentials(Clavinetal.,2007).
Althoughtheexac tmechanismofactionofantiarthriticactivityof
EESPCSandAESPCShasnotbeenelucidated,itissuggestedthat
these phytochemicals individually or in synergy could be responsi‐
ble for this particular pharmacological action.
Rat adjuvant‐induced ar thritis is the most frequently employed
anim almode lforassessingtheef f icac yofNSAIDSa nddis eas e‐mod‐
ifyingantirheumaticdrugs(DMARDS)insubclinicalstudiesanditis
proposed to be most suitable model for assessing drugs af fecting
human arthritis. The development of arthritis in rats using complete
Freund's adjuvant could be divided into three stages just as in human
rheumatoidarthritis,viz.: inductionstage without proofof synovi‐
tis, ear ly synovitis, and l ate synovitis and any e ffective antir heu‐
maticagentshouldbeabletooccludeanyofthesestages(Woode,
Boakye‐Gyasi,Danquah,Ansah,&Duwiejua,2009).
Alterationsinbodyweighthavebeenemployedinevaluatingthe
course of disease and the response to treatment of antiinflammatory
drugs (Winder et al., 2005). Inthe current study,the reductionin
thebodyweightofR A‐inducedratsmightbeduetothechangesin
their met abolic act ivities (Kore, S hete, & Desai, 2 011).Th is might
equally be attributed to reduction of absorption of 14C‐glucose and
14C‐leucinein the intestineof inflamed rats (Somasundaram et al.,
1983). Additionally, it has been suggested that administration of
complete Freund's adjuvant stimulate the production of increased
levelofleptin(acytokinehormone)whichfacilitatelossofappetite
andhence,weightloss(Mariam2016).Theincreasedbodyweightof
thearthritic ratsonadministrationoftheextractscouldbedueto
restoration of the decreased absorption capacity of intestine during
inflammationupontreatmentwiththeextracts.
The paw size was s uppressed by admi nistration of dif ferent
doses of th e extract s as well as indomet hacin in compar ison to
untreated control group. This suggests that the mechanism of
action of EESPCS and AESPCS might be linked to stoppage of
prostaglandin and histamine and may be leukotriene production.
These me tabolites p roduced thro ugh cyclooxy genase (COX) and
lipoxygenase (LOX) pathways represent two crucial classes of
FIGURE 3 (a–d)EffectofEESPCSandAESPCSoninflammatoryparametersofadjuvant‐inducedarthriticrats.
EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda;CRP,C‐reactive
protein;ESR,Erythrocytesedimentationrate;RF,Rheumatoidfactor;ADA,Adenosinedeaminase;Norm.C,normalcontrol;Neg.C,negative
control;Pos.C,positivecontrol;EE,ethanolextract;AE,aqueousextract
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inflammatory mediators (Sunita, Jha, & Pattanayak, 2011).It has
been reported that inflammation is the initial present ation of the
ailmentatthesiteofinjectionofCFAinthehindpawwhichculmi‐
nateinpawswelling(Prajapati,Shah,&Sen,2011).CRP isoneof
the acute phase proteins whose concentration rises during inflam‐
matory reactions (McConkey, Crockson, Crockson, & Nilkinson,
1973).Treatment withethanol andaqueous extracts of SPCS as
well as indomethacin considerably caused diminution in the con‐
centrationof CRP.Enhancedproduction ofendogenous proteins
like fibrinogen and α/βglobulincouldaccountforariseintheESR
and such an elevation suggests an active but obscure disease pro‐
cess(Patiletal.,2011).Inthesamevein,ESRexhibitsthecharac‐
teristic of displaying elevations in the amount in reaction to stress
or inflammations such as injury, injections, surgery, and tissue
death (Nai r, Singh, & Gup ta, 2012). The ext racts admini stration
abrogate d the elevated E SR close to norm al and attenu ated the
activityofADA.
Notable immune reaction disorders that may be relevant in de‐
velopmentofRA areimmunecomplexesthatareseeninjointfluid
cells and in vasculitis. Antibodies, for instance, IgM produced by
plasmacellsplayapartinformationofthesecomplexes.SerumRF
givesanindexofIgMtiterfoundintheserum(Prajapatietal.,2011).
RF is the immunological manifestation of one's immune response to
the presence of an antigen. This reaction to the antigen culminates
intheformationofimmunecomplexes;whichsuccessivelycombine
with the complement and may eventually result in damage of sy‐
novium, c artilage, and b one. The develop ment of inflammatio n is
directlyproportionaltotheamountofserumRF(Viswanathaetal.,
2011).Thus,investigationoftheamountofserumRFlevelsinrheu‐
matoid arthritis is important in elucidating and evaluating the dis‐
ease progression and to enhance the production of new therapeutic
agents for rheumatoid arthritistreatment.Serum RF is amarker of
systemic inflammation and antibody production against the injected
adjuvant . The increased levels of RF in the adjuvant‐induced arthritic
rats was due to activationofCD4+Tcellswhich stimulate Bcells
toproduceimmunoglobulins,that areassociated withelevationof
serum RF (Nielen, Horst‐Bruinsma, Koning, Habibuw, & Dijkmans,
2006;Yeh,2004).
FIGURE 4 (a–c)EffectofEESPCSandAESPCSoninflammatorycytokinesofadjuvant‐inducedarthriticrats.
EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda; IL‐1β,
interleukin‐1beta;IL‐6,interleukin‐6;TNF‐α,TumorNecrosisFactor‐alpha;Norm.C,normalcontrol;Neg.C,negativecontrol;Pos.C,positive
control,EE,ethanolextract;AE,aqueousextract
ϮϬϬ
ϮϮϬ
ϮϰϬ
ϮϲϬ
ϮϴϬ
ϯϬϬ
ϯϮϬ
ϯϰϬ
ϯϲϬ
ϯϴϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
/>Ͳϭɴ;ƉŐͬŵůͿ
d/D
(a)
(c)
(b)
ϮϬϬ
ϮϮϬ
ϮϰϬ
ϮϲϬ
ϮϴϬ
ϯϬϬ
ϯϮϬ
ϯϰϬ
ϯϲϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
/>Ͳϲ;ƉŐͬŵůͿ
d/D
ϭϳϬ
ϭϵϬ
ϮϭϬ
ϮϯϬ
ϮϱϬ
ϮϳϬ
ϮϵϬ
ϯϭϬ
ϯϯϬ
ϯϱϬ
ϯϳϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
dE&Ͳɲ;ƉŐͬŵůͿ
d/D
EŽƌŵ͘ EĞŐ͘ WŽƐ͘ ϰϬϬϲϬϬ
ϴϬϬϰϬϬϲϬϬϴϬϬ
8 of 13
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ALOKE Et AL.
TNF‐α a nd IL‐1 β have a significant function in the devel opment of
adjuvantarthritis,inadditiontomediatorslikeIL‐6,IL‐15,IL‐18,and
leukotrieneB4,withsomefunctioningincarryingneutrophilsduring
immune inflammation. TNF‐α is the principal agent in the process
ofinflammationanditisimplicatedin T and Bcells differentiation,
proliferationandinboneerosion(Rioja,Bush,Buckton,Dickson, &
Life,2004),whileIL‐1β is involved in damage of car tilage and bone
(Cuzzocreaetal.,2000).Ithasbeen reported thatTNF‐α blockage
reduces inflammationand alleviatecartilage damage(Cuzzocreaet
al.,2000).Besides,itfunc tionsinleukocyterecruitmenttotheartic‐
ulationsandequallyintheregulationofnitricoxidesynthase‐2and
cyclooxygenase‐2expressioninthesynovial tissueandcartilageof
arthriticrats(Amin,Attur,&Abramson,1999,Issekutzetal.,1994).
ThisprocessisremarkableasnitricoxideandprostaglandinE2,have
an important role in the development of the inflammatory process
(Fahmi, 2004). IL‐6has beenimplicated in jointdamage, leukocyte
recruitment,apoptosis,andT‐cellactivationusingmodelsofexper‐
imenta l arthritis i n subclinical t rials (Schelle r,Oh nesorge, & Rose‐
John,2006).
Anemia is often observed in subjects with severe arthritis
(Glen,Bowman,Ronloff,&Seely,1977).Thiscouldbeaccounted
for by the gastrointestinal blood loss due to rheumatoid ar thri‐
tis drugs and alteration in bone marrow in inflammatory ar thritis
subjects that hinders the liberation of iron for use in synthesis of
red bloo d cells (Allar, O'Dr iscoll, & Laur ie, 1977;Mo wat, 1971).
Bhujade,Talmate,andPatil(2015)suggestedthatdecreaseinHb
during ar thritis may be ascribed to reduced er ythropoiten level
or lowered response of the bone marrow erythropoiten and de‐
structionofredbloodcellsprematurely.Mostcasesofanemiain
rheumatoid arthritis subjects could be as a result of the depletion
FIGURE 5 (a–e)EffectofEESPCSandAESPCSonhematologicalparametersofadjuvant‐inducedarthriticrats.
EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda;RBC,Redblood
cell;PCV,Packedcellvolume;Hb,Hemoglobin;WBC,whitebloodcell;Norm.C,normalcontrol;Neg.C,negativecontrol;Pos.C,positive
control;EE,ethanolextract;AE,aqueousextract
ϭϴϬ
ϮϬϬ
ϮϮϬ
ϮϰϬ
ϮϲϬ
ϮϴϬ
ϯϬϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
Z;yϭϬϭϮͬ>Ϳ
d/D
(a)
(c)
(e)
(d)
(b)
ϭϱ
ϮϬ
Ϯϱ
ϯϬ
ϯϱ
ϰϬ
ϰϱ
ϱϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
Ws;йͿ
d/D
ϱ
ϭϬ
ϭϱ
ϮϬ
Ϯϱ
ϯϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
,ď;ŵŐͬĚůͿ
d/D
ϭϯϬ
ϭϰϬ
ϭϱϬ
ϭϲϬ
ϭϳϬ
ϭϴϬ
ϭϵϬ
ϮϬϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
WůĂƚĞůĞƚŽƵŶƚ;yϭϬϵͬ>Ϳ
d/D
ϭϬϬϬϬ
ϭϮϬϬϬ
ϭϰϬϬϬ
ϭϲϬϬϬ
ϭϴϬϬϬ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰ ĂLJϯϭ
tŽƵŶƚ;yϭϬ
ϵͬ>Ϳ
d/D
EŽƌŵ͘EĞŐ͘WŽƐ͘ϰϬϬ ϲϬϬ
ϴϬϬ ϰϬϬϲϬϬϴϬϬ
|
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ALOKE Et A L.
ofironthatisstoredinthebody(Punnonenetal.,2000).Ironde‐
ficienc y may also be caused by inflamed synovial tissue preferring
totakeupiron(Giordano,Floravanti,Sancasciani,Marcolongo,&
Borghi,1984). ThelevelofIL‐6is significantly higherinrheuma‐
toid arthritis patients that are anemic and hemoglobin levels have
inverserelationshipwithIL‐6(Nikolaisen,Figenschau,&Nossent,
20 08).IL‐6isimplicatedi nt heind uct ionofhepcidindur inginfl am‐
mation and hence brings about low iron level in blood (Nemeth et
al., 20 04). Hepcidin is a n iron regulator y hormone pr oduced by
the liver cells and inhibits the release of iron from macrophages in
thespleenandironuptakefromtheduodenum(Ganz,2003).Our
resultsrevealedthattheextractssignificantly(p<0.05)elevated
the levelsofHb and RBCin thetreatedarthritic rats relative to
the untreated ones. This can be attributed to the phytochemical
constituentoftheextract.Alowhematocritlevelisanindication
of low red blood cell count. There was a significant increase in
thePCVlevelsofthearthriticratstreatedwithindomethacinand
varie ddo sesoft hee xtrac t sat400,600,a nd8 0 0mg / kgbo dy.Th e
effectwasbothtime‐anddose‐dependent. However,treatment
with the ex tracts and indomet hacin boosted the PCV levels in
comparison to the negative control.
Previous authors have shown that platelets count and plate‐
let‐derived proteins are elevated within the synovium and synovial
fluid in RA (Gasparyan, Stavropoulos‐Kalinoglou, Sandoo, & Kitas,
2010).IL‐6indirectlymediatesthrombocytosis, throughthe induc‐
tionofthrombopoietin,andiscommontootherinflammatorycyto‐
kines(Pablos‐Álvarez,2009).Gasparyanetal.(2010)hasshownthat
pleiotropic cytokines of rheumatoid arthritis have megakaryotopoi‐
etic/thrombopoietic activities. Thrombocytosis is a characteristic
ofsharp and sluggish blood failure and in R A it might be ascribed
toanemiaseeninthedisease(MowatandHothersal,1968).Choie
etal.(1974)hasrevealedthatbothchronic anemiaandnormoblast
deprivation of iron are implicated in the thrombocytosis production.
The homeostasis of platelet is mediated by thrombopoietin (a hu‐
moralregulatoryfactor)thathaseffectonsize,number,ploidy,and
maturationrateofmegakaryocyte(Ebbe,1974).Itissuggestedthat
there is chemical correlation between thrombopoietin and erythro‐
poietin in chronically anemic subjects with persistent reticulocytosis
FIGURE 6 (a–f)EffectofEESPCSandAESPCSonoxidativestressmarkersandantioxidantstatusofadjuvant‐inducedarthriticrats.
EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda;CAT,catalase;
SOD,superoxidedismutase;GPx,Glutathioneperoxidase;GSH,reducedglutathione;MDA,Malondialdehyde;NO,Nitricoxide;Norm.C,
normalcontrol;Neg.C,negativecontrol;Pos.C,positivecontrol;EE,ethanolextract;AE,aqueousextract
Ϭ
Ϭ͘ϱ
ϭ
ϭ͘ϱ
Ϯ
Ϯ͘ϱ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰĂLJϯϭ
d;ƵͬŵŐͿ
d/D
(a)
(c)
(e) (f)
(d)
(b)
Ϯϱ
ϯϱ
ϰϱ
ϱϱ
ϲϱ
ϳϱ
ϴϱ
ϵϱ
ĂLJϭϬĂLJϭϳĂLJϮϰĂLJϯϭ
^K;ƵͬŵŐͿ
d/D
ϯϬ
ϯϱ
ϰϬ
ϰϱ
ϱϬ
ϱϱ
ϲϬ
ϲϱ
ϳϬ
ϳϱ
ĂLJϭϬ ĂLJϭϳ ĂLJϮϰĂLJϯϭ
'Wdž;ƵͬůͿ
d/D
ϱ
ϭϬ
ϭϱ
ϮϬ
Ϯϱ
ϯϬ
ϯϱ
ĂLJϭϬĂLJϭϳĂLJϮϰĂLJϯϭ
'^,;hŵŽůͬůͿ
d/D
ϭϬ
ϮϬ
ϯϬ
ϰϬ
ϱϬ
ĂLJϭϬĂLJϭϳĂLJϮϰĂLJϯϭ
EK;ŶŵŽůͬŵůͿ
d/D
EŽƌŵ͘ EĞŐ͘ WŽƐ͘ ϰϬϬϲϬϬ
ϴϬϬϰϬϬϲϬϬϴϬϬ
Ϯ
ϯ
ϰ
ϱ
ϲ
ϳ
ĂLJϭϬ ĂLJϭϳĂLJϮϰĂLJϯϭ
DĞƋƵŝǀĂůĞŶƚͿ
d/D
10 of 13
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ALOKE Et AL.
and elevated ery thropoietin levels with increased platelet counts
(Hutchins on et al., 1976).E xtract s administ ration to the ad juvant‐
induced rats mitigated the platelet counts relative to the untreated
ones.
TheincreaseintotalWBCcountinarthriticratsmaybeattributed
to immune system stimulation against foreign microorganisms as
demonstrated by the infiltration of inflammatory mononuclear
cells in the a rthriti c rats’ joint (Ma ria, Engeniusz , Miroslaw, Maria,
&Iwona, 1983). The WBC of the arthriticrats treated with varied
dosesofethanolandaqueousextractsofseedpodofC. salikounda
was reduced significantly ( p<0.05)relativetotheuntreatedones.
Treatmentwithethanol and aqueousextracts ofseedpodofC. sa‐
likoundarestored thelevels of Hb, RBC, andWBCwhich mightbe
attributed to inhibition of migration of leucocytes into the site of in‐
flammationbytheextract(Suralkar,Jadhav,Gupta,&Bhoite,2015)
suggesting that these extracts may have antiarthritic potentials
which are perhaps mediated by immune‐suppressant mechanism.
Withinthebody,reactiveoxygenspecies(ROS)/reactivenitro‐
genspecies(RNS)/freeradicalarefrequentlyproducedasaresultof
exposureofthe bodytochemicals generated internallyand exter‐
nally. There is normally a balance between these free radic als and
antioxidants as the generation of ROS/RNS is stabled by endoge‐
nousantioxidants. Anyimbalancebetweenthegenerationof ROS/
RNSspecies anddeactivationprocessculminates tocellularabnor‐
mality anduntowarddisease conditionsincludingRA (Ali, Barakat,
& Hassan, 2015). The ROS/RNS species bring about removal of
FIGURE 7 (a–e)EffectofEESPCSandAESPCS(800mg/kg)onhistopathologyofjointsofadjuvant‐inducedarthriticrats.H&E.Mag.
×100.EESPCS,ethanolextractofseedpodof Copaifera salikounda;AESPCS,aqueousextractofseedpodof Copaifera salikounda;A,normal
control;B,negativecontrol;C,Positivecontrol;D,TreatedwithEESPCS;E,TreatedwithAESPCS.Yellowarrow,multiplesynoviocytesand
giantcellswithoutevidenceofinflammation(Figure7a),blackarrowin7B:destructionofepidermallayer,lossofthicknessandhyperplasia
of synoviocytes. Quick onset of stratum corneum and stratum granulosum regeneration with intact collagen fibers and diffuse synoviocytes
attheepidermalregioninindomethacin‐treatedgroup(Figure7c).Regenerationofstratumcorneumwithstratumgranulosumevident
inEESPCS‐treatedgroup(Figure7d).Formationofstratumcorneum,Sratumgranulosum,andstratumbasaleinAESPCS‐treatedgroup
(Figure7e)
D E
F
H
G
|
11 of 13
ALOKE Et A L.
synovial fluid and facilitate the degradation of hyaluronic acid which
leads to loss of viscosity injoints(Arnett et al., 1988). Thehuman
system has an effective way of avoiding and mopping up free radical
usingitsendogenousantioxidantmechanism.
TheresultsinFigure7(a‐e)showedsignif icantelevatio nsinthe
activitiesofSOD,CAT,GPx,andGSHanddecline intheconcen‐
tration of MDAinthetreatedarthriticratswhencomparedwith
the negat ive control. Thi s indicates tha tE ESPCS‐ and A ESPCS‐
treated arthritic rats were effectively shielded from deleterious
effectoffreeradicals.Thephytochemicalconstituentsoftheex‐
tractsmighthaveplayedacontributoryantioxidantfunctioninad‐
ditiontotheendogenousantioxidantdefensemechanism,hence
curbingtheoxidativestressinducedintherats.Previousauthors
have reported the involvement of oxidative stress inthe patho‐
genesis of R A partic ularly in proli feration of syn ovitis (Maurice ,
Nakamura,&vanderVoort, E.A., vanVliet,A., Staal,F.J.,Tak,P.P.,
Breedveld, F.C., & Verweij, C.L., 1997; Tak, Zvaifler, Green, &
Firestein, 2000). Pro‐inflammatory mediators such as cy tokines
andprostaglandins, in addition toreactiveoxygenspecies(ROS)
andnitricoxide (NO)areliberated at inflammatory sitesinmost
joint disorders (Sakurai et al., 1995). These mediators are re‐
latedtodiminutionof SOD activityduringinflammation(Afonso,
Champy,Mitrovic,Collin,&Lomri,20 07 ).Thismayaccountforthe
continueddeclineintheactivityofSODinnegativecontrolgroup
relat ivetoEESP C S‐a ndA E SPCS‐tr eate dar t hrit icr ats. SODacti on
isanessentialintegral partofthecellularantioxidant mechanism
thatshieldcellsandtheextracellularmatrixfromthedeleterious
consequencesofsuperoxideanion(
O−
2
)anditsderivativeslikehy‐
droxyl radical(OH−) (Afonso et al., 2007). Superoxidedismutase
transforms
O−
2
toH2O2(Afonsoetal.,2007).CATmakestheH2O2
harmlessbytransformingitintowaterandoxygen.Inthiscurrent
study, the increase inserumactivity of CATactivity mightsafe‐
guard against reactive oxygen species‐mediated inflammation in
EESPCS‐ and AESPCS‐treated groups, while the diminution of
serumCATactivityinnegativecontrolgroupwouldinevitablyac‐
centuate the oxidativestressin tissues. This stemsfromthefact
thattheH2O2producedwouldbetransformedintoHOCl,anin‐
flammatory factor which is capable of reacting with
O−
2
to produce
OH‐thatmayreactandcausecellinjuryinlivingcells(Hitchon&
Ei‐Gabalawy,2004).Ithas earlier beenreported that GSHcould
functionasasulfhydrylbuffertosafeguardthethio(–SH)groups
ofbiomolecules,particularly,proteinfromthe harmfuleffectsof
ROS (Singh, Rajasekar, Raj, & Paramaguru, 2011). The reduction
in serum concentration of MDAin EESPCS and AESPCSas well
as indomethacin‐treated groups equally demonstrated that the
extracts could have protective potential against harmful effect
ofROS mediatedlipid peroxidation in tissuebiomolecules(Andy,
Gabriel,&Onyebuguwa,2004).
Theresult ofhistopathologicalexaminationswasin conformity
with the result of biochemical findings as there was restoration of
the rats’ joint architecture near normal control in the adjuvant‐in‐
ducedratsonwithtreatmentwiththeextracts.
5 | CONCLUSION
Inviewoftheresultsofthisstudy,itisinferredthebothEESPCSand
AESPCSattenuatedadjuvant‐inducedrheumatoidarthritisinratsby
modulating inflammatory mediators, hematological, and oxidative
stress parameters. This could be attributed to the phy tochemicals
therein.
ACKNOWLEDGMENT
WeexpressourprofoundgratitudetoMrAlfredOziokoforhelping
usin theidentification and authenticationoftheplant.Weequally
appreciate Dr. C.E.C.C. Ejike and Dr. I.I. Ekpe for their insightful com‐
ments and contributions.
CONFLICT OF INTERESTS
We wish to confir m that there are n o known conflic ts of interes t
associated with this publication and there has been no significant fi‐
nancial support for this work that could have influenced its outcome.
ORCID
Chinyere Aloke https://orcid.org/0000‐0002‐8929‐6754
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How to cite this article:AlokeC,IbiamUA,ObasiNA ,etal.
Effectofethanolandaqueousextractsofseedpodof
Copaifera salikounda(Heckel)oncompleteFreund’sadjuvant‐
induced rheumatoid arthritis in rats. J Food Biochem.
2019;e12912. https ://doi.org/10.1111/jfbc.12912
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