Rev. bras. farmacogn. vol.27 número5

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Anti-caries

activity

of

selected

Sudanese

medicinal

plants

with

emphasis

on

Terminalia

laxiflora

Ebtihal

Abdalla

M.

Mohieldin

_,

_Ali

_M.

_Muddathir

_,

_Kosei

_Yamauchi

_,

_Tohru

_Mitsunaga

b_{FacultyofPharmacy,UniversityofScienceandTechnology,Omdurman,Sudan}

c_{DepartmentofHorticulture,FacultyofAgriculture,UniversityofKhartoum,KhartoumNorth-Shambat,Khartoum,Sudan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27November2016

Accepted19April2017

Availableonline11July2017

Keywords:

Glucosyltransferase

Dentalcavity

Antibacterial Terchebulin

a

b

s

t

r

a

c

t

InSudan,somemedicinalplants,suchasAcaciaseyal,CalotropisproceraandBalanitesaegyptiacahavebeen usedtopreventortreatoralhealthproblems.ThestemandstembarkofTerminalialaxifloraEngl., Combre-taceae,areusedasantisepticsformouthwashtopreventgingivitisandthrushinAfrica.Methanoland50% hydroethanolicextractsof25plantsthatareusedintraditionalSudanesemedicineforseveraldiseases andcavitydisorderswerescreenedforanti-cavityactivities.T.laxifloramethanolicwoodextracts,which exhibitedsuchactivity,wereinvestigated.Thecrudeextractswereassayedfortheirantimicrobial activi-tiesagainstStreptococcussobrinusintermsofminimuminhibitoryconcentrationandglucosyltransferase inhibition.TheactiveextractofT.laxiflorawoodwassubsequentlyfractionatedbydifferent chromato-graphictechniques.IsolatedcompoundswereidentifiedbyspectroscopicmethodsandassessedforS. sobrinusandglucosyltransferaseinhibitoryeffects.MethanolicextractsofTerminaliabrownii(bark),T. laxiflora(wood),A.seyal(bark),Persicariaglabra(leaves)andTamarixnilotica(stem)showedgood activi-tiesagainstbothS.sobrinusandglucosyltransferase(MIC≤1mg/ml,IC50values<50␮g/ml).Overallplant extracts,T.laxiflorademonstratedthegoodcombinedactivities(MIC0.5mg/ml,glucosyltransferase,IC50 10.3␮g/ml);therefore,itsmethanolicwoodextractswereselectedforfurtherphytochemicalstudies. Fourconstituentswereisolatedbychromatographictechniquesandidentifiedbyspectroscopic tech-niques.Pharmacologicalevaluationoftheobtainedcompoundsshowedthatflavogallonicaciddilactone hadcomparativelygoodantibacterialactivity.Intheglucosyltransferaseinhibitorytest,terchebulin dis-playedpotentactivitywithanIC50of7.5␮M.Thescreeningpresentedinthisstudyshowedthatmethanol extractsofT.laxiflorawoodpossessedpromisinganti-cavityeffects.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Dentalcariesisdefinedasanirreversiblemicrobialdiseaseof thecalcifiedtissuesoftheteeth,characterizedbydemineralization oftheinorganicportionanddestructionoftheorganicsubstance ofthetooth(Rajendranetal.,2009).

Bacterial plaque composed of native oral flora accumulated ondentalsurfacesembeddedinanextracellular polysaccharide (EPS)matrixand istheprimary etiologicagentofdental caries (Kolenbranderetal.,2006;Kooetal.,2009).Outofthesevenspecies ofmutansstreptococcigroup,Streptococcussobrinushavebeenone ofthemostcommonlyimplicatedinthepathogenesisofdental cavity;moreover,itproducesexoenzymesnamed glucosyltrans-ferases(GTF),whichplaycriticalrolesinthesynthesisofglucan

∗ _{Correspondingauthor.}

E-mail:muddathir@uofk.edu(A.M.Muddathir).

andEPStoprovidingsitesondentalsurfacesformicrobial coloniza-tion,inadditiontoadherentglucanforbacterialcoherence(Paes Lemeetal.,2006;BowenandKoo,2011;Nishimuraetal.,2012; Hashizume-Takizawaetal.,2014).

Medicinalplantshavebeenagreatsourceofnoveldrug com-poundsfromlongtime.Plantderivedproductshavemadelarge contributionstothewellbeingofhumanhealth.Scientistsacross theglobehavereportedantimicrobialpropertiesofseveral medic-inalplantsbutstillaveryfewofthisenormouspotentialdrughas beenscientificallyscreened(SiqueiraandRocas,2005;Karuppiah andRajaram,2012;GauniyalandTeotia,2014).InSudan,various medicinalplantshavebeenusedtopreventor treatoralhealth problems.Thisstudyinvestigatedsomeplantsthatareusedin tra-ditionalSudanese medicineasmouthdetergentssuchasAcacia seyal,CalotropisproceraandBalanitesaegyptiaca(ElGhazalietal., 2003; Khalidetal., 2012).Different parts ofTerminalialaxiflora Engl.,Combretaceae,areusedtopreventgingivitisandthrushin Congo;thestemsareusedaschewingsticksandalsomacerated

http://dx.doi.org/10.1016/j.bjp.2017.04.002

0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

stem barkare usedas antiseptic towash mouth (Fasolaet al., 2013).

Inthepresentstudy,25plantspecieswereselectedand eval-uatedfortheiranti-cariogenicactivityintermsofinhibitionofS. sobrinusbacterialgrowthandGTFinhibitoryeffectsbyusingtheir methanolicand50%hydroethanolicextracts.Methanolicextracts ofT.laxiflorawooddemonstratedsignificantcombinedactivities; thus,itwasfurtherfractionatedinordertoidentifytheactive com-poundsresponsibleforthebiologicalactivities.

Materialsandmethods

Reagents

All materials were purchased from Wako, Japan except p -iodonitrotetrazolium(INT)violet,whichwasfromSigma-Aldrich Co.Ltd,Japan.

Plantmaterialsandextraction

The plants were collectedfrom Khartoum state (Khartoum, Omdurman and Shambat cities) and Elgadarif state of Sudan. VoucherspecimensaredepositedintheHorticulturalLaboratory, DepartmentofHorticulture,FacultyofAgriculture,Universityof Khartoum(Table1).

Plantmaterialswereshadedriedandpowderedbefore extrac-tion;theywereeachextractedthreetimesfor12h,withmethanol and50%hydroethanol.Theextractswerefilteredandthesolvent wasremovedunderreducedpressureusingrotaryevaporator.The concentratedextractswerethendriedwithafreezedryer.

Fractionation,purificationandidentificationofpurecompounds fromTerminalialaxiflora

Fractionationandisolation ofTerminalialaxifloraEngl., Com-bretaceae, wood were performed by the method described by Muddathiretal.(2013).Methanoliccrudeextracts(5g)were chro-matographedonmediumpressureliquidchromatography(MPLC) usingoctadecyl-silica(ODS)column(YMC-DispoPackATODS-25, particlesize25␮m;columnsize120g,(40mm×188mm),Japan), chromatographypump(Co.No.540Yamazen,Osaka,Japan)with pressure of 1.2MPa, UV detector at 280 wavelength (UV-10V Yamazen,Osaka,Japan)andfractioncollector(SF-2120,Advantec TokyoLtd,Japan).

The column was conditioned with the first eluent used for separation for30minwithflow rateof 0.5ml/min. Thenwater containingincreasingproportionsofmethanolstepwiseelutionto obtaintwofractions(F1andF2).F1(0.89g)waspassedthrough columnchromatography(40mm×430mm)onaSephadexLH-20 (18–111␮m,GEHealthcareBio-SciencesCorp,Tokyo,Japan)that

revealedfoursubfractions.Thesefoursubfractions,F(1a,1b,1c and1d)introducedthroughpreparativehighperformanceliquid chromatography(HPLC)withreversedphaseInertsilODS-3column (10mm×250mm,GLSciencesInc.,Tokyo,Japan)andmonitoredat 280nm.Solventsystemused10–100%gradientmethanolinwater with0.05%TFAprogrammedfor60minataflowrateof5ml/min togivecompound(1)(14mg),compound(2)(8.5mg)and com-pound(3)(5mg).F2(0.50g) was alsosubjected topreparative HPLCunderthesameconditiontoisolatecompound(4)(17.5mg). ThecompoundspuritywasconfirmedusinganalyticalHPLC (Shi-madzuSIL-20A)withreversedphaseInertsilODS-3Vcolumn(5␮m

(4.6mm×250mm), GL Sciences Inc., Tokyo, Japan), flow rate: 1ml/min,wavelength:280nm,gradientprogram:methanol:0.05% TFAaqueoussolutionfor60min(Fig.1).

The isolated compounds from T. laxiflora wood methanol extract,exactlycompounds(1)and(2)wereidentifiedusingthe

1_H,13_{Cnuclearmagneticresonance(NMR).Spectrawererecorded}

in methanol-d4 with a JEOL EC600MHz NMR (Tokyo, Japan).

Additionally, ultra-performanceliquid chromatography-time-of-flightmassspectrometry(UPLC-TOFMS,WatersXevoTM_QTofMS,

Waters, Milford,MA, USA) was performed using a C18 column

(2.1mm×100mm,Waters)withMeOH/H2O=5/95(30min),100/0

(10min)withalineargradientaseluent.TheUPLC-TOFMSdata werecollectedinnegativeionizationmode(Fig.2).

Flavogallonicaciddilactone(1) Tanpowder.1_HNMR(inCD

3OD):ı7.26(s),7.50(s).13CNMR(in

CD3OD):ı108.1(C-1,1′),110.1–114.4(C-6,6′),112.8(C-5),113.3

(C-6′′_{),117.5–120.2(C-5}′_,2′′_),124.9(C-1′′_{),135.7(C-2),136.3} (C-3),136.5(C-4),137.8(C-2′_),139.2(C-3′_),143.2(C-4′_),144.1(C-3′′_), 145.9(C-4′′_),147.8(C-5′′_{),158.9–160.4(C-7,7}′_),168.9(C-7′′_); UPLC-TOFMSm/z469[M−H]−_(calcd.forC

21H10O13470.2963).

Terchebulin(2)

Tanpowder.1_HNMR(inCD

3OD):ı3.04(t,J=11.6Hz,oneof

theH-6′′_{),4.21(t,J=10.3Hz, H-5}′′_{),4.48(t,J=8.9Hz,oneofthe} H-6′′_{),4.78(t,J=11.0Hz,H-4}′′_{),4.98(dd,J=3.5,9.7Hz,H-2}′′_),5.23 (d,J=2.8Hz,H-1′′_{),5.64(t,J=9.6Hz,H-3}′′_{),6.37(s,H-B6),6.42(s,} H-D6),6.56(s, H-A2),6.79(s,H-C2),7.48(s,H-5).13_{C NMR(in}

CD3OD):ı63.4(C-6′′),68.5(C-4′′),69.0(C-5′′), 74.1(C-3′′), 74.2

(C-2′′_),90.2(C-1′′_{),106.4(C-B6),106.5(C-D6),106.8(C-A2),108.5} (C-C2),112.0–114.0(C-A6,B2,5,5′_,1,1′_,2,2′_,6,6′_{),116.0(C-C6),} 122.2(C-D1),123.5(C-B1,C1),125.1(C-A1),135.9(C-B4),136.1 (C-A4),137.5(C-C4),137.6(C-D4),138.4(C-3),139.1(C-D3),140.7 (C-3′_{),141.7(C-D2),143.4–143.6(C-A5,B3,C5),144.5–144.6(C-A3,} B5,C3,D5),147.4(C-4′_{),150.3(C-4),158.3(C-7}′_{),159.5(C-7),166.9} (C-D7),167.0(C-C7),168.9(C-A7),169.5(C-B7);UPLC-TOFMSm/z 1083.07[M−H]−_(calcd.forC

48H28O301084.7179).

Table1

Minimuminhibitoryconcentration(MIC)andglucosyltransferase(GTF)inhibitoryactivitiesofselectedSudanesemedicinalplantextracts.

No. Botanicalname Family Examinedpart Voucherspecimen Collectionplace Extracts MIC(mg/ml) GTF(%)

1 AristolochiabracteolataLam. Aristolochiaceae Wholeplant SD-SH-04 Shambat M –a _–b

2 E 1.0 08.0±4.3h

3 Calotropisprocera(Aiton) Dryand.

Apocynaceae Leaves SD-SH-11 Shambat M 4.0 –

4 E – 06.7±8.9h

5 AmbrosiamaritimaL. Asteraceae Aerialpart SD-SH-03 Shambat M 4.0 –

6 E – 08.9±7.5h

7 XanthiumbrasilicumVell. Asteraceae Leaves SD-SH-12 Shambat M – –

8 E 4.0 –

9 Balanitesaegyptiaca(L.)Delile Zygophyllaceae Leaves SD-KH-15 Khartoum M 4.0 –

10 E 4.0 –

11 Bark M 4.0 –

12 E – –

13 AdansoniadigitataL. Malvaceae Fruitpulp SD-OD-27 Omdurman M – 57.0±1.0d

14 E – 13.7±4.8g,h

15 GuierasenegalensisJ.F.Gmel. Combretaceae Leaves SD-OD-40 Omdurman M 2.0 70.4±1.0c,d

16 E – 69.6±3.0c,d

17 TerminaliabrowniiFresen. Combretaceae Bark SD-GF-02 Elgadarif M 0.5 88.5±2.1a,b

18 E 4.0 90.1±2.1a,b

19 TerminalialaxifloraEngl. Combretaceae Wood SD-KH-03 Khartoum M 0.5 87.2±2.6a,b

20 E 4.0 80.5±1.7a,b,c

21 VernoniaamygdalinaDelile Asteraceae Leaves SD-KH-19 Khartoum M 4.0 –

22 E 4.0 –

23 EuphorbiahirtaL. Euphorbiaceae Aerialpart SD-SH-37 Shambat M – –

24 E – –

25 RicinuscommunisL. Euphorbiaceae Leaves SD-SH-36 Shambat M 4.0 –

26 E 4.0 –

27 Acaciatortilis(Forssk.)Hayne Fabaceae Bark SD-KH-07 Khartoum M 2.0 –

28 E 4.0 –

29 Wood M – –

30 E 4.0 11.2±9.0h

31 Acaciaseyalvar.fistula

(Schweinf.)Oliv.

Fabaceae Bark SD-KH-06 Khartoum M – 82.9±3.0a,b,c

32 E – 89.4±0.1a,b

33 Wood M – –

34 E – 50.3±1.0e

35 AcaciaseyalDelile Fabaceae Bark SD-GF-05 Elgadarif M 1.0 91.5±5.1a,b

36 E 1.0 85.2±3.1a,b,c

37 Wood M – –

38 E 4.0 34.8±4.3f

39 CassiaacutifoliaDelile Fabaceae Leaves SD-SH-24 Shambat M 2.0 –

40 E 2.0 –

41 ParkinsoniaaculeataL. Fabaceae Leaves SD-SH-02 Shambat M 4.0 –

42 E 4.0 07.1±5.0h

43 Khayasenegalensis(Desr.)A.Juss. Meliaceae Bark SD-SH-14 Shambat M 4.0 88.7±3.0a,b

44 E – 77.8±3.8b,c,d

45 PeganumharmalaL. Nitrariaceae Seed SD-OD-20 Omdurman M 2.0 –

46 E – –

47 ArgemonemexicanaL. Papaveraceae Leaves SD-KH-39 Khartoum M 4.0 –

48 E – –

49 Seed M 1.0 –

50 E – –

51 Persicariaglabra(Willd.)M. Gómez

Polygonaceae Leaves SD-SH-A-03 Shambat M 1.0 87.2±9.0a,b

52 E – 78.2±0.6b,c,d

53 Ziziphusspina–christi(L.)Desf. Rhamnaceae Bark SD-SH-06 Shambat M 4.0 94.5±2.3a

54 E 4.0 27.0±5.0f,g

55 Leaves M 2.0 –

56 E – –

57 SolanumdubiumDunal Solanaceae Fruits SD-SH-34 Shambat M – –

58 E – –

59 Tamarixnilotica(Ehrenb.)Bunge Tamaricaceae Stems SD-OD-10 Omdurman M 1.0 85.4±0.3a,b

60 E – 91.4±2.2a,b

61 TribulusterrestrisL. Zygophyllaceae Aerialpart SD-SH-33 Shambat M – –

62 E – –

M,methanol;E,50%hydroethanol.Valueswereexpressedasmean±SD;n=3.Meanswithdifferentlettersinthesamecolumnweresignificantlydifferentatthelevel

(p<0.05).

a_{Noinhibitoryactivityatconcentrationof4mg/ml.}

4 2

1 3

1

2

3

4

0 10 20 30 40 50 60

Fig.1.HPLCfingerprintofmethanolextractofTerminalialaxifloraandtheirisolatedcompounds.(1)Flavogallonicaciddilactone[retentiontime:13.6min],(2)terchebulin

[retentiontime:17.3min],(3)gallicacid[retentiontime:8.8min]and(4)ellagicacid[retentiontime:26.7min].

Biologicalactivitiesofthecrudeextractsandisolatedcompounds

Determinationoftheminimuminhibitoryconcentration(MIC) MICwasdeterminedbythebrothdilutionmethodaccordingto Iwakietal.(2006).S.sobrinus6715wasculturedinaBrain-Heart InfusionBroth.Thecrude extracts,fractionsorpurecompounds weretestedforantibacterialactivityinsterile96-wellplates.The inoculumswerepreparedbydilutingthebrothcultureto approx-imately 106_{cells/ml. To each wellcontaining sample,100}

␮l of

microbialinoculumswereadded,followedbyadditionofmedium toachieveafinalvolumeof200␮l.Thetestedsamplewasprepared

inaconcentrationrangeof4000–31␮g/mlusingatwo-fold

dilu-tionmethod.Solventandmediumcontrolswereincludedineach testplate.Inordertodissolvethesampleextracts,20%dimethyl sulfoxide(DMSO)wasusedinthisstudy.Thefinalconcentrationof DMSOaloneinthewellshowednoinhibitoryeffectonS.sobrinus growth.Theexperimentswereperformedintriplicate. Chlorhex-idinewasincludedintheassaysaspositivecontrol.Thecultures wereincubatedfor24hat37◦_{Cunderanaerobicconditions.} Micro-bialgrowthwasindicatedbytheadditionof50␮lof(0.2mg/ml)

INTtotheculture andincubated at37◦_{C for2h.The MICwas} definedasthelowestconcentrationthatinhibitedthecolorchange ofINT(Eloff,2001).

AssayforGTFinhibitoryactivity

Streptococcussobrinus6715wasculturedfor20hat37◦_Cin4l ofToddHewittbroth.Aftercentrifugationofthecultureat1300×g

for10minat4◦_{C,thecellswerecollectedandthenextractedwith} 8Mureafor1hwhilestirring.Thecrudeenzymesolutionwas dia-lyzedagainst10mMsodiumphosphatebuffer(pH6.0).Thecrude enzymesolutionwasstoredinafreezerat−80◦_C.

GTFwereincubatedin300␮lof0.1Mphosphatebuffer(pH

6.0)containing1%sucrose,0.5%dextranT-10,inthepresenceor absenceofasampleat37◦_{Cfor3h.ThevolumeofthecrudeGTF} solutionusedintheassaywasdeterminedbymeasuredturbidity around1.0absorbanceat590nm(Mitsunagaetal.,1997).

Inhibition (%)

=Absorbanceofcontrol−Absorbanceofsample

Absorbanceofcontrol ×100

Statisticalanalysis

TheinhibitorypercentageandIC50valuesofGTFwereexpressed

asthemean(mean±standard deviation).Thesignificant differ-ences between samples were assessed by one-way analysis of variance(ANOVA)followedbypairwisecomparisonofthemean usingTukey’smultiplecomparisontest.Valuesweredetermined tobesignificantwhenpwaslessthan0.05(p<0.05).

Results

A

20120901_1-4 110 (0.861)

3W 17

20120901_1-5 111 (0.868)

01-Sep-201220:21:10 1: TOF MS ES-3.33e4

01-Sep-201221:13:08 1: TOF MS ES-3.58e3 0

100

100

0

%

425.016

163.037

169.013

177.021298.985 301.000

423.018

392.996 425.018

450.997

495.023

539.011

595.093 626.112

627.129

729.207 781.069 861.258 993.304 1035.321 1081.055

1085.081 1084.073 1083.071

625.108

1086.073 1106.038

1167.355 245.155 249.041

335.024397.024 423.002

469.007

470.011 471.013

993.308

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

m/z

%

B

Table2

IC50values(␮g/ml)obtainedbythepotentmethanolicextractsagainst

glucosyl-transferase(GTF)enzyme.

Botanicalname Partused GTFIC50(␮g/ml)

A.seyal Bark 03.8±1.7a

T.brownii Bark 09.2±3.4a

T.laxiflora Wood 10.3±2.2a

K.senegalensis Bark 23.6±2.6b

P.glabra Leaves 27.1±4.1b,c,d

A.digitataL. Fruitpulp 35.9±3.2c,d,e

Z.spina–christi Bark 38.8±3.5d,e,f

A.seyalvar.fistula Bark 47.2±3.6e,f

T.nilotica Stems 49.8±4.9f

Meanswithdifferentlettersinthesamecolumnweresignificantlydifferentatthe level(p<0.05);n=3.

hydroethanolic extracts were prepared; and their antibacterial activitiesagainstS.sobrinusandGTFenzymeinhibitoryactivities wereinvestigated.

Antibacterialassay

To evaluate the antibacterial activity of selected Sudanese medicinalplantextractsagainstS.sobrinususingdilution meth-ods.TheMICweredeterminedinTable1.Among62plantextracts 35 extracts showed antibacterial activity. Alsonoteworthy the methanolicextractsofCombretaceae family;Terminaliabrownii (bark)andT.laxiflora(wood)demonstratedhighestantibacterial activity(MICof0.5mg/ml)amongthem.

GTFenzymeinhibitoryactivity

Inhibitoryeffectsover55%onGTFenzymeactivitywere demon-stratedby18ofthe62plantextractsat100␮g/ml(Table1);of

thesethirteenextractsexhibitedinhibitoryactivitymorethan80%. Table2showstheIC50 valuesof GTFinhibitoryactivityof nine

methanolicextractsrangedbetween3.8and49.8␮g/ml.A.seyal

(bark),T.brownii(bark)andT.laxiflora(wood)displayasignificant inhibitoryactivity.

Combinedactivitiesofthecrudeextractsandisolatedcompounds

Methanolicextracts of T. brownii (bark),T. laxiflora, A. seyal (bark), Persicaria glabra (leaves) and Tamarix nilotica (stems) demonstratedMIC≤1mg/mlagainstS.sobrinusandIC50lessthan

50␮g/mlagainstGTFenzyme.

Methanolicwoodextractsof T. laxiflorawhich showedgood combinedactivities(MIC0.5mg/ml,GTF,IC5010.3␮g/ml),were

selectedfor furtherpurification,crudemethanolicextractsafter subjectedtoMPLCresultedin twofractions F1(MIC0.5mg/ml, GTF96.4%) and F2(MIC1mg/ml, GTF93.1%); moreover purifi-cationrevealingthepresence offlavogallonicaciddilactone(1), terchebulin(2), gallicacid(3),and ellagicacid(4).Antibacterial andGTFinhibitoryactivitiesforisolatedcompoundsareshownin Table3.Flavogallonicaciddilactone(1)demonstratedrelatively goodantibacterialactivity,withMICof0.5mg/ml.Terchebulin(2) andellagicacid(4)showedmoderateantibacterialactivity, how-ever,terchebulin(2)displayedpotentactivityagainstGTFenzyme.

Discussion

AntibacterialandGTFenzymeinhibitoryactivitiesofextracts

Inthisstudysolventselectionreliedonpreviousstudies men-tioningthatmethanolisclassifiedasa polarsolventdue tothe presence of hydroxyl group.Nevertheless, there is alsomethyl

Table3

Anti-S.sobrinusandglucosyltransferase(GTF)inhibitoryactivityofcompounds

iso-latedfromT.laxiflorawood.

Compound MIC(mg/ml) GTFIC50(␮M)

Terchebulin 1 7.5±2.7a

Gallicacid –a _NC

Ellagicacid 1 1017.5±4.1c

Flavogallonicaciddilactone 0.5 149±1.6b

Chlorhexidineb _{0.0004 5.8±3.3a}

NC,theIC50couldnotbecalculatedbecausetheactivitywaslessthan50%athighest

concentration.Meanswithdifferentlettersinthesamecolumnweresignificantly

differentatthelevel(p<0.05);n=3.

a_{Noinhibitoryactivityatconcentrationof4mg/ml.}

b_{Positivecontrol.}

group presence in methanol, which is sort of non-polar, so it hasability toextract varioustypes ofcompounds and increase extract’syield.Furthermore,itcangivehigherconcentrationsof bioactivemoleculesfromplantssuchasdifferentclassesof phe-noliccompounds.50%hydroethanolyieldhighcontentofphenolic andflavonoidcompounds(Ahmadetal.,2009;Cauniietal.,2012; Thanhetal.,2016).Somestudiesmentionedthatpolyphenols,such as flavonoids,phenolic acidsand tanninsshowed anti-enzyme, antibacterialand/oranti-biofilmactivities(Gulatietal.,2012;Livia etal.,2016).

TheMICvaluesofthemethanolextractswererelativelylower thanthoseofthe50%hydroethanolicextracts,implyingmoreactive antibacterialcompositioninmethanolthanin50% hydroethano-lic extracts. These findings were similar to those reported by Samuelsen (2000).Ncube et al. (2012) believed that the crude extractwillbeactivewhenhavingMICvalueslessthan8mg/ml, whilst Gibbons (2005) suggested that isolated phytochemicals shoulddemonstrateatleastMIC<1mg/ml.Inthisstudy,MICvalues of0.5mg/mlwereconsideredanindicationofgoodantibacterial activity.

Zhietal.(2016)statedthatinhibitionofGTFactivityandthe con-sequential polysaccharidesynthesis maydiminishthevirulence ofcariogenicbiofilms, whichcouldbeanalternativestrategyto eradicatedentalcaries.MethanolicextractsofA.seyal(bark),T. brownii(bark)andT.laxiflora(wood)showedsignificantinhibitory activitieson GTF.A previous studymentioned that methanolic extractsoftheseplantscontaincondensedandhydrolysable tan-nins (Muddathir and Mitsunaga, 2013).Yamauchi et al. (2016) reportedthatT.brownii(bark)containedgallicacid,punicalagin, terchebulin,ellagicacid4-O-␣-l-rhamnopyranoside,ellagicacid and3,4,3′_{-tri-O-methylellagicacid.Plantpolyphenolssharedwith} catechin-basedoligomericforms(condensedtannins)and/or gal-lateesterformcompounds(hydrolysabletannins)displaystrong anti-GTFactivities(Yanagidaetal.,2000).

BiologicalactivitiesofcompoundsisolatedfromTerminalia laxiflorawood

OurchemicalprofilingofthemethanolextractsoftheT.laxiflora wooddisclosedthepresencesoffourcompoundsaswedescribed inexperimentalpart.Flavogallonicaciddilactone(1)showed1_H

NMRspectral patternwithtwo1_{Hsinglets(ıH7.26and7.50).} 13_{CNMRspectrumdisplayedsignalsofninenon-oxygenated}

excellentlyreportedbyseveralauthors(GrimshawandHaworth, 1956;Tanakaetal.,1986,1996;Kinjoetal.,2001;Hiranoetal., 2003;Shuaibuetal.,2008;Ibrahimetal.,2014;Orabietal.,2015). Flavogallonicaciddilactone(1)wasisolatedearlierfrom Termina-liacatappainfreeformandasasingle-bondedacylunitontheir tanninpyranosecores,suchasterflavinsA–D(Tanakaetal.,1986). The1_Hand13_{CNMRdataofterchebulin(2)weresimilartothose}

reportedbyLinetal.(1990).TheUPLC-TOFMS(m/z1083[M−H]−₎ datawasinagreementwithSilvaetal.(2000).PreviouslySilvaetal. (2000)reportedthatterchebulinwasthemaincompoundpresent inTerminaliamacropteraroot.Gallicacid(3)andellagicacid(4) werealsodetectedin T.laxiflorawoodextract;thesetwo com-poundswereisolatedfromdifferentplantsofthegenusTerminalia (Silvaetal.,2000;Shuaibuetal.,2008).

Data in Table3 showedthat flavogallonicaciddilactone (1) exhibitedgood antibacterialactivity(0.5mg/ml).From previous study,theactivityofPropionibacteriumacneswasinhibitedat con-centration0.25mg/ml(MuddathirandMitsunaga,2013).TheMIC forgallicacid(3)hasnoactivitytowardS.sobrinusupto4mg/ml. Kangetal.(2008)statedthattheMICofgallicacidagainstS. sobri-nuswas8mg/ml.TheMICofellagicacid(4)was1mg/ml.Sarabhai etal.(2013)mentionedthatpureellagicaciddidnotexertmuch biofilminhibitingeffect.

Accordingtoourfindings, ellagicacid(4)showedweakGTF inhibitoryactivity(IC501017.5␮M).EventhoughSawamuraetal.

(1992)suggestedthat theuseofellagicacidwillnotaffectthe ecologicalbalanceoforalbacterialflora,butitcouldbeapossible anti-carriesagentthroughitsGTFinhibitoryaction.

Terchebulin(2)exhibitedsignificanceactivityagainstGTF(IC50

7.5␮M),whencomparedtothepositivecontrolofchlorhexidine

(IC505.8␮M).Oolongteafractionrichinhighmolecularweight

polyphenolsinhibitedthesynthesisofglucannon-competitively (Matsumotoetal.,2003).

Chlorhexidinedemonstrated thestrongest activity againstS. sobrinus and in addition, it showed good activity against GTF (Table 3).Nonetheless, it hasbeenreported that chlorhexidine wasfoundtobeacytotoxicagenttomurinefibroblastcelllines, humanalveolarbonecellsandhumanosteoblasticcellline.These resultsapprovethatchlorhexidineisnotcelltypespecific(Cabral andFernandes,2007;Giannellietal.,2008;Fariaetal.,2009;Li etal.,2014).Toxicityassaywithmousefibroblastsshowedthat ellagicacid,terchebulinandflavogallonicacidisolatedfrom Termi-naliaavicennoidesstembark,hadIC50≥1500␮g/mlaswellasthey

didnotaffecttheintegrityofhumanerythrocytemembraneofthe human(Shuaibuetal.,2008).

Conclusion

Inthepresentstudy,T.laxiflorawooddemonstratedsignificant anti-cavityactivity.Theseresultsjustifytheuseofthisplantfor oral carein traditional African medicine.The promising results ofantimicrobialandGTFinhibitoryactivityshownhere,suggests terchebulin(2)andflavogallonicaciddilactone(1)couldbe con-sideredforfurtherpharmacologicalstudies,evaluatingthetoxicity anddevelopmentofanaturalanticariogenicagentfordentalcaries.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

EAMM,AMM,KYandTMallcontributedtothewritingofthis article.EAMMandAMMobtainedsamples.TMdesignedthestudy andsupervisedthelaboratorywork.EAMMandAMMperformed thedifferentassaysandstatisticalanalysis.KMcontributedto com-poundidentification.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authorsthank Dr.Ashraf Mohamedfrom theFaculty of Forestry,Mrs.HamzaTagEL-Sir,Botanist,FacultyofAgriculture, DepartmentofBotany(Herbarium),UniversityofKhartoum,Sudan fortheirassistanceintheplantsidentificationandauthentications. EAMMacknowledgeforUniversityofGifu,Japan.

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