Rev. bras. farmacogn. vol.27 número6

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

Original

Article

In

vitro

antitubercular

activity

of

extract

and

constituents

from

the

stem

bark

of

Disthemonanthus

benthamianus

Jean

Noel

Evina

_,

_Dominique

_Serge

_Ngono

_Bikobo

_,

_Auguste

_Abouem

_A.

_Zintchem

_,

Norbert

Mbabi

Nyemeck

II

_,

_Esther

_Del

_Florence

_Moni

_Ndedi

_,

_Patrick

_Hervé

_Betote

_Diboué

_,

Maximilienne

Ascension

Nyegue

_,

_Alex

_de

_Théodore

_Atchadé

_,

_Dieudonné

_Emmanuel

_Pegnyemb

_,

Ulrich

Koert

_,

_Christian

_G.

_Bochet

a_{DepartmentofOrganicChemistry,FacultyofScience,UniversityofYaoundéI,Yaoundé,Cameroon}

b_{DepartmentofChemistry,HigherTrainingCollege,UniversityofYaoundéI,Yaoundé,Cameroon}

c_{FacultyofChemistry,Philipps-UniversitätMarburg,Marburg,Germany}

d_{DepartmentofMicrobiology,FacultyofScience,UniversityofYaoundéI,Yaoundé,Cameroon}

e_{DepartmentChemie,UniversitätFribourg,Fribourg,Switzerland}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10May2017 Accepted12September2017 Availableonline4November2017

Keywords:

Fabaceae

Antitubercularactivity Distemonanthoside Flavonoids Phenolicacids

a

b

s

t

r

a

c

t

A new C-glycosylflavone, apigenin7-methylether 6-C-[␤-xylopyranosyl-(1→3)-␤-glucopyranoside]

named distemonanthosidewas isolatedfromthestem barkofDistemonanthusbenthamianus Baill.,

Fabaceae,alongwithsixknowncompounds,sitosterol3-O-␤-d_{-glucopyranoside,4-methoxygallicacid,}

syringicacid,quercetin,6′′_{-O-acetylvitexin,quercetin3-O-␤-}d-glucopyranoside.Thestructuresofthose

compoundsandothersweredeterminedthroughspectralanalyses.Compoundsdistemonanthoside,

sitosterol3-O-␤-d_{-glucopyranoside,4-methoxygallicacidandquercetinweretestedagainstaclinical}

iso-latestrainofMycobacteriumtuberculosisAC45;theyexhibitedgoodtomoderateantitubercularactivities

withMICvaluesrangedfrom31.25to125␮g/ml.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Tuberculosis (TB) is a chronic contagious disease caused by severalspeciesofMycobacterium.Duetothefactthatthereisa doubtnowadaysontheefficiency ofcurrent antibiotics forthe treatmentoftuberculosis,micro-organismsdevelopedresistance inducinganincreaseofthenumberofpatientswiththedisease inworldwide(WHO,2016).ThisincreasingofMDR-TBincidence hasledtoanurgentneedforthediscoveryofnewplantnatural productsthatmaypotentiallyeradicateTB.Severalinvitrogrowth inhibitionofdifferentstrainsofM.tuberculosisbyplantextracts havebeenreported(Okunadeetal.,2004;CoppandNorriePearce, 2007;Gautametal.,2007;McGawetal.,2008).The Cameroo-nianmedicinalplantDistemonanthusbenthamianusBaill.,Fabaceae, isa largerainforesttreewidely distributedin Africa, especially inequatorialregion.Thisessenceishighlyappreciated industri-allyforheavyconstructionandsomecountriesusetoexportitas

∗ Correspondingauthor.

E-mail:ngonos@uy1.uninet.com(D.S.Bikobo).

“Movingi”.InMayombéregion(Congo),traditionalhealersemploy thestembarkinthetreatmentofseveraldiseasesas:parasitic, der-matitis,furuncles,acesandchancres.IntheChailluregion(Congo), thatplantisusedtocurebronchitisaffectionsandchildrenfever (Bouquet,1969).In previousworkscarried onD. benthamianus, mainlymethoxylatedflavonolsandflavoneswereisolated(King etal.,1952;Kingetal.,1954;MalanandRoux,1979;Happiand Mpondo,1994);this paperdescribestheisolation andstructure elucidationofconstituentsfromstembarkofD.benthamianus.The evaluationofantitubercularactivitiesofcompounds distemonan-thoside(1),sitosterol3-O-␤-d-glucopyranoside,4-methoxygallic acid(2)andquercetinagainstresistantstrainofM.tuberculosiswas alsoexamined.

Materialandmethods

Generalprocedures

Meltingpointswereuncorrectedandweremeasuredona Met-tlerToledoinstrument.IRspectrawererecordedonanAlphaFT-IR SpectrometerfromBruker,while1D and2DNMR spectrawere

https://doi.org/10.1016/j.bjp.2017.09.006

obtainedonaBrukerDRX500(500MHzfor1_{Hand125MHzfor} 13_{C spectra)spectrometer(Bruker,Rheinstetten,Germany)with}

chemicalshiftsreportedinı(ppm)usingTMS(ıH)asan inter-nalstandard.TheHR-ESI-MSwasobtainedonLTQ-FTinstrument (ThermoScientific).UPLC–MSwasmeasuredbyaShimadzu UPLC-MSsystem.OpticalrotationsweremeasuredonaPerkin-Elmer 341polarimeter.Silica gel 60 (230–400 meshE. Merck, Darm-stadt,Germany)wasemployedforcolumnchromatography,the solventmixingsystemsforelutionweremainlyCH2Cl2/MeOHwith increasingpolarityeach.

Plantmaterial

Stem bark of Distemonanthus benthamianus Baill., Fabaceae, werecollectedatEséka(Koumoul)nearYaoundé(3◦₃₈′_60.00′′_N,

10◦₄₆′_0.00′′_{E)intheCentreRegionofCamerooninMarch2014}

andidentifiedbyVictorNana.Avoucherspecimen(No.45488HCN) wasdepositedattheNationalHerbariuminYaoundé,Cameroon.

Extractionandisolation

DriedandpowderedstembarkofD.benthamianus(254g)were extractedfor48hwithMeOH(3×1l)atroomtemperature.After

filtrationandevaporationofsolvent,thecrudeMeOHextract(16g) wassubjectedtoCC(150×3cm)[(SiO2),elutingwithagradient solventsystem(CH2Cl2/MeOH)]givingfourmainfractions:I(1.9g), II(3.8g),III(3.6g)andIV(6.7g).Fractions(100ml)werecollected andgroupedonthebasisofTLCanalysis.FractionII(3.8g)was sub-mittedtoCC(SiO2,100×1cm)usingsolventsystemCH2Cl2/MeOH

(50/1)togivesitosterol3-O-␤-d-glucopyranoside(65mg).Fraction III(3.6g)wassubmittedtoCC(SiO2,100×1cm)usingsolvent

sys-temCH2Cl2/MeOH(60/1to5/1)togivefoursub-fractions(IIIa,IIIb, IIIcand IIId).Sub-fractionIIIc(1g) waschromatographed(SiO2, 50×1cm)usingCH2Cl2/MeOH(40/1–15/1)toafford compound

2(480mg)andcompound3(3mg).Sub-fractionIIId(0.65g)was subjectedtoasilicagelcolumningradientelutionmixturesolvent composedofCH2Cl2/MeOH(25/1–5/1)toaffordquercetin(8mg) andquercetin3-O-␤-d-glucopyranoside(11mg).Usingthesame process,fractionIV(6.7g)gavethreesub-fractions(IVa,IVbandVc). Sub-fractionIVa(0.98g)wasfurtherchromatographedonasilica gelcolumn(100×1cm)usingCH2Cl2/MeOH(10/1)toafford com-pound4(4mg).Sub-fractionIVb(2.8g)waspurifiedbyrepeated CConsilicagel(100×1cm)withthesolventsystemCH2Cl2/MeOH (10/1–1/1)toprovidecompound1(28mg).

Structuralcharacterizationofdistemonanthoside(1)

Yellowsolid;[˛]25_D =−54◦ _{(c0.05, MeOH);m.p.285–287}◦_C;

IRmaxKBr_cm−1_{:3267,2923,2853,1595,1512,1226,1159;TLC,R} f: 0.28(CH2Cl2/MeOH:90/10);ESI-MSm/z:ESI-MS:577.4[M−H]−•,

LC–MS:m/z579[M+H]+_{andESI-MS:m/z601.5[M+Na]+(Calcdfor} C27H30O14Na=601.5);1_{HNMR(500MHz,DMSO-d}

6),ıH:8.09(2H,

d,J=8.8Hz,H-2′_andH-6′_{),6.96(2H,d,J=8.8Hz,H-3}′_and5′_),6.82

(1H,s,H-3),6.44(1H,s,H-8),4.81(1H,d,J=10.0Hz,H-1′′_),4.01b

(1H,H-3′′_{),3.86(3H,s,–OCH3),3.82(1H,d,J=7.0Hz,H-1}′′′_),3.72

(1H,dd,J=11.3;2.4Hz,H-6′′_),3.42b _(1H,H-6′′_),3.39b_(1H,H-2′′_),

3.36b_(1H,H-4′′_),3.21b_(1H,H-5′′_{),2.83(1H,dd,J=11.5;4.0Hz,}

H-5′′′_),2.81b_(1H,H-3′′′_),2.78b_(1H,H-2′′′_),2.39b_(1H,H-5′′′_);13_CNMR

(125MHz,DMSO-d6):ıC:182.1(C-4),164.1(C-2),163.4(C-7),161.4 (C-9),161.2(C-4′_{),155.5(C-5),128.9(C-2}′_andC-6′_)121.3(C-1′_),

115.8(C-3′_andC-5′_),105.3(C-1′′′_{),104.7(C-6),104.1(C-10),102.3}

(C-3),94.8(C-8),81.7(C-5′′_),80.4(C-3′′_),78.1(C-2′′_),76.4(C-3′′′_),

75.6(C-2′′′_),73.5(C-1′′_),71.3(C-4′′_),69.2(C-4′′′_),65.3(C-5′′′_),60.8

(C-6′′_{),56.5(–OCH3).}

b_{Signalpatternsareunclearduetooverlap.}

Antitubercularactivity

MICvaluesweredeterminedfortheextractagainstM. tuberculo-sisstrainAC45(clinicalisolateobtainedfromSangmelimadistrict’s HospitalinSouthRegionofCameroon)employingthemicroplate AlamarBlueassay,usingRifampicinasreference.The96wellsplate received100␮lofMiddlebrook7H9mediumsupplementedwith 10%OADC(oleicacid,albumin,dextrose,catalase)2%glyceroland 0.05%v/voftween80.Brothandserialdilutionofcompoundswere madedirectlyontheplatewithdrugconcentrationsof0.244to 250␮g/ml.Plateswerecoveredandsealedwithparafilmand incu-batedat37◦_{Cfor14days.Then,40␮lAlamarBluesolutionwas}

addedtotheplateandincubatedfor24h.Abluecolourinthewell wasinterpretedasnobacterialgrowthandpinkcolourwasscored asgrowth.TheMICwasdefinedasthelowestdrugconcentration, whichpreventedcolour changefrombluetopink.Theresultof antitubercularactivitydepictedinTable1.TheMICandMBCwere determinedaccordingtotheguidelinesofCLSI(2011).Each exper-imentwasperformedatleasttwiceaccordingtotheguidelinesof theClinicalandLaboratoryStandardsInstitute(CLSI,2011).

Acidhydrolysisof1

Compound 1 (8mg) wasdissolved in7% H2SO4 (0.5ml) and heatedonanaqueousbathat100◦_{Cfor4h.Thereactionmixture}

wasdilutedwithH2OandextractedwithCH2Cl2.TheCH2Cl2layer wasevaporatedtodrynessandpurifiedbypreparativeTLCover silicagelwithCH2Cl2–MeOH(5/1)aseluent.Apigenin-7-methyl ether 6-C-glucoside (3mg) was isolated and identified through directcomparisonwithauthenticsamples(TLC,MP,andIR).The neutralizedand lyophilizedaqueuoushydrosylatesof the aque-oussolutiongaveonlyxylose.GC-MS(Column:5%phenyland95% methylsiliconeonultra2,0.2×46m,columntemp.:250◦_C,

car-riergas:He0.8ml/min,sample:trimethylsilylderivatives:tR(min) xylose(19.29for1).

Table1

MICandMBCvaluesofthemethanolextractandtheisolatedcompoundsagainstclinicalisolatestrainofMycobacteriumtuberculosis(AC45).

Plantspecies/compounds MICa_{(␮g/ml) MIC}a_{(␮M) MBC}b_{(␮g/ml) MBC/MIC}

D.benthamianus 1250 ndc _{2500 2}

1 125 216.3 125 1

Sitosterol3-O-␤-d-glucopyranoside 62.5 108.5 125 2

2 31.25 169.8 125 4

Quercetin 62.5 207.0 125 2

RMP 0.976 ndc _{7.8125 8}

RMP,Rifampicin.

Resultsanddiscussion

The detailed investigation of methanol extract of the stem barkofD.benthamianusledtotheisolationofsevencompounds. Six of them were identified as the known sitosterol 3-O-␤-d -glucopyranoside (Ngono Bikobo et al., 2014), 4-methoxygallic acid(2)(Ouyangetal.,2007),syringicacid(3)(BayihaBaNjock et al., 2011), quercetin (Güvenalp and Demirezer, 2005), 6′′

-O-acetylvitexin (4) (Bayiha Ba Njock et al.,2011), quercetin 3-O-␤-d-glucopyranoside(Muraietal.,2014).Thestructuresofthese compoundswereelucidatedbyNMRspectroscopyanalysis, includ-ing1Dand2Dtechniquesandalsobycomparingexperimentaldata withrespectiveliteraturedata

Compound 1 was obtained as yellow amorphous powder, [˛]25_D =−54◦ (c=0.05,MeOH).Its molecularformula, C27H30O14 wasestablishedbynegative-ionHR-ESI-MS(Fig.S4).Thespectrum displayedthedeprotonatedmoleculepeak[M−H]−atm/z=577.4

inagreementwiththeaboveformula(calcd,577.44).TheIR spec-trum of 1 showed absorption bands characteristic of hydroxyl groups(3219cm−1_{),conjugatedcarbonylgroups(1652cm}−1_)and aromaticrings(1603and1572cm−1_{).UVspectralpropertiesof1}

showedabsorptionmaximaatmax340nmand268nminMeOH, characteristic for a substituted flavone (Mabry et al., 1970). In addition,acidhydrolysisof1gaveapigenin7-methylether 6-C-glucosideand␤-xylosewhichwereidentifiedbyTLCanalysisand comparisonwithauthenticsamples(GC;tR19.29min).Inthe1H NMRspectrum(Table2)thesetofortho-coupledAA′_BB′_type

pro-tonsatıH8.04(2H,d,J=8.8Hz)and6.96(2H,d,J=8.8Hz),was respectivelyassignedtoH-2′_/6′_andH-3′_/5′_{protonsoftheB-ringof}

themolecule, whileanisolatedaromaticprotonappearedatıH 6.44(s, H-8)fromAring.Thespectrum alsorevealedthe pres-enceofamethoxylgroupatıH3.86and twosignalsassignable toanomericsugarprotons,whichwereidentifiedtobeaninner ␤-glucopyranoseandaterminal␤-pyranosestructure ofxylose. Thiswasstrengthenedbytheobservationin13_CNMRandDEPT spectraofelevencarbonsignals(Table2)amongwhichtwoare anomericcarbonsignalsatıC73.5and105.3,sevenmethinecarbon signals,twooxymethylenecarbonsatıC60.8and65.3.Sincethe anomericprotonsofglucoseandxyloseatıH4.81and3.82 exhib-itedlargecouplingconstants(J=10.0and7.0Hz),thesugarswere consideredofthe␤-pyranosetype.TheHMBCspectrumof com-pound1revealedcorrelationsoftheanomericprotonatıH4.81 (H-1′′_{)andcarbonsatıC161.3(C-7),155.6(C-5)104.7(C-6)and}

81.7(C-5′′_{)(Fig.1),indicatingtheC-Cbondbetweentheinner␤}

-glucopyranosylmoietyandtheaglyconeat6-position.Inaddition, H-1′′′_{atıH3.82correlatestobothC-3}′′_{(ıC80.4)andC-5}′′′_(ıC65.3)

Table2

1_Hand13_{CNMRspectroscopicdataofcompound1(500and125MHzinDMSO-d6)} ıinppm.

Position ıC DEPT ıH(JinHz) HMBC(C→H)

Apignenin

2 164.1 C H-C(3);H-C(2′₎

3 102.3 CH 6.82(s)

4 182.1 C H-C(3)

5 155.5 C H-C(1′′₎

6 104.7 C H-C(1′′₎

7 163.4 C H-C(1′′_);CH3

O-8 94.8 CH 6.44(s)

9 161.4 C H-C(8)

10 104.1 C H-C(3)

1′ _{121.3 C H-C(3);H-C(2}′_,

6′₎

2′ _{128.9 CH 8.04(d,8.8) H-C(3}′_,5′₎

3′ _{115.8 CH 6.96(d,8.8) H-C(2}′_,6′₎

4′ _{161.2 C H-C(2}′_);

H-C(5′₎

5′ _{115.8 CH 6.96(d,8.6) H-C(2}′_,6′₎

6′ _{128.9 CH 8.04(d,8.6) H-C(3}′_,5′₎

7-OCH3 56.5 CH3 3.86(s)

Innerglucose

1′′ _{73.5 CH 4.81(d,10) H-C(2}′′_);

H-C(5′′₎

2′′ _{78.1 CH 3.39}a _H-C(1′′₎

3′′ _{80.4 CH 4.01}a _H-C(1′′₎

4′′ _{71.3 CH 3.36}a _H-C(2′′_);

H-C(5′′₎

5′′ _{81.7 CH 3.21}a _H-C(1′′_);

H-C(6′′₎ 6′′ _{60.8 CH2 3.72(dd,11.3;2.4)}

3.42a

H-C(5′′₎

Terminalxylose

1′′′ _{105.3 CH 3.82(d,7.0) H-C(2}′′′_); H-C(5′′′₎

2′′′ _{75.6 CH 2.78}a _H-C(1′′′₎

3′′′ _{76.4 CH 2.81}a _H-C(1′′′_);

H-C(5′′′₎

4′′′ _{69.2 CH 2.89}a _H-C(5′′′₎

5′′′ _{65.3 CH2 2.83(dd,11.5;4.0)} 2.39a

H-C(1′′′_); H-C(4′′′₎ a_{Signalpatternsareunclearduetooverlap.}

revealingthatthe␤-xylopyranosylmoietywaslinkedtoC-3′′_at ıC80.4,showingthatglucoseandxylosearelinkedthrougha1→3

type.ThiswasstrengthenedbytheNOESYcrosspeaksoftheprotons H-3′′_{(ıH4.01)withH-1}′′′_{(ıH3.82)confirmingtheaforementioned}

bonding.Theattachmentofamethoxylgrouptothe7-positionwas shownbytheobservationofthecrosspeaksatıH3.86(3H,s,OMe) andıC164.1(C-7)inthelong-rangeHMBCspectrum.Moreover theNOESY(Fig.1)experimentconfirmedthispositionthroughthe correlationbetweenH-8(ıH6.44)andthemethoxylproton sig-nalsatıH3.86.Thecompleteassignmentofallprotonandcarbon resonanceswasachievedaftercarefulanalysisofCOSY,HSQCand HMBCtechniques.

SomesignificantHMBCcorrelationsareshowninFig.1andin Table2.Compound1iscloselyrelatedtothepreviouslyreported swertisin 2′′_{-O-arabinoside from thetall beardediris (Takayuki}

etal.,2012);meanwhile,differencesoccurinthesequenceofsugar moietiesandthisisexemplifiedbythevaluesoftheretentiontimes ofxylose[whichisclosetoreporteddata(Liuetal.,2009)].This assertionisalsostrengthenedbytheupperchemicalshiftvaluesof protonsofthexylosemoietiescomparedtothoseofarabinose(Gu etal.,2011).

Accordingly,1wasdefinedasapigenin7-methylether6-C-[␤ -xylopyranosyl-(1→3)-␤-glucopyranoside]named

O O

OH O

OH

O

O

HO HO

O HO

HO

H H

H H

H H

H

C H

H H

2

4 6

8

10

9

1' 3'

4'

6'

1'' 3''

6''

5''' 1'''

3'''

H

H OH

OH 4'''

2'' 4''

2'''

Fig.1. SelectedHMBC( )andNOESY( )correlationsofcompound1.

Accordingto Cantrell et al. (2001) isolated compounds that exhibitaMICof64␮g/mlorlowerareconsideredpromising.For crudeextracts,theMICshouldbeequaltoorlowerthan125␮g/ml (Guetal.,2004).Thus,thevaluesof125,62.5,31.25and62.5␮g/ml for 1, sitosterol 3-O-␤-d-glucopyranoside, 4-methoxygallicacid andquercetin,respectivelyobtainedhere,areasgoodasa promis-ingisolatedcompoundsexceptforcompound1(Table1).According toGuetal.themethanolextractofD.benthamianusshowedpoor inhibitoryactivityagainstM.tuberculosis,exhibitingaMICandMBC of1250and2500␮g/mlrespectively,suggestingthelow lipophilic-ity of its constituents (more polar compounds) when they act mutuallyinsynergy.AccordingtoPetersonandShanholtzer(1992) bacteriostaticactivityhasbeendefinedasaratioofMBCtoMICof >4.Thus,alltestedcompoundsexhibitedbactericidalactivity.The resultsofthepresentstudyareinaccordancewithpreviousreport regardingthevaluesofMICofisolatedcompounds(Guetal.,2004; Jiménez-Alleranesetal.,2007).

Conclusion

Thespecies D. benthamianus,is known asabundant sources offlavonoids.Compounds1,sitosterol3-O-␤-d_{-glucopyranoside} and 4-methoxygallicacid wereisolated for the first time from thisspecies.Thebioactivitystudyoftheisolatedcompounds indi-catedthatthreecompounds(sitosterol3-O-␤-d_{-glucopyranoside,} 4-methoxygallicacidandquercetin)exhibitedinteresting antitu-bercularactivity.

Authors’contributions

JNE(PhDstudent)contributedrunningthelaboratorywork,and draftedthepaper;EFMN,PHBDandMANcontributedto biologi-calstudies,runningthelaboratorywork,analysisofthedataand draftedthepaper;NMNcontributedtoanalysisofthedataand draftedthepaper;UK,ATA,DEPandCBcontributedtocritical read-ingofthemanuscript;AAZandDSNBcontributedincollectingplant samples,supervisedthelaboratorywork,didtheNMR investiga-tionsand revisedthepaper.Alltheauthorshaveread thefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorsgratefullyacknowledgefinancialsupportfromthe SwissNationalScience Foundation(SNSF)(No: IZK0Z2-157272) forresearchfellowshipsinSwitzerlandtoD.S.NgonoBikobo.We thank MrV. Nanafor the collectionand identification ofplant material.WethankMrFelixFehrofDepartmentofChemistryof UniversityofFribourgandKoert’steam,particularlyMrOliverBorn ofPhilipps-UniversitätMarburgforspectralanalysis.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.09.006.

References

BayihaBaNjock,G.,Bartholomeusz,T.A.,Foroozandeh,M.,Pegnyemb,D.E., Chris-tens,P.,Jeannerat,D.,2011.NASCA-HMBC,anewNMRmethodologyforthe resolutionofseverelyoverlappingsignals:applicationtothestudyof agathis-flavone.Phytochem.Anal.23,126–130.

Bouquet, A., 1969. Fetish and traditional medicine of Congo (Brazzaville). O.R.S.T.O.M.Paris36,177–178.

Cantrell, C.L.,Franzblau,S.G., Fischer,N.H.,2001. Antimycobacterialplant ter-penoids.PlantaMed.67,685–694.

CLSI,2011.SusceptibilityTestingofMycobacteria,Nocardiae,andOtherAerobic Acti-nomycetes,2nded.ClinicalandLaboratoryStandardsInstitute(CLSI),Wayne, PA,USA,ApprovedStandardM24-A2.

Copp,B.R.,NorriePearce,A.,2007.Naturalproductgrowthinhibitorsof

Mycobac-teriumtuberculosis.Nat.Prod.Rep.24,278–297.

Gautam,R.,saklani,A.,Jachak,S.M.,2007.Indianmedicinalplantsasasourceof antimycobacterialagents.J.Ethnopharmacol.110,200–234.

Gu,J.Q.,Wang,Y.,Franzblau,S.G.,Montenegro,G.,Yang,D.,Timmermann,B.N.,2004. AntitubercularconstituentsofValerianalaxiflora.PlantaMed.70,509–514. Gu,X.,Lee,S.G.,Bar-Peled,M.,2011.BiosynthesisofUDP-xyloseandUDP-arabinose

inSinorhizobiummeliloti1021:firstcharacterizationofabacterialUDP-xylose

synthase,andUDP-xylose4-epimerase.Microbiology157,260–269. Güvenalp,Z.,Demirezer,O.,2005.FlavonolglycosidesfromAsperulaarvensisL.Turk.

J.Chem.29,163–169.

Happi,E.N.,Mpondo,T.N.,1994.Twopolymethoxylatedflavonesfrom

Distemonan-thusbenthamianus.J.Nat.Prod.57,291–293.

Jiménez-Alleranes,A.,Meckes,M.,Torres,J.,Herrera-Luna,J.,2007. Antimycobacte-rialtriterpenoidsfromLantanahispida(verbenaceae).J.Ethnopharmacol.111, 202–205.

King,F.E.,King,T.J.,Sellars,K.,1952.Thechemistryofextractivesfromhardwoods. PartV.Theisolationof3,7,4′_{-trimethylquercetin(ayanin)fromtheheartwood}

ofDistemonanthusbenthamianus.J.Chem.Soc.0,92–95.

King,F.E.,King,T.J.,Stokes,P.J.,1954.Thechemistryofextractivesfromhardwoods. PartsXIX.Thestructuresoffurthernewflavonesoccurringinayan(

Distemo-nanthusbenthamianus).J.Chem.Soc.0,4587–4594.

Liu,R.,Ma,S.,YU,S.,Pei,Y.,Zhang,S.,Chen,X.,Zhang,J.,2009.Cytotoxicoleanane triterpenesaponinsfromAlbiziachinensis.J.Nat.Prod.72,632–639.

Malan, E., Roux, D.G., 1979. Flavonoids from Distemonanthus benthamianus

Baillon.Methoxylatedflavonesandinter-relationshipsofbenthamianin,a [2]-benzopyrano-[4,3-b][I]-benzopyran.J.Chem.Soc.1,2696–2703.

McGaw,L.J.,Lall,N.,Meyer,J.J.M.,Eloff,J.N.,2008.ThepotentialofSouthAfrican plantsagainstMycobacteriuminfections.J.Ethnopharmacol.119,482–500. Murai,Y.,Kitajima,J.,Iwashina,T.,2014.FlavonoidsfromtwoalpineCampanula

speciesinJapan.Bull.Natl.Mus.Nat.Sci.40,113–118.

NgonoBikobo,D.S.,Mosset,P.,Abouem,A.,Zintchem,A.,Atchadé,A.T.,Balemaken Missi,M.,MbabiNyemeckII,N.,Pegnyemb,D.E.,2014.Campylospermine,anN -hydroxyalkaloidfromtheleavesofCampylospermumdensiflorum(Ochnaceae). Int.J.Pharm.Phytopharmacol.Res.6,719–728.

Okunade, L., Elvin-Lewis, M.P., Lewis, W.H., 2004. Natural antimycobacterial metabolites:currentstatus.Phytochemistry65,1017–1032.

Ouyang,M.A.,Wein,Y.S.,Su,R.K.,Kuo,Y.H.,2007.RhusemialinsA-C,newcyclolignan estersfromtherootsofRhusjavanicavar.roxburghiana.Chem.Pharm.Bull.55, 804–807.

Peterson,L.R.,Shanholtzer,C.J.,1992.Testsforbactericidaleffectsofantibacterial agents:technicalperformanceandclinicalrelevance.Clin.Microbiol.Rev.5, 420–432.

Takayuki,M.,Tsutomu,Y.,Nobuhiro,S.,Tsukasa,I.,2012.Phenoliccompounds, includingnovelC-glycosylflavone,fromtheflowersofthetallbeardedIris cul-tivar‘VictoriaFalls’.Nat.Prod.Commun.7,1591–1594.