The Root Aqueous Extract of Entada africana Guill. et Perr.(Mimosaceae) Inhibits Implant Growth ...

16 Pages·2017·4.36 MB·English

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Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 8563909, 15 pages
https://doi.org/10.1155/2017/8563909
Research Article
Entada africana
The Root Aqueous Extract of
Guill. et Perr. (Mimosaceae) Inhibits Implant Growth,
Alleviates Dysmenorrhea, and Restores Ovarian Dynamic in
a Rat Model of Endometriosis
MarieAlfredeMvondo,1StéphaneMinkoEssono,1FrancisDésiréBombaTatsinkou,1
SylvinBenjaminAteba,2andDieudonnéNjamen2
1LaboratoryofAnimalPhysiologyandPhytopharmacology,DepartmentofAnimalBiology,FacultyofScience,UniversityofDschang,
P.O.Box67,Dschang,Cameroon
2LaboratoryofAnimalPhysiology,DepartmentofAnimalBiologyandPhysiology,FacultyofScience,UniversityofYaoundeI,
P.O.Box812,Yaounde,Cameroon
CorrespondenceshouldbeaddressedtoMarieAlfredeMvondo;[email protected]
Received 20 October 2017; Revised 24 November 2017; Accepted 11 December 2017; Published 31 December 2017
AcademicEditor:JairoKennupBastos
Copyright©2017MarieAlfredeMvondoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Entadaafricana(Mimosaceae)wasreportedtohaveanalgesicandantioxidantproperties.Thepresentstudyisaimedatinvestigating
theeffectsoftherootaqueousextractofEntadaafricana(EA)onanexperimentalmodelofendometriosis.Thestudywasperformed
inratsorallytreatedwithEAatdosesof127.5,255,and510mg/kg.Microgynon(cid:2)30servedasthereferencesubstance.Estradiol
valerateandoxytocinwereusedtoinducedysmenorrhea.Endometrialimplantlevelsofcatalaseandmalondialdehyde(MDA)
allowedestimatingtissueoxidativestatus.Ovariandynamicandratsexualbehaviorwereassessedthroughhistologicalanalysis
ofovaries,uterus,andvagina.EAdecreaseddysmenorrheaattesteddosesfollowinga7-daytreatment(𝑝 < 0.001).Endometrial
implantvolumedecreasedfollowingthethreetreatmentperiods(𝑝 < 0.05).Catalaseactivity(𝑝 < 0.001)andMDAlevel(𝑝 <
0.01)increasedonlyfollowinga3-daytreatment.EAalsoincreasedantralfollicles,reducedluteinizedunrupturedfolliclenumber
(𝑝 < 0.001),andinducedanimalstobeintheestrusphase.Inconclusion,EApreventedtheprogressofendometriosis,reduced
dysmenorrhea,promotedovarianfolliclegrowth,preventedanovulation,andstimulatedthespecialperiodofratsexualdesire.
TheseresultssuggestthatEntadaafricanacouldbeapromisingalternativeoptionforthetreatmentofendometriosis.
1.Introduction fullyeffectiveandareassociatedwithsubstantialsideeffects
andfrequentrecurrences[4],hencetheurgencytodevelop
Endometriosis is a chronic estrogen-dependent condition innovative active substances that are better tolerated and
that causes dysmenorrhea, nonmenstrual pelvic pain, dys- more efficient than the currently applied pharmacological
pareunia, and infertility [1]. Due to its pain symptoms and andsurgicalapproaches.
highrecurrencerate,endometriosisisoftenassociatedwith There are scientific evidences to suggest that medicinal
severely altered quality of patients’ private and professional herbs with analgesic and fertilizing properties are potential
life [2]. The treatment options are diverse and consist of alternatives to treat endometriosis [5, 6]. Entada africana
analgesictherapies,hormonaltherapiesthatfocusonreduc- Guill. et Perr. (Mimosaceae) for instance is an African
ing systemic levels of estrogen (e.g., progestins, androgens, medicinal plant traditionally used to treat various ailments
gonadotropin-releasinghormone(GnRH)agonists,oralcon- including female infertility, malaria, and rheumatism [7,
traceptives, and aromatase inhibitors), surgery, or a combi- 8]. The root bark decoction of this plant is recommended
nation of these [3]. Unfortunately, these treatments are not in Tchabal (Adamaoua region, Cameroon) as a cure for
2 Evidence-BasedComplementaryandAlternativeMedicine
severalcomplaintsincludinglowerabdominalpain(Table1). women with endometriosis is chronic pelvic pain [17], we
Moreoverthisplanthasbeensubjecttonumerousscientific adoptedtherootdecoctionofEntadaafricanaasextraction
investigationsreportinganalgesic[9],anti-inflammatory[8, protocol.
9], and antioxidant [10] properties. Apigenin, a flavonoid TherootaqueousextractofEntadaafricanawasprepared
isolatedfromtherootsofEntadaafricana[11],wasreported followingtherecommendationsofthetraditionalpractition-
todisplayantioxidantactivity[12],toinhibitcellproliferation ers consulted for treating pelvic pain. Slight modifications
[13, 14], and to have anti-inflammatory activity [15]. The were applied to improve the yield of extraction. One kg of
presence of this compound in Entada africana roots may freshandcleanEntadaafricanarootbarkswascutintosmall
accountforitsscientificprovenproperties[8–10].Although pieces, air-dried (under shade), and ground. The resulting
bearing all these properties, Entada africana has not yet powder(257g)wasboiledin5Lofdistilledwaterfor1hour
beeninvestigatedforendometriosisanditsrelatedpainand andthenfilteredwithWhatmanpapernumber4.Thefiltrate
ovarian dysfunction. The present study therefore is aimed wasfreeze-driedandatotaldrymassof39goftheaqueous
∘
at investigating the effects of the root aqueous extract of extract was obtained. This extract was kept at 4 C in an
Entada africana in different doses and different treatment airtightcontainertilluse.
regimens,onendometriosisrelateddysmenorrheaandovar- Threedosesofthisextractwereadministeredtoanimals:
ian dynamic (this refers to follicle development, ovulation, 127.5, 255, and 510mg/kg. After lyophilizing a little volume
formation,andregressionofthecorporalutea)inanexper- (250mL) of the decoction, obtained from the traditional
imental rat model. Dysmenorrhea was evaluated through practitioners, a human dose of 41.1mg/kg was extrapolated
abdominal writhing response, and ovarian dynamic was fromthetraditionalposology.Animalequivalentdose(AED)
evaluated through the modulation of the number of antral was determined using the method described by Nair and
follicles, luteinized unruptured follicles (these are mature Jacob [18]. The human dose was therefore multiplied by
ovarianfolliclesthatfailtoruptureandundergoluteinization 6.2 to obtain an AED of 255mg/kg. Low and high doses
with trapped oocytes [16]), and corpora lutea. Rat sexual were obtained by dividing and multiplying 255mg/kg by 2,
behavior was estimated through histological analysis of the respectively.
uterusandthevagina.
2.2. Animals. Young adult female Wistar rats aged 10 to
2.MaterialandMethods 12 weeks (150g) were obtained from the breeding facility
of the Animal Physiology and Phytopharmacology Labora-
2.1. Plant Material and Preparation of the Aqueous Extract. tory, University of Dschang (Cameroon). They were bred
TherootsofEntadaafricanawerecollectedinApril2016in and kept under a standard soy-free rat diet in order to
Tchabal (Cameroon Adamaoua region) where it is known eliminateexposuretoexogenousestrogeniccompounds.All
as «Padde wandu» in Foulfoulde´, «We´le´on» in Toupouri, rats were given free access to diet and water ad libitum.
or«Missisiriwe´»inGuisiga,threeCameroonianvernacular Animal handling and in vivo experiments were carried out
languages. The plant was identified and authenticated in in conformity with the European Union on Animal Care
∘
comparison with the botanical sample of C. Geerling N (CEECouncil86/609)guidelinesadoptedbytheInstitutional
5188registeredattheCameroonNationalHerbarium,where Ethics Committee of the Cameroon Ministry of Scientific
a voucher specimen has been deposited under the number ResearchandTechnologyInnovation.
36694/HNC.
To carry out the present study, a small ethnobotanical 2.3. Study Design. Before establishing the model, estradiol
survey was conducted in Tchabal (Cameroon Adamaoua valerate (Progynova(cid:2) 2mg purchased from DELPHARM
region) in order to gain insight into the traditional uses of (Lille, France)) at 0.5mg/kg was orally administered to rats
Entadaafricana,whichallowedustoestablishanappropriate for3consecutivedaystobeintheestrusperiod,aspreviously
extraction protocol, to determine the doses to be tested, described[19].Exceptfortheshamgroup(𝑛 = 3),otherrats
andtodefinetreatmentperiods.Thisethnobotanicalsurvey (𝑛 = 50)underwentautotransplantationsurgerytoestablish
wasconductedusingastructuredinterviewwithsome(five) endometriosis as described in the literature [19]. Briefly, all
traditional healers. They were interviewed individually and ratswereanesthetizedwithanintraperitonealadministration
theapproachwasbasedonadialogueusingoneofthefour of10mg/kgdiazepamand50mg/kgketamine.Usingaseptic
languages(Foulfoulde´,Toupouri,Guisiga,orFrench)accord- technique, an abdominal incision was made to expose the
ing to the traditional healers’ choice. A local person was uterus. The left uterine horn was ligated at the cervical end
actingasaguide.Thetraditionalhealerswerefirstinformed using a 4-0 silk and then excised and placed in an isotonic
abouttheobjectivesofthestudybeforethebeginningofthe saline. Two segments (1cm each) of the excised horn were
interview. The information sought were the local names of cut open longitudinally and attached to the intimal surface
the plant, plant parts used, and medical practices such as totherightsideoftheabdominalwallwiththeendometrium
drugpreparationandadministrationmethodsforthelisted facingtheaforesaidwall.A4-0absorbablethreadwasusedto
diseases. The information we collected is summarized in attachthegraft.Shamsurgerieswereperformedonthesham
Table 1. It comes out from this table that the treatment of group using the same steps as the endometriosis surgeries,
lowerabdominalpainconsistsofdrinkingaglass(250mL) but no tissue was sutured to the abdominal wall. After
of the root decoction of Entada africana once a day for 7 confirming hemostasis, the midventral abdominal incision
consecutive days. Since the most pronounced complaint of was sutured closed in layers and penicillin was injected
Evidence-BasedComplementaryandAlternativeMedicine 3
nt g
e n
eatm heali
noftr mplete 7days 1day 4days 3days 5days 1day 7days
o
ati co
Dur Till
amaouaregion). PosologyPoulticeonthewoundonceaday. Aglass(250mL)onceaday. Adropintotheeye.Twoglasses(200mLperglass)twiceaday.Aglass(250mL)twiceaday.Threeglasses(200mL)threetimesperday.5mLofthedecoctioninsinglecatch. Aglass(250mL)onceaday.
d
A
n n, n
Tchabal(Camerou Diseases Burnwounds werabdominalpaihemorrhoidsEyedisease Malaria Diarrhea Cough Intestinalworms werabdominalpai
of Lo Lo
y
localit rs. hour.1 for1 1
Entadaafricanainthe n5Lofwaterfor2hou inaliterofwaterfor1in500mLofwaterfor rksin500mLofwater arksin5Lofwaterfor
alsurveyof mode kgofbarki 00gofbark50gofbark gofrootba kgofrootb
Ethnobotanic Preparation Leafpowder Decoction:1 Decoction:2Decoction:2hour.Decoction:2hour.Decoction:1hour.
1:
e d k
abl use ves bar ots
T art Lea em Ro
P St
”
´e
d
ul a”
o g
oulfuri”uisi
FoG
“p“
me duin“Tou´wein
rna wanninsiri
Vernacula (i)Padde´´(ii)Weleo(iii)Missi
4 Evidence-BasedComplementaryandAlternativeMedicine
for 3 days at 400,000 units per rat to prevent infection. −20∘C till use. This experiment was performed twice to
Afterarecoveryperiodof28days,ratsunderwentasecond ensuretheaccuracyoftheresultsweobtained.
exploratorylaparotomytoexamineifmodelsofexperimental
endometriosishavebeensuccessfullyestablished.Ofthe50 2.4.BiochemicalAnalysis. Catalaseactivitywasestimatedby
experimentalrats,11didnotdevelopanysignsofendometrio- themethodofSinha[22]whichisbasedonthedecomposi-
sis,andtheywereexcludedfromthestudy.Thepretreatment tionofH2O2intowater.Theconcentrationofundecomposed
implantvolumeswerecalculatedbymeasuringtheirdimen- H2O2 was evaluated using a calibration curve established
sions(length,width,andheight,inmillimeters).Forvolume from a standard solution (50mMH2O2). Tissue catalase
calculations, the ellipsoid volume formula (𝜋/6 × length × activitywasdeterminedasfollows:
width×height)wasused[20].
Following a recovery period of 2 weeks, animals were ΔDO
C.A= , (1)
assignedtothefollowinggroups: 𝑎⋅𝑡⋅𝑚
(1):shamcontrolgroup(𝑛 = 3):normalcontrolanimals
where
treatedwithE2V(0.5mg/kg)andthevehicle.
(2):control(𝑛 = 3):animalswithendometriosistreated
C.Aisthecatalaseactivity(mMofH2O2/min/mgof
withE2V(0.5mg/kg)andthevehicle. tissue);
(3): micro (𝑛 = 9): animals with endometriosis treated
ΔDOistheabsorbanceofthesample−absorbanceof
withE2V(0.5mg/kg)andthereferencesubstance,Microgy-
thereagentblank;
non 30 (one tablet (levonorgestrel (0.15mg) + ethinylestra-
diol (0.03mg))/rat/day). This dosage for Microgynon was 𝑎istheslopeofthecalibrationcurve;
adopted with reference to an experimental study using 𝑡isthereactiontime(1minute);
ethinylestradiol and levonorgestrel (in female rats) in the
𝑚isthemassofthetissuecollectedforhomogeniza-
massproportionoccurringinMicrogynon,todeterminethe
tion(mg).
mechanismwherebyemotionalandsexualdisordersoccurin
hormonalcontraceptiveusers[21].
Malondialdehyde (MDA) level was determined by the
(4): EA (𝑛 = 27): animals with endometriosis treated
methodofWilburetal.[23]whichisbasedonthereaction
withE2V(0.5mg/kg)andtherootaqueousextractofEntada with thiobarbituric acid (TBA) at 90–100∘C. In the TBA
africanaatthedoseof127.5(EA127.5,𝑛 = 9),255(EA255,
test reaction,MDA or MDA-like substances and TBA react
𝑛=9),or510(EA510,𝑛=9)mg/kg.
withtheproductionofapinkpigmenthavinganabsorption
Ineachgroup,E2Vwasadministereddailyfor14consecu- maximum at 532nm. Tissue level of MDA was determined
tivedays,6hoursfollowingtheadministrationofthevehicle,
usingthefollowingformula:
Microgynon,ortherootaqueousextractofEntadaafricana.
MicrogynonandtherootaqueousextractofEntadaafricana ΔDO
[MDA]= , (2)
wereadministeredtoanimalsfor3,7,and14consecutivedays 𝜀⋅𝐿⋅𝑚
asfollows:
(i)Fora3-daytreatment,thevehiclewasadministeredfor where
11days;MicrogynonandtherootaqueousextractofEntada
[MDA] is the concentration of MDA (nM/mg of
africanawereadministeredduringthelast3days.
tissue);
(ii) For a 7-day treatment, the vehicle was administered
for 7 days; Microgynon and the root aqueous extract of ΔDOistheabsorbanceofthesample−absorbanceof
Entadaafricanawereadministeredduringthelast7days. thereagentblank;
(iii) For a 14-day treatment, Microgynon and the root 𝜀 is the molar extinction coefficient
aqueousextractofEntadaafricanawereadministeredfor14 (1.56⋅10−4nM−1cm−1);
days. 𝐿isthepathlength(1cm);
All treatments were orally administered. On day 14,
each rat received an intraperitoneal injection of oxy- 𝑚isthemassofthetissuecollectedforhomogeniza-
tocin (2U/rat) 1h after the last administration of E2V to tion(mg).
induce writhing response. Thirty minutes after observing
the writhing response, animals were sacrificed under anes- 2.5.HistologicalAnalysis. Histologicalanalysisoftheovaries,
thesia (diazepam + ketamine). Laparotomy was performed uterus, and vagina were assessed from 5-𝜇m sections of
and the volumes of ectopic foci were measured again as paraffinembeddedtissues.Followingthehematoxylin-eosin
previously described [20]. A difference between both vol- staining,uterineandvaginalepithelialheightswereassessed
umes (𝑉2 − 𝑉1) was made to evaluate volume change and on microphotographs using the complete Zeiss equipment
to compare the effect obtained in the control group to consisting of a microscope Axioskop 40 connected to a
that obtained in treated groups. Following measurements, computerwheretheimagewastransferredandanalysedwith
ectopic foci were excised and homogenated in 10mM Tris the MRGrab1.0 and Axio Vision 3.1 software, all provided
(tris(hydroxymethy)aminomethane) buffer (0.1g per 1mL). by Zeiss (Hallbergmoos, Germany). Ovarian follicles were
Tissue homogenates were centrifugated at 3000rpm for 10 countedonfoursectionsofthesameovarybytwoinvestiga-
∘
minutes at 5 C. The resulting supernatants were stored at tors at different times (with one month of interval between
Evidence-BasedComplementaryandAlternativeMedicine 5
the first and the second observation) and the final result 14-daytreatment,thisparameterremainedelevatedfollowing
foreachovaryrepresentsthemeanofthetwoobservations. treatment with Entada africana at 255 (𝑝 < 0.01) and 510
Luteinizedunrupturedfollicles(LUFs)wereidentifiedbythe (𝑝<0.05)mg/kg(Figure1(c)).
presence of oocytes not surrounded by cumulus oophorus
cellswithinmatureantralfollicles[16,24]. 3.2. Comparison of Endometrial Implant Volume, Catalase
Activity, and Malondialdehyde Level. In the control group,
2.6.StatisticalAnalysis. Dataarepresentedasmean±SEM. implant volume was on average 22.24 ± 0.76mm3 before
Statisticalsignificanceandthedifferenceamonggroupswere treatment and 112.78 ± 36.41mm3 14 days following daily
evaluatedbyone-wayanalysisofthevariance(ANOVA)fol- administrationofdistilledwater,avolumechangeof90.54±
lowedbyTukeytestformultiplecomparisons,anddifferences 37.16mm3(Figure2(a)).Thisvolumegainwasreversedwith
wereconsideredstatisticallysignificantat𝑝<0.05. Microgynonfollowingthethreetreatmentperiods(147,122,
and 175% (𝑝 < 0.05) induction, respectively). A similar
3.Results atrophy of the implants was observed with Entada africana
following the three treatment periods at doses of 255 and
3.1. Comparison of Writhing Response among Groups. 510mg/kg(𝑝 < 0.05and𝑝 < 0.01).Thedoseof127.5mg/kg
Endometriosis induced a 2.6-fold increase in writhing was not able to reverse swelling of implants following the
frequency (𝑝 < 0.01 and 𝑝 < 0.001) as compared to sham three treatment periods and rather induced a swelling of
operatedanimals.Comparedtocontrolanimals,Microgynon 35% lower than that of the control group following a 3-day
reduced writhing frequency by 91 and 94% (𝑝 < 0.001) treatmentandasimilarswellingfollowinga14-daytreatment.
followinga3-dayanda7-daytreatment.Followinga14-day Microgynon increased catalase activity following the
treatmentwrithingfrequencywascomparabletothatofthe threetreatmentperiods(𝑝 < 0.001) (Figure2(b)). A 3-day
control group. The root aqueous extract of Entada africana treatment with the root aqueous extract of Entada africana
reducedwrithingfrequencyatalltesteddosesfollowingthe increased catalase activity by 39.37% at the dose of
three treatment periods. We thus noticed that following a 127.5mg/kg. At doses of 255 and 510mg/kg, catalase
3-day treatment, writhing frequency decreased by 42% at activityincreasedby2-and5-fold(𝑝 < 0.001),respectively.
127.5mg/kg(𝑝 < 0.05),72%at255mg/kg(𝑝 < 0.001),and Followinga7-dayanda14-daytreatment,thelevelofactivity
67%at510mg/kg(𝑝 < 0.001).Followinga7-daytreatment, of catalase induced by the root aqueous extract of Entada
writhing frequency was reduced by 99 and 85% at 127.5 africana was comparable to that observed in the control
and 255mg/kg, respectively (𝑝 < 0.001), and completely group.
abolished at 510mg/kg (100% induction; 𝑝 < 0.001). This Treatment with Microgynon did not significantly affect
parameter remained reduced after 14-day administration of malondialdehyde (MDA) level in endometrial implants fol-
theaqueousextractofEntadaafricanaespeciallyatdosesof lowingthethreetreatmentperiods(Figure2(c)).Incontrast,
255and510mg/kg(𝑝<0.001)(Figure1(a)). the root aqueous extract of Entada africana significantly
Thedurationofanabdominalwrithewaslengthenedby increasedMDAlevelsinendometrialimplantsonlyfollowing
3.5-fold(𝑝 < 0.05)incontrolanimalsascomparedtosham a3-daytreatment.Thisincreasewas261%at127.5mg/kg(𝑝<
operatedanimals.Comparedtocontrolanimals,thetimeof 0.001),169%at255mg/kg(𝑝<0.01),and165%at510mg/kg
writhingwasreducedwithMicrogynonby39%followinga (𝑝<0.01).Followinga7-dayanda14-daytreatment,thelevel
3-daytreatment,59%(𝑝<0.05)followinga7-daytreatment, ofMDAwascomparableandevenlowerthanthecontrol.
and63%(𝑝 < 0.05)followinga14-daytreatment.Asimilar
reduction was observed with the root aqueous extract of 3.3. Ovarian Dynamic. Antral follicles were 75% higher in
Entadaafricanafollowinga7-dayanda14-daytreatment.At the ovaries of control animals than in the ovaries of sham
the dose of 127.5mg/kg, this extract decreased the duration operatedanimals,althoughthiseffectdidnotreachthelevel
of an abdominal writhe by 57% (𝑝 < 0.05) following a of statistical significance (Table 2). Compared to control
7-day treatment and 35% following a 14-day treatment. At animals,Microgynondidnotsignificantlyaffectthenumber
255mg/kg,thedurationofanabdominalwrithewasreduced ofantralfollicles.Entadaafricanaincontrastincreaseditby
by 66% (𝑝 < 0.05) following a 7-day treatment and 44% 2.7-fold at 127.5mg/kg (𝑝 < 0.001), 29% at 255mg/kg, and
followinga14-daytreatment.Thisparameterwascompletely 2.2-foldat510mg/kg(𝑝<0.01)followinga3-daytreatment.
abolished(100%reduction;𝑝<0.001)at510mg/kgfollowing Antralfolliclesremainedsignificantlyelevatedfollowinga7-
a 7-day treatment while no significant effect was observed dayanda14-daytreatmentatthethreetesteddoses.
followinga14-daytreatment(Figure1(b)). Thenumberofcorporaluteawas2timesmoreelevatedin
Regardingwrithinglatency,endometriosisreduceditby controlanimalsthaninshamoperatedanimals(𝑝 < 0.001).
75% as compared to sham operated animals. Compared to Compared to control animals, Microgynon decreased the
controlanimals,aprominentincreaseinwrithinglatencywas number of corpora lutea by 53 (𝑝 < 0.001) and 43% (𝑝 <
observedfollowinga7-daytreatmentwithbothMicrogynon 0.05) following the three treatment periods. At 127.5mg/kg
andtherootaqueousextractofEntadaafricanaatalltested Entadaafricanadecreasedthenumberofcorporaluteaby16
doses. This parameter was increased by 8.5-fold following and 18% following a 3-day and a 7-day treatment, although
treatment with Microgynon while Entada africana length- this effect did not reach the level of statistical significance.
ened it by 26-fold at 127.5mg/kg (𝑝 < 0.001), 23-fold at At 255mg/kg, an increase of 29% (𝑝 < 0.05) was observed
255mg/kg,and30-foldat510mg/kg(𝑝<0.001).Followinga following a 3-day treatment while no significant difference
6 Evidence-BasedComplementaryandAlternativeMedicine
60 20
Writhing frequencyer of abdominal writhesper 30 minutes) 2400 B ∗ ∗∗∗ C C C B n of an abdominal writhe(seconds) 1105 A C C A B ∗ A A
numb ∗∗∗ A ∗∗∗ ∗∗∗ uratio 5 ∗ ∗ ∗
( ∗∗∗ ∗∗∗B B A D
0 ∗∗∗ ∗∗∗ ∗∗∗ 0 ∗∗∗
3 7 14 3 7 14
Treatment periods (days) Treatment periods (days)
SHAM EA 127.5 SHAM EA 127.5
Control EA 255 Control EA 255
Micro EA 510 Micro EA 510
(a) (b)
40
C
nutes) 30 ∗C∗∗∗C∗∗∗∗∗ A∗∗
mi ∗
y (
nc 20
e
at
g l
n
hi
Writ 10
0
3 7 14
Treatment periods (days)
SHAM EA 127.5
Control EA 255
Micro EA 510
(c)
Figure1:Comparisonofwrithingresponse((a)writhingfrequency;(b)durationofanabdominalwrithe;(c)writhinglatency)amonggroups.
Dataarepresentedasmean±SEM;𝑛 = 3;A𝑝 < 0.05,B𝑝 < 0.01,andC𝑝 < 0.001versusSHAM;∗𝑝 < 0.05,∗∗𝑝 < 0.01,and∗∗∗𝑝 < 0.001
versuscontrol;treatedgroupswerecomparedtoSHAMandtocontrolwithinthesametreatmentperiodusingone-wayANOVA+Tukey’s
multiplecomparisontest;SHAM:shamoperatedanimals;control:animalswithendometriosisreceivingthevehicle;micro:animalswith
endometriosistreatedwiththereferencesubstanceMicrogynon;EA:animalswithendometriosisreceivingtheaqueousextractofEntada
africanaat127.5,255,or510mg/kgBW/day.
was noticed following a 7-day and a 14-day treatment. At identified: Graafian follicle, tertiary follicle, corpus luteum,
510mg/kg, the number of corpora lutea decreased by 20% andluteinizedunrupturedfollicle.
following a 3-day treatment and 32% (𝑝 < 0.01) following
a7-daytreatment(Table2). 3.4. Relative Uterine Weight, Uterine, and Vaginal Epithelial
Luteinized unruptured follicles (LUFs) were more ele- Heights. No significant difference of the relative uterine
vated in control animals (6.5 per rat, 𝑝 < 0.001) than weight, as well as the uterine and the vaginal epithelial
in sham operated animals (2.7 per rat). Microgynon and heights, was observed between sham and control groups.
EntadaafricanareducedthenumberofLUFstothelevelof Similarly, the relative uterine weight and the uterine and
shamoperatedanimalsfollowingthethreetreatmentperiods the vaginal epithelial heights of treated groups were not
(Table2). significantlydifferentfromthecontrolgroup(Table3).
Figure 3 shows microphotographs of the ovaries of Figure4showsmicrophotographsoftheuterineepithe-
experimental animals where the following follicles are lium. The uterus of sham operated animal was lined by
Evidence-BasedComplementaryandAlternativeMedicine 7
180 300
3Ｇ) 19305 ometrialg of tissue) ∗∗∗ ∗∗∗ ∗∗∗
m dm 200
ge ( 45 n enO2/
an 0 y iH2
Volume ch −−−1943055 ∗ ∗ ∗ ∗∗ Catalase activitplants (M of 100
m
−180 i 0
3 7 14 3 7 14
Treatment periods (days) Treatment periods (days)
Control EA 255 Control EA 255
Micro EA 510 Micro EA 510
EA 127.5 EA 127.5
(a) (b)
50
∗∗∗
alondialdehyde levels inendometrial implants(nM/mg of tissue) 12340000 ∗∗∗∗
M
0
3 7 14
Treatment periods (days)
Control EA 255
Micro EA 510
EA 127.5
(c)
Figure 2: Graphical representation of volume change (a), catalase activity (b), and malondialdehyde (c) levels in endometrial implants
followingtreatments.Dataarepresentedasmean±SEM;𝑛=3;∗𝑝<0.05,∗∗𝑝<0.01,and∗∗∗𝑝<0.001versuscontrol;treatedgroupswere
comparedtocontrolwithinthesametreatmentperiodusingone-wayANOVA+Tukey’smultiplecomparisontest;control:animalswith
endometriosisreceivingthevehicle;micro:animalswithendometriosistreatedwiththereferencesubstanceMicrogynon;EA:animalswith
endometriosisreceivingtheaqueousextractofEntadaafricanaat127.5,255,or510mg/kgBW/day.
a tall columnar epithelium showing cellular degeneration eithermitotic(3-daytreatment)ornecrosisfigures(7-dayand
or necrosis. In control animals, both mitotic and necrosis 14-daytreatment).
figures were seen on the tall columnar uterine epithelium. Microphotographs of the vaginal epithelium are shown
In Microgynon-treated animals, the uterus was lined by a in Figure 5. It comes out from this figure that the vaginal
tallcolumnarepitheliumwithnecrosisfigurefollowinga3- epitheliumofshamoperatedanimalswaslinedbyashedding
day and a 14-day treatment. Following a 7-day treatment, cornified layer. In control animals, the vaginal epithelium
fewmitoticfigureswereseeninatallcolumnarepithelium. was made up of a thicker stratum germinativum with a
At 127.5mg/kg, the uterus of animals treated with the root shedding stratum granulosum. In Microgynon-treated ani-
aqueous extract of Entada africana was lined by a tall mals, the vagina was lined by a shedding cornified layer
columnarepitheliumwithnecrosisfiguresfollowinga3-day following a 3-day and a 14-day treatment. Following a 7-
treatment.Followinga7-dayanda14-daytreatmentthetall day treatment, the vaginal epithelium consists of a basic
columnar epithelium showed only few mitotic figures. At stratumgerminativum,anintermediatestratumgranulosum,
255mg/kg,theuteruswaslinedbyatallcolumnarepithelium andawell-attacheduppercornifiedlayer.At127.5mg/kg,the
with necrosis figures following the three treatment periods. vaginalepitheliumofanimalstreatedwiththerootaqueous
At 510mg/kg, the tall columnar uterine epithelium showed extract of Entada africana was lined by a shedding (3-day
8 Evidence-BasedComplementaryandAlternativeMedicine
14-daytreatment CL/ratLUFs/rat 7.67±0.892.67±0.33bc17.00±1.006.50±0.29∗∗∗∗9.67±1.761.33±0.67a∗∗∗16.33±1.202.33±0.33aa,∗∗∗15.33±2.190.33±0.33ba,∗∗∗19.00±1.160.50±0.29 groupswerecomparedtoSHAMandtoosisreceivingthevehicle;micro:animalsmg/kgBW/day;CL:corpusluteum;LUFs:
m,andluteinizedunrupturedfolliclesinratovaries. 7-daytreatmentAntralCL/ratLUFs/ratfollicles/rat7.67±0.892.67±0.331.33±0.33cb17.00±1.006.50±0.292.33±0.67∗∗∗∗∗8.00±0.582.67±0.332.33±0.67c∗∗∗14.00±0.582.33±0.673.67±1.20c∗∗∗a16.67±0.672.00±0.586.00±0.58a,∗∗∗∗a11.50±0.872.67±0.675.67±1.33 ∗∗∗∗∗𝑝𝑝𝑝<0.05<0.01<0.001,,andversuscontrol;treatedSHAM:shamoperatedanimals;control:animalswithendometriceivingtheaqueousextractofEntadaafricanaat127.5,255,or510
2:Numberofantralfollicles,corpusluteu AntralLUFs/ratfollicles/rat2.67±0.331.33±0.33c6.50±0.292.33±0.67∗∗∗2.33±0.672.00±0.58∗∗∗a2.67±0.334.33±0.67∗∗∗a1.50±0.294.67±0.88∗∗a3.50±0.294.33±0.33 bc∗𝑝𝑝5<0.01<0.001,,andversusSHAM;yANOVA+Tukey’smultiplecomparisontest;Microgynon;EA:animalswithendometriosisre
Table 3-daytreatmentAntralCL/ratfollicles/rat1.33±0.337.67±0.89SHAMc2.33±0.6717.00±1.00Control∗∗∗2.50±0.298.00±0.58Microc,∗∗∗b6.50±0.2914.33±1.20EA127.5c,∗3.50±0.5822.00±0.58EA255c,∗∗b5.33±0.3313.67±1.20EA510a𝑝±𝑛=3<0.0ResultsarepresentedasmeanSEM;;controlwithinthesametreatmentperiodusingone-wawithendometriosistreatedwiththereferencesubstanceluteinizedunrupturedfollicles.
Evidence-BasedComplementaryandAlternativeMedicine 9
ndepithelialheightsoftheuterusandthevagina. 𝜇𝜇rineepithelialheight(m)Vaginalepithelialheight(m)7-daytreatment14-daytreatment3-daytreatment7-daytreatment14-daytreatment1.28±0.311.28±0.315.28±1.355.28±1.355.28±1.351.51±0.211.51±0.215.31±0.935.31±0.935.31±0.931.86±0.151.69±0.175.23±0.307.38±0.706.35±0.221.25±0.352.01±0.415.48±0.355.7±0.706.22±0.321.63±0.131.14±0.095.05±0.284.92±0.055.33±0.842.03±0.231.77±0.124.67±0.435.29±0.305.31±0.46 olwithinthesametreatmentperiodusingone-wayANOVA+Tukey’smultiplecomparisontest;SHAM:shamndometriosistreatedwiththereferencesubstanceMicrogynon;EA:animalswithendometriosisreceivingthe
Table3:Relativeweightoftheuterusa uterusweight(mg/kgBW)Ute7-daytreatment14-daytreatment3-daytreatment1548.02±26.091548.02±26.091.28±0.311425.53±99.361425.53±99.361.51±0.211637.30±240.631756.26±101.942.35±0.141226.79±100.341387.03±216.931.25±0.161393.96±98.561588.09±18.422.06±0.231458.43±79.861297.75±76.101.92±0.33 𝑛=3M;;treatedgroupswerecomparedtoSHAMandtocontrwithendometriosisreceivingthevehicle;micro:animalswitheat127.5,255,or510mg/kgBW/day.
ve SEalsna
Relati3-daytreatment1548.02±26.09SHAM1425.53±99.36Control1246.53±186.04MICRO1422.60±177.96EA127.51084.67±68.35EA2551385.12±151.50EA510±Resultsarepresentedasmeanoperatedanimals;control:animaqueousextractofEntadaafrica
10 Evidence-BasedComplementaryandAlternativeMedicine
The root aqueous extract of Entada africana induced
Microgynon-like effects by increasing writhing latency and
by reducing writhing frequency and abdominal writhing
duration. These effects on dysmenorrhea were found sig-
nificant at all tested doses following a 7-day treatment.
TheseresultssuggestthatEntadaafricanawouldhaveinhib-
LUF
ited the signaling pathway mediated by estradiol resulting
CL in a decreased dysmenorrhea. These analgesic effects of
Entada africana on the dysmenorrhea rat models support
the traditional use of Entada africana for the treatment of
GF pelvic pain and corroborate the observations of Ezenyi et
al. [9] who reported analgesic effects of Entada africana
in acetic acid-induced nociceptive responses in mice. The
CL
increase in writhing frequency observed with Microgynon
following a 14-day treatment suggests a downregulation of
progesteronereceptorsasaresultofanhyperstimulationby
TF levonorgestrel (0.15mg), a progestogen (a compound that
inducesprogesterone-likeeffects),whoseamountis5times
Figure3:Microphotographs(×400,hematoxylinandeosinstain- more abundant than that of ethinylestradiol (0.03mg). The
ing)ofexperimentalratovaries.GF:Graafianfollicle;TF:tertiary literature reports that progesterone inhibits the effects of
follicle;LUF:luteinizedunrupturedfollicle;CL:corpusluteum.
estradiol[30].Therefore,thedownregulationofprogesterone
receptors would have allowed the stimulation of estradiol
pathway by ethinylestradiol and therefore the increase in
treatment) or a well-attached (7-day and 14-day treatment) writhingfrequency.
cornified layer. At 255mg/kg the vaginal epithelium was The analgesic effects of Entada africana were associated
delimited by a shedding cornified layer following the three with a reduced size of endometrial implants, especially
treatmentperiods.At510mg/kgthevaginalepitheliumwas following a 7-day treatment where the dose of 255mg/kg
delimited by a well-attached cornified layer following a 3- inducedamoreprominentandsignificanteffect.Thisresult
day treatment. Following a 7-day and 14-day treatment, the indicates a positive correlation between the progress of
vaginal epithelium was delimited by a shedding cornified endometriosis and the severity of pelvic pain. To reduce
layer. endometrialimplantsize,Entadaafricanamayactbyinduc-
ing ectopic endometrium cell damage as indicated by an
4.Discussion increasedlevelofmalondialdehyde(MDA),anendproduct
oflipidperoxidation.Indeed,oxidativestresswasassociated
Clinicalsymptomsofendometriosisincludedysmenorrhea, withthedevelopmentofendometriosis[31,32].Otherstudies
nonmenstrualpelvicpain,andinfertility[1].Dysmenorrhea reported that oxidative stress leads to necrosis and cell
isdefinedasacrampingpaininthelowerabdomenoccurring death [33–35]. Chemotherapy is known to resort to the
justbeforeorduringmenstruation[25]asaresultoftheinter- oxidative stress mechanism which provokes cancerous cell
actionofuterineproinflammatoryprostaglandins(especially death. Indeed, patients under chemotherapy were found to
PGF2𝛼, released by disintegrating endometrial cells) with haveelevatedlevelofoxidativestressduetothedestructionof
theirreceptors[26].Inrodents,dysmenorrheaisassessedas cancerouscells[36].However,achronicoxidativestresswas
abdominal writhing responses following an intraperitoneal foundtoinduceaninverseeffectbypromotingangiogenesis
injectionof2Uoxytocin,onehourafterthelastadministra- and tumor development [37]. A transient oxidative stress
tionofestradiol[19,27].Estradiolisreportedtoupregulate may then be crucial for the destruction of tumor cells. In
oxytocin and prostaglandin F2𝛼 (PGF2𝛼) receptors in rats agreementwiththishypothesis,ourresultsshowedthatthe
uterus [28]. Following binding to their G protein-coupled root aqueous extract of Entada africana increased MDA
receptors,oxytocinandPGF2𝛼stimulateuterinecontraction only following a 3-day treatment. This effect was reversed
2+
through activating the phospholipase C/Ca dependent following a 7-day treatment and values began to be lower
pathway [29]. The presence of an additional endometrial thanthoseofthecontrolgroupfollowinga14-daytreatment.
tissue outside the uterine cavity during endometriosis may Previous animal studies reported decreased levels of MDA
resultinmoreseveredysmenorrhea.Inagreementwiththis in endometrial implants following a 7-day treatment with
hypothesis,ourresultsshowedthatendometriosisincreased resveratrol[20]or5-daytreatmentwithozone-oxygenmix-
writhingfrequency,decreasedwrithinglatency,andlength- ture[38].Allthesetreatmentsdecreasedendometrialimplant
enedthedurationofanabdominalwritheinratswithectopic volumesuggestingthatfollowingtreatments,therewouldbe
endometrium compared to the normal control, suggesting atransientincreaseintheoxidativestressofectopictissues
that besides the normal uterine production of proinflam- thatdoesnotexceed3days.Resultsalsoshowedthatcatalase
matory prostaglandins, there is additional release of these activity increased in endometrial implant following a 3-day
uterotonicagentsbytheectopicendometrium,whichwould treatment. We can therefore hypothesize like Aktun et al.
haveincreaseddysmenorrhea. [38]thatEntadaafricanamediatedoxidativepreconditioning