Investigations into the therapeutic effects of aerial and stem parts of Buxus papillosa CK Schneid.: In vitro chemical, biological and toxicological perspectives

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l oc a t e / j p b a

Investigations

into

the

therapeutic

effects

of

aerial

and

stem

parts

of

Buxus

papillosa

C.K.

Schneid.:

In

vitro

chemical,

biological

and

toxicological

perspectives

Hammad

Saleem

a,b

_,

_Thet

_Htar

a

_,

_Rakesh

_Naidu

c

_,

_Irshad

_Ahmad

d

_,

_Gokhan

_Zengin

e

_,

Manzoor

Ahmad

f

_,

_Nafees

_Ahemad

a,g,∗

a_School_of_Pharmacy,_Monash_University_Malaysia,_Jalan_Lagoon_Selatan,_47500,_Bandar_Sunway,_Selangor_Darul_Ehsan,_Malaysia b_Institute_of_{Pharmaceutical}_Sciences_(IPS),_University_of_Veterinary_&_Animal_Sciences_(UVAS),_Lahore,_54000,_Pakistan

c_Jeffrey_Cheah_School_of_Medicine_and_Health_Sciences,_Monash_University_Malaysia,_Jalan_Lagoon_Selatan,_47500,_Bandar_Sunway,_Selangor_Darul_Ehsan,

Malaysia

d_Department_of_Pharmacy,_The_Islamia_University_of_Bahawalpur,_Bahawalpur,_63100,_Pakistan e_Department_of_Biology,_Faculty_of_Science,_Selcuk_University,_Campus,_Konya,_Turkey f_Department_of_Chemistry,_University_of_Malakand,_Chakdara,_Dir_(L),_Pakistan

g_Tropical_Medicine_and_Biology_{Multidisciplinary}_Platform,_Monash_University_Malaysia,_Jalan_Lagoon_Selatan,_47500,_Bandar_Sunway,_Selangor_Darul

Ehsan,Malaysia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received9October2018

Receivedinrevisedform4January2019 Accepted6January2019

Availableonline7January2019 Keywords: Buxuspapillosa Phytochemicals Enzymeinhibition Antioxidant Cytotoxicity

a

b

s

t

r

a

c

t

Inthisstudy,differentsolventextracts(methanol,dichloromethaneandn-hexane)fromaerialandstem partsofBuxuspapillosaC.K.Schneid(Buxaceae)wereinvestigatedforapanoplyofbioassays.Biological profileswereestablishedbydeterminingantioxidantandenzymeinhibitionprofiles.Toxicitywastested usingMTTcellviabilityassayonfivedifferenthumancancercelllinesi.e,MCF-7,MDA-MB-231,CaSki, DU-145andSW-480.Forchemicalfingerprinting,totalbioactivecontentsandUHPLC-MSsecondary metabolitesprofileweredetermined.Generally,bothaerialandstemmethanolextractshadhighest totalbioactivecontents,radicalscavengingandreducingpowerpotential.DCMandn-hexaneextracts werefoundtobemostactivefortotalantioxidantandmetalchelatingactivity.TheUHPLC-MSanalysis ofmethanolextractsrevealedthepresenceofseveralphenolic,flavonoid,alkaloid,saponinand dep-sipeptidederivatives.Alltheextractsweresignificantlyactiveagainstbutyrylcholinesterase,whereas moderateinhibitionwasobservedforacetylcholinesterase,␣-glucosidaseandurease.Similarly,a con-siderablelevelofcytotoxicitywasobservedagainstallthetestedcelllineswithIC50valuesrangingfrom

26to225.9␮g/mL.Aerialmethanolandstemn-hexaneextractswerefoundtobemostcytotoxic. Princi-palcomponentanalysiswasalsoperformedtoﬁndanypossiblecorrelationbetweenbiologicalactivities andtotalbioactivecontents.Onthebasisofourﬁndings,B.papillosamaybeconsideredaspromising sourceofbioactivemolecules.

1. Introduction

Bloomingresearchanddevelopmentinventivenessemphasized

thepolypharmacologicalprospectiveofplantsecondary

metabo-litesforthetreatment,preventionand/ormanagementofseveral

healthproblems [1,2].Indeed, theuseofherbalmedicationsto

curediseasespredatesrecordedhistoryofmankind.Advancesin

analyticaltechniqueshavestimulatesubstantialresearchforthe

∗ Correspondingauthorat:SchoolofPharmacy,MonashUniversityMalaysia,Jalan LagoonSelatan,47500,BandarSunway,SelangorDarulEhsan,Malaysia.

E-mailaddress:nafees.ahemad@monash.edu(N.Ahemad).

developmentofnewpharmacophoresandscaffoldsfordesigning

efﬁcaciousagentsforseveraldiseases[1,3].

Inspiteofthefactthat,thepharmaceuticalindustrialrevolution

andadvancementoforganicchemistryresultedintheinclination

forsyntheticmedications.However,theassociatedsideeffectsand

high-costofthesesyntheticdrugsonhumanhealthhaverenewed

theinterestintheinvestigationofnaturalproductsfordrug

discov-eryanddevelopment[4].Plantshavealwaysservedhumanswith

allhisneedsintermsofshelter,clothing,food,ﬂavours,fragrances

and most importantly,medicines [5]. Nowadays, knowledge of

ancientbotanicalmedicinalpracticesandapplicationofmodern

phytochemicaltechniqueshaveprovided excellenttoolsforthe

https://doi.org/10.1016/j.jpba.2019.01.007

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puriﬁcationandstructuralelucidationofvariousphytochemicals,

which,inturn,hasgiveninsightsintotheirmodeofactiononthe

humanbody.Infact,11%ofthe252drugs,appraisedasbasicand

essentialbytheWorldHealthOrganisation(WHO),areexclusively

ofplantoriginandanumberofsyntheticdrugsareobtainedfrom

naturalprecursors[4].

ThegenusBuxusbelongstofamilyBuxaceaeandcomprisesof

approximately70speciesaroundtheworldwhichhavebeenused

intheindigenoussystemsofmedicineforanticholinesterase[6],

anti-HIV,immunosuppressive andcytotoxicityactivities[7,8].B.

papillosa,locallyknownasshamshad,oneofthecommonplant

ofthis genusis traditionallyusedtocure malaria,rheumatism,

skindiseases,headacheandalsoreportedtobeusedas

antidiar-rheal, antisecretory,cardiotonic and neurotonicagent agent [9].

However, previously nomuch research hasbeen done for this

speciesregardingitschemicalcomposition,toxicologyand

phar-macologicalactivities.Toaddressthislacuna,thisstudyaimedat

investigatingthetotalbioactivecontents,secondarymetabolomics

proﬁlingaswellantioxidantpotentialofdifferentsolventextracts

of B. papillosa aerial and stem parts. The inhibitory potential

of all the extracts was tested on acetylcholinesterase,

butyryl-cholinesterase,_{␣-glucosidase}andurease.Moreover,aspecialaim

ofthisstudywastoassessthecytotoxicityagainstbreast,cervix,

prostateandcoloncancersinordertoestablishascientiﬁcbasisfor

furtherapplicationsofthisplantasafunctionalfood/ingredient.

Inaddition,forassessingthesimilarityofthebiologicalactivities

expressedbytheextractsandtoanalyzethecorrelationsbetween

themeasuredvariables,aprincipalcomponentanalysis(PCA)was

also performed.As faras our knowledge, this will be theﬁrst

reportonsuchcomprehensivebiological,chemicaland

toxicolog-icaldetailingofB.papillosa.

2. Materialandmethods

2.1. Plantcollectionandextraction

B.papillosaaerialandstempartswerecollectedfromthe

north-ernareasofPakistan andidentiﬁedby Dr.Abdul-RehmanNiazi

(InchargeHerbarium),Department ofBotany,Punjab University

Lahore,Pakistan.Inaddition,aVoucherspecimennumber(LAH

#7517)wasalsodepositedin theherbariumof Departmentof

Botany,PunjabUniversityLahore,Pakistan.Theshade-driedaerial

andstempartsweresubjectedforextractionbymaceration(72h)

successivelywithDCMandmethanolatroomtemperaturewith

occasionallyshaking.Theresultantextractswereconcentratedby

Rotavapor-R20at35◦C.

2.2. Totalphenolic,ﬂavonoidsandsecondarymetaboliteproﬁle

Total phenolic content assay was done by utilizing

well-established Folin–Ciocalteu reagent method [10]. The phenolic

contents wereexpressedasmg gallicacidequivalentpergram

offreshsample(mgGAE/gextract).Totalﬂavonoidcontentswere

determinedwiththealuminiumchloridecolorimetricmethod[11].

Theﬂavonoidconcentrationwasexpressedasmgquercetin

equiv-alentpergramsample(mgQE/gextract).

Secondary metabolites were evaluated by RP-UHPLC-MS.

UHPLCofAgilent1290InﬁnityLCsystemcoupledtoAgilent6520

Accurate-Mass Q-TOF mass spectrometer withdual ESI source

was used [12,13]. Column speciﬁcations were as: Agilent

Zor-baxEclipseXDB-C18,narrow-bore2.1x150mm,3.5micron(P/N:

930990-902).Columnandauto-samplertemperaturewere

main-tainedat25◦Cand4◦C,respectively.Flowratewas0.5mL/min.

Mobilephasesusedwere:A-0.1%formicacidinwater,B-0.1%

formicacidinacetonitrile.Injectionvolumewas1.0␮L.Runtime

was25minandpost-runtimewas5min.FullscanMSanalysiswas

doneoverarangeofm/z100–1000usingelectrosprayionsource

innegativemode.Nitrogenwassuppliedasnebulizinganddrying

gasatﬂowratesof25and600L/hour,respectively.Thedryinggas

temperaturewas350◦C.Thefragmentationvoltagewasoptimized

to125V.Analysiswasperformedwithacapillaryvoltageof3500V.

DatawasprocessedwithAgilentMassHunterQualitativeAnalysis

B.05.00(Method:Metabolomics-2017-00004.m).Identiﬁcationof

compoundswasdonefromSearchDatabase:METLINAM

PCDL-N-170502.cdb,withparametersas:Matchtolerance:5ppm,Positive

Ions:+H,+Na,+NH4,NegativeIons:-H[14].

2.3. Antioxidantactivities

1,1-Diphenyl-2-picrylhydrazylradical(DPPH) assaywas

per-formed as described by Kolevaet al [15]. ABTS (2, 2-azino-bis

(3-ethylbenzothiazoline-6-sulphonicacid)radicalcation

scaveng-ingactivity,CUPRAC(Cupricionreducingantioxidantcapacity)and

metalchelatingactivitywasperformedasdescribepreviously[16].

TheFRAP(ferricreducingantioxidantpower)assayestimatesthe

abilityoftheplantextracttoreduceFe3+_to_Fe2+_and_performed

accordingtostandardmethodasdescribedpreviously[17].Total

antioxidantcapacity(TAC)ofextractswasevaluatedby

phospho-molybdenummethodaccordingtoPrietoetal[18].Thereducing

powerandtotalantioxidantcapacitywasexpressedasequivalent

ofgallicacid(mgGAE/g).TheABTSandCUPRACantioxidant

activ-itieswerereportedastroloxequivalents,whereasEDTAwasused

asreferencestandardformetalchelatingassay.

2.4. Enzymeassays

Chloinesterase(acetylcholinesteraseandbutyrylcholinesteras),

␣-glucosidase and urease inhibition activities were

deter-minedspectrophotometricallyaccordingtostandardmethodsas

describedpreviously[14].EserinewasusedascontrolforAChEand

BChE,acarbosefor␣-glucosidaseandkojicacidforurease.EZ–Fit

EnzymekineticssoftwarewasusedtocalculateIC50values

(Per-rellaScientiﬁcInc.Amherst,USA).Thepercentageinhibitionwas

calculatedas

Inhibition(%)=Control−Test_Control ×100

2.5. MTTcytotoxicityassay

MCF-7,MDA-MB-231(breastcancer),CaSki(cervicalcancer),

DU-145(prostatecancer)andSW-480(Colon)celllineswereused

totestcytotoxicity.Thebreastcancercelllinesweremaintainedin

DMEMculturemediumwhileRPMI-1640mediawasusedforCaSki,

DU-145andSW-480cells.Bothmediaweresupplementedwith

10%FBS(foetalbovineserum)and1%P/S(penicillin-streptomycin)

environment.Cellswereculturedat37◦Cinahumidiﬁed

atmo-spherein5%CO2incubator.Thecytotoxicityofalltheextractswas

doneusingMTTassay[14].Thepercentagecellviability(%)was

calculatedbyusingfollowingformula

Cellviability(%)=Abss–Absc×100

Alloftheexperimentswerecarriedoutintriplicatedandthe

IC50values(48htreatmentwithcancercells)werealsocalculated

usingGraphpadprismsoftware(V7).

2.6. Statisticalanalysis

Alltheexperimentswererepeatedthreetimesandanalysiswas

doneintriplicates.Theobtainedresultswereexpressedasmean

value andstandard deviation(mean_±SD).One-way analysisof

variance(ANOVA)wasusedtocalculatethedifferences,followed

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Table1

TotalbioactivecontentsofB.papillosaaerialandstemextracts.

Plant Solvents Abbreviations Totalphenoliccontent

(TPC)(mgGAE/g)

TotalFlavonoidcontent

(TFC)(mgQE/g)

B.papillosaaerial Methanol BA-M 36.27±0.57a _40.48_±_0.93a

DCM BA-D 23.77±2.37b _21.75_±_0.3b

n-hexane BA-H 11.81±1.34c _20.71_±_2.06b

B.papillosastem Methanol BS-M 24.68±4.63b _47.10_±_1.51a

DCM BS-D 20.64±0.95b _24.53_±_1.53b

n-hexane BS-H 10.80±1.45c _4.56_±_0.64c

Datafromthreerepetitions,withmean±standarddeviation;meanswithdifferentsuperscriptlettersinthesamecolumnaresigniﬁcantly(p<0.05)different.GAE:gallic acidequivalent;QE:quercetinequivalent.

Fig.1.TotalionchromatogramsofB.papillosaaerialandstemmethanolextracts.

PrismsoftwarewasusedtocalculateIC50.SPSSv22.0softwarewas

usedtocarryoutallexperimentalanalysis.

3. Resultsanddiscussion

3.1. TotalbioactivecontentsandUHPLC-MSanalysis

TotalbioactivecontentsofdifferentsolventextractsofB.

papil-losaweredeterminedintermsoftotalphenolicandtotalﬂavonoid

contentsandtheresultsareshowninTable1.

The highest TPC were observed in BA-M (36.27±0.57mg

GAE/g), followed by BS-M (24.68±4.63mg GAE/g), BA-D

(23.77±2.37mgGAE/g) andBS-D (20.64±0.95 mg GAE/g).The

totalﬂavonoidcontentsof B.papillosarangedfrom47.10±1.51

to4.56±0.64mgQE/gextract.Thehighestvalueswereobtained

for BS-M and BA-M i.e., 40.48±0.93 and 47.10±1.51mg QE/g,

respectively.Bothn-hexaneextractsfromaerialand stemparts

hadlowesttotalbioactivecontents.Overall,highertotalbioactive

contentswerefoundinpolartonon-polarsolvent.Similarly,the

total phenolic contents of different solvent extracts of B.

sem-pervirensasreportedbyOrhanetal[19],alsoshowedmethanol

extractto exhibithigher phenolic content(63.85mgGAE/g). In

anotherstudy,B.hyrcanaleavesmethanolextract(148.2±4.1mg

GAE/g)wasfoundtohavehighest TPC ascompared withethyl

acetate(101.6±5.1mgGAE/g)andn-hexane(15.3±3.3mgGAE/g)

extracts[20].

Liquidchromatographymassspectrometryanalysisoftheaerial

andstemmethanolextractsofB.papillosawasperformedtohave

aninsightintothepossiblesecondarymetabolitecomponents.A

lineargradientelutionwithwaterandmethanolcontaining0.1%

fromicacidasthemobilephaseofferedthebestresolution.A

typ-icalchromatogramofboththeextractswithmassspectrometric

detectioninnegativeionmodeexhibitedquitecomplexpatterns

ofpeaksasshowninFig.1.TheUHPLC/MSanalysisofB.papillosa

aerialmethanolextracthasrevealedthepresenceof16different

compoundsandgiveninTable2.

Mostofthesecompoundsbelongtotypicalphenolicacids,

cin-namic acid and ﬂavonoid derivatives. The ﬂavonoids present

were diffutin, depressonol A, kaempferol

3-lathyroside-7-rhamnoside, kaempferol 3-(2G-glucosylrutinoside), quercetin

3-alpha-arabinopyranosyl-(1->2)-glucoside, robinin,

isoori-entin 2’-O-arabinoside, robinetin 3-rutinoside, quercetin

3-galactoside-7-rhamnoside,laricitrin3-rhamnosideandluteolin

3,4-diglucuronide. Gallic acid was the major phenolic

identi-ﬁed. Moreover, coriandrone C (oumarin), Citrusin A (lycoside),

LucyosideJ(saponin)andAntanapeptinA(Ddepsipeptide)were

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Fig.2. CytotoxicityofB.papillosaaerialextracts.

Thegraphsshowscytotoxiceffectsas(1)MCF-7(BA-M),(2)MCF-7(BA-D),(3)MCF-7(BA-H),(4)MDA-MB-231(BA-M),(5)MDA-MB-231(BA-D),(6)MDA-MB-231(BA-H), (7)CaSki(BA-M),(8)CaSki(BA-D),(9)CaSki(BA-H),(10)DU-145(BA-M),(11)DU-145(BA-D),(12)DU-145(BA-H),(13)SW-480(BA-M),(14)SW-480(BA-D)and(15) SW-480(BA-H)****indicatessigniﬁcantdifferencewhencomparedwithuntreated(control)cells(pvalue<0.05).

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Table 2 UHPLC-MS of B. papillosa aerial methanol extract (negative ionization mode). S. No. RT (min) Base peak (m/z) Peak height AUC Proposed compounds Compound class Mol. formula Mol. mass MFG Diff (ppm) Vol (%) 1 0.63 245.04 90238 230847 Coriandrone C Coumarin C13 H10 O5 246.05 0.58 0.08 2 1.77 169.01 49829 380500 Gallic acid Phenolic C7 H6 O5 170.02 1.41 0.13 3 7.55 463.15 50991 419706 Diffutin Flavonoid C23 H28 O10 464.16 3.3 0.14 4 8.25 537.19 44308 387064 Citrusin A Glycoside C26 H34 O12 538.20 − 1.23 0.13 5 8.35 741.19 962282 9380450 Depressonol A Flavonoid C32 H38 O20 742.19 − 3.1 3.1 6 8.55 725.19 1419855 10045164 Kaempferol 3-lathyroside-7-rhamnoside Flavonoid C32 H38 O19 726.20 − 2.13 3.32 7 8.56 755.20 338457 2521837 Kaempferol 3-(2G-glucosylrutinoside) Flavonoid C33 H40 O20 756.21 − 1.13 0.83 8 8.60 595.13 362708 3165513 Quercetin 3-alpha-arabinopyranosyl-(1->2)-glucoside Flavonoid C26 H28 O16 596.13 − 2.22 0.57 9 8.61 739.20 182405 1291070 Robinin Flavonoid C33 H40 O19 740.21 − 0.02 0.43 10 8.82 579.13 372101 2260134 Isoorientin 2”-O-arabinoside Flavonoid C26 H28 O15 580.14 − 2.27 0.75 11 8.85 609.14 160543 1129312 Robinetin 3-rutinoside Flavonoid C27 H30 O16 610.15 − 2.83 0.37 12 8.98 609.14 51756 330836 Quercetin 3-galactoside-7-rhamnoside Flavonoid C27 H30 O16 610.15 0.79 0.11 13 9.72 477.10 49062 293647 Laricitrin 3-rhamnoside Flavonoid C22 H22 O12 478.11 − 5.12 0.1 14 10.07 809.43 66914 547995 Lucyoside J Saponin C42 H66 O15 810.44 − 2.21 0.18 15 10.31 635.12 71913 537819 Luteolin 3’,4’-diglucuronide Flavonoid C28 H28 O17 636.13 0.83 0.18 16 12.84 795.45 111349 1093479 Antanapeptin A Depsipeptide C41 H60 N4 O8 736.44 0.24 0.36 RT: retention time; AUC: area under curve.

Similarly, the stem methanol extract was found to

pos-sess 20 secondary metabolite compounds as presented in

Table3, andits MStotal ion chromatogramis shownin Fig.2.

Polyphenolics present were found to be beta-glucogallin, 2, 4,

6-trihydroxybenzoic acid, 4-O-methyl-gallate, ferulic acid

4-O-glucuronide,demethyloleuropein,p-salicylicacidandgrossamide.

Whereas,ﬂavonoidsidentiﬁedweredepressonolA,kaempferol

3-lathyroside-7-rhamnoside,kaempferol3-(2G-glucosylrutinoside),

isoorientin2’-O-arabinoside,robinetin3-rutinoside,quercetin

3-galactoside-7-rhamnoside,jaceidin5-glucosideandwightin.Two

saponins(lucyoside J and phytolaccosideA), one iridoid

glyco-side(verbenalin),oneglycoside(citrusinB)andonedepsipeptide

(antanapeptin A) were alsopresent. This is the ﬁrst report on

thepreliminaryUHPLC-MSanalysisofB.papillosaaerialandstem

methanolextracts.

3.2. Antioxidantcapacity

Determinationofantioxidantactivityusingonlyonemethod

isnotreliablesinceantioxidantsexhibittheirprotectiveroleby

threemainproposedmechanisms,namely,hydrogentransfer,

elec-trontransfer,andmetalchelation.Inthisrespect,theantioxidant

potentialoftheB.papillosaplantextractswasevaluatedusing

dif-ferent spectrophotometricmeasurements including,freeradical

scavenging(DPPHandABTS),reducingpower(CUPRACandFRAP),

phosphomolybdenumandmetalchelatingassaysandtheresults

arepresentedinTable4

IntheDPPHradicalassay,theBA-M(IC50=65.70±1.90␮g/mL),

BA-D(IC50=165.38±2.22␮g/mL)andBS-M(IC50=185.32±2.48

␮g/mL) displayed more potent radical scavenging potential

amongst allextracts.Moreover,in ABTS assay,similartoDPPH

results,theBA-M, BS-Mand BA-Dhasthehighest radical

scav-enging potential with values of 95.21_±1.74, 86.29_±1.16 and

73.51±0.48mgTE/g,respectively.Whereas,BA-H,BS-Dand

BS-Hwereleastactiveforboth DPPHand ABTSradicalscavenging

assays.Similartoourﬁndings,previousresearchershadreported

theB.hyrcanaleavesmethanolextract(47.8±0.8%)tohave

high-estDPPHinhibitionascomparedwithethylacetate(33.8±0.1%)

andn-hexane(20.8±0.6%)extracts[20].Similarly,anotherstudy

reported the methanol extract of B. wallichiana exhibited the

highestDPPHinhibition(IC50;29.2±2.10␮g/mL)amongdifferent

extracts[21].

FRAPandCUPRACassayswereutilizedtomeasurethe

reduc-ing potential of B. papillosa aerial and stem extracts. BA-M

(FRAP: 47.33±0.14mg GAE/g and CUPRAC: 134.26±10.02mg

TE/g) revealed more prominent reducing power capacity as

compared to BS-M (FRAP: 41.93±0.11mg GAE/g and CUPRAC:

98.42±1.20mgTE/g)andBA-D(FRAP:35.02±0.08mgGAE/gand

CUPRAC:81.55±2.33mgTE/g).Allotherthreeextractsexhibited

lowerreducingpowercapacities.

Similarly,phosphomolybdenumassaywasusedtodetermine

the total antioxidant capacity and theresults are presented in

Table4.BS-D(58.19±2.52mgGAE/g)andBA-D(42.32±1.65mg

GAE/g)weremostactiveforTAC,ascomparedwithBA-M,BA-H

andBS-Mwhich showsTACvalues of42.32±1.65, 31.19±1.38

and19.29±1.50mgGAE/g,respectively.Inmetalchelatingassay,

BS-D(46.64±7.53mgEDTA/g),BA-H(41.20±1.15mgEDTA/g)and

BA-D(37.17±2.94mgEDTA/g)werethemostactive,whereas

BA-M(8.32±0.99mg EDTA/g)wasleastactive.Takentogether,our

resultsindicatedthatthestudiedplantextractsexhibited

signif-icantantioxidantproperties.Overall,aerialmethanolextractwas

themostactiveforantioxidantcompounds.Clearly,theobtained

resultsareassociatedwiththelevelsoftotalbioactivecomponents

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pre-Table 3 UHPLC-MS of B. papillosa stem methanol extract (negative ionization mode). S. No. RT (min) Base peak (m/z) Peak height AUC Proposed compounds Compound class Mol. formula Mol. mass MFG Diff (ppm) Vol (%) 1 0.68 387.13 268534 968683 Verbenalin Iridoid C17 H24 O10 388.13 0.03 0.21 2 1.3 331.07 546101 6069407 beta-Glucogallin Phenolic C13 H16 O10 332.07 − 4.83 1.34 3 1.76 169.02 253254 2126693 2,4,6-Trihydroxybenzoic acid Phenolic C7 H6 O5 170.02 − 1.96 0.47 4 7.16 183.03 299456 2947017 4-O-Methyl-gallate Phenolic C8 H8 O5 184.03 1.9 0.65 5 7.31 369.09 116400 751668 Ferulic acid 4-O-glucuronide Phenolic C16 H18 O10 370.09 − 6.01 0.17 6 7.44 525.17 113880 813974 Demethyloleuropein Polyphenol C24 H30 O13 526.17 − 3.21 0.18 7 8.33 741.19 699589 5336061 Depressonol A Flavonoid C32 H38 O20 742.19 − 2.3 1.18 8 8.54 567.21 99618 550841 Citrusin B Glycoside C27 H36 O13 568.21 − 1.82 0.12 9 8.54 725.20 370427 2560362 Kaempferol 3-lathyroside-7-rhamnoside Flavonoid C32 H38 O19 726.20 − 1.41 0.56 10 8.55 755.21 216328 1509661 Kaempferol 3-(2G-glucosylrutinoside) Flavonoid C33 H40 O20 756.21 − 1.72 0.33 11 8.82 579.14 98463 602503 Isoorientin 2”-O-arabinoside Flavonoid C26 H28 O15 580.14 1.29 0.13 12 8.84 609.15 304301 2085273 Robinetin 3-rutinoside Flavonoid C27 H30 O16 610.15 − 1.37 0.46 13 8.97 609.15 84059 560970 Quercetin 3-galactoside-7-rhamnoside Flavonoid C27 H30 O16 610.15 1.78 0.12 14 9.93 137.03 104194 878880 p-Salicylic acid Phenolic C7 H6 O3 138.03 3.62 0.19 15 10.06 809.44 341457 3141468 Lucyoside J Saponin C42 H66 O15 810.44 − 4.7 0.69 16 10.46 521.13 147738 939907 Jaceidin 5-glucoside Flavonoid C24 H26 O13 522.13 − 2.7 0.21 17 11.83 647.38 124942 1398703 Phytolaccoside A Saponin C36 H56 O10 648.38 − 2.13 0.31 18 11.98 623.24 172617 2320697 Grossamide Phenolic C36 H36 N2 O8 624.24 − 0.48 0.51 19 12.88 735.44 679669 7137133 Antanapeptin A Depsipeptide C41 H60 N4 O8 736.44 0.3 1.57 20 12.98 343.09 166817 1430549 Wightin Flavonoid C18 H16 O7 344.09 − 3.82 0.32 RT: retention time; AUC: area under curve.

viousstudies,whichreportedastrongcorrelationbetweentotal

bioactivecomponentsandantioxidantproperties[22]

3.3. Enzymeinhibition

Increasingevidencestendtoadvocate thelinkbetween

dia-betesandtheoccurrenceofneurodegenerativedisorders.Diabetes

sequelaeaffectthebrainovertimeuntilcognitivedeclinebecomes

clinically apparent. Cholinesterases have been targeted for the

managementofneurodegenerativedisorderssuchasAlzheimer’s

disease. On the other hand, ␣-glucosidase are key players in

thecontrolofglycaemiclevel.Interestingly,phytochemicalshave

provedtobeinhibitorsofthesekeyenzymesandhaveusheredto

thedevelopmentofnovelpharmaceuticals[23].Differentextracts

ofB.papillosaaerialandstempartsweretestedforAChE,BChE,

␣-glucosidaseandureaseinhibitionactivityandtheresultsareshown

inTable5.

AlltheextractswereleastactiveagainstAChEwithIC50values

morethan500␮g/mL.However,withtheonlyexceptionofBS-H,all

otherextractsweresigniﬁcantlyactiveagainstBChEwithIC50

val-uesrangingfrom21.27±0.59to26.45±0.47␮g/mL.Theobserved

higherbutyrylcholinesteraseinhibitionissupportedbysome

pre-vious researchers who had reported the presence of different

secondarymetabolitesfrombuxusspeciesforexample,B.hyrcana

andB.natalensistobepotentinhibitorsforcholinesterases[24–26].

Incaseof␣-glucosidaseinhibition,BS-HandBS-Mshowedhighest

inhibition (IC50 = 16.29±0.37 and 42.53±2.28␮g/mL,

respec-tively),but,BA-H(IC50 =113.24±2.15␮g/mL)and BS-D(IC50 =

175.46±3.29␮g/mL)werealsomoderatelyactiveforthisenzyme.

However,BA-MandBA-Dwereleastactivefor␣-glucosidase.The

resultsof␣-glucosidaseinhibitionareinaccordancewiththe

pre-viousstudieswhich had alsoreportedthehighestinhibition of

then-hexaneextract(42.9±2.3%)ofB. hyrcanaascompared to

methanolextract(12.3±1.1%)[20].Forurease,BA-M,BS-Mand

BS-HshowedmoderateinhibitionwithIC50valuesof223.74±0.48,

228.56±0.59and425.62±0.3528␮g/mL,respectively.Theother

threeextracts,BA-D,BA-HandBS-Dwerefoundtobeleastactive

againsturease.Ingeneral,BS-MandBS-Hweresigniﬁcantlyactive

againstallenzymes(exceptAChE)andoverall,theB.papillosastem

extractsweremoreactiveascomparedwithaerialextracts.This

istheﬁrstreportoncomprehensiveenzymeinhibitionproﬁlesof

differentsolventextractsofB.papillosaaerialandstemparts.

3.4. Cytotoxicity

Majorissues concerningconventional anticancer

chemother-apyaretheoccurrenceofsideeffectsinducedbythenon-speciﬁc

targetingofbothnormalandcancercells,andtheemergenceof

drug-resistantcancercells.Basedonthis,therehasbeengrowing

interestintheuseofnaturallyoccurringmoleculeswith

chemo-preventiveandchemotherapeuticpropertiesincancertreatment

[27].Themethanol,DCMandn-hexaneextractsofB.papillosaaerial

andstempartsweretestedforcytotoxicityagainstﬁvehuman

car-cinomacellsi.e.,breast(MCF-7andMDA-MB-231),cervix(CaSki),

prostate(DU-145)andcolon(SW-480).TheIC50valuesofthetested

extractsaregiveninTable6andthepercentagecellviabilityat

dif-ferentconcentrations(500-15.62␮g/mL)isshowninFigs.3and4.

Theaerialmethanolextractdisplayedthehighestlevelof

cyto-toxicityagainstthebreastcancercellline(MDA-MB-231;IC50 =

14.19␮g/mL),butitwasalsoconsiderablyactiveagainsttwoother

celllines,MCF-7andCaski(IC50=50.34and82.18␮g/mL,

respec-tively).BA-Dextractshowedmostsigniﬁcantcytotoxicityagainst

MCF-7(IC50=56.54␮g/mL)andconsiderabletoxicityagainstCaSki

andDU-145,withIC50 valuesof72.46and78.86␮g/mL,

respec-tively.TheBA-DextractwasalmostaspotentasBA-Mextractin

(7)

BA-Fig.3.CytotoxicityofB.papillosastemextracts.

Thegraphsshowscytotoxiceffectsas(1)MCF-7(BS-M),(2)MCF-7(BS-D),(3)MCF-7(BS-H),(4)MDA-MB-231(BS-M),(5)MDA-MB-231(BS-D),(6)MDA-MB-231(BS-H), (7)CaSki(BS-M),(8)CaSki(BS-D),(9)CaSki(BS-H),(10)DU-145(BS-M),(11)DU-145(BS-D),(12)DU-145(BS-H),(13)SW-480(BS-M),(14)SW-480(BS-D)and(15)SW-480 (BS-H).

(8)

Table4

AntioxidantactivitiesofB.papillosaaerialandstemextracts.

Samples Radicalscavengingactivity Reducingpower Totalantioxidant

capacity(TAC) Ferrouschelating %RSC (1mg/mL) DPPHIC50 (␮g/mL) AAEAC (mgAAE/g) ABTS (mgTE/g extract) FRAP (mgGAE/g) CUPRAC (mgTE/g extract) Phosphomolybdenum (mgGAE/g) MetalChelating (mgEDTA/g) BA-M 82.43±0.19 65.70±1.90 58.99±1.72 95.21±1.74 47.33±0.14 134.26±10.02 42.32±1.65 8.32±0.99 BA-D 79.34±0.41 165.38±2.22 23.40±0.31 73.51±0.48 35.02±0.08 81.55±2.33 51.16±3.12 37.17±2.94 BA-H 54.38±0.18 287.98±3.18 13.44±0.14 15.54±1.77 15.61±0.14 67.35±1.46 31.19±1.38 41.20±1.15 BS-M 86.16±0.41 185.32±2.48 20.88±0.27 86.29±1.16 41.93±0.11 98.42±1.20 19.29±1.50 14.98±0.52 BS-D 74.68±0.27 591.38±1.81 6.54±0.02 44.60±2.69 15.74±0.22 91.31±2.09 58.19±2.52 46.64±7.53 BS-H 61.33±1.05 920.21±35.52 4.21±0.16 5.64±1.44 11.64±0.29 65.19±3.26 26.85±1.77 31.43±2.56 Ascorbicacid 89.96±1.60 16.82±069 nt nt nt nt nt nt

Datafromthreerepetitions,withmean±standarddeviation;RSC:radicalscavengingcapacity;AAEAC:Ascorbicacidequivalentanti-oxidantcapacity;FRAP:ferricreducing anti-oxidantpower;TAC:totalantioxidantcapacity;nt:nottested.

Table5

EnzymeinhibitioneffectsofB.papillosaaerialandstemextracts.

Samples AChE BChE ␣-glucosidase Urease

Inhibition(%) IC50 (␮g/mL) Inhibition(%) IC50 (␮g/mL) Inhibition(%) IC50 (␮g/mL) Inhibition(%) IC50 (␮g/mL) BA-M 47.32±0.35 >500** _83.54_±_0.75 _22.82_±_0.63 _24.53_±_1.26 _>500** _76.85_±_0.63 _223.74_±_0.48 BA-D 16.74±0.23 >500** _82.35_±_0.56 _26.45_±_0.47 _8.47_±_1.27 _>500** _35.26_±_0.45 _>500** BA-H 5.36±0.14 >500** _79.42_±_0.45 _25.63_±_0.38 _87.62_±_2.53 _113.24_±_2.15 _15.38_±_0.26 _>500** BS-M 25.25±0.46 >500** _84.73_±_0.75 _23.54_±_0.63 _91.35_±_2.53 _42.53_±_2.28 _75.25_±_0.74 _228.56_±_0.59 BS-D 24.53±0.35 >500** _87.24_±_0.63 _21.27_±_0.59 _85.62_±_3.74 _175.46_±_3.29 _45.53_±_0.35 _>500** BS-H 29.85±0.42 >500** _81.26_±_0.45 _152.65_±_0.38 _89.74_±_0.45 _16.29_±_0.37 _54.37_±_0.49 _425.62_±_0.35 Eserine 91.27±1.17 0.04±0.01 82.82±1.09 0.85±0.01 nt nt nt nt Acarbose nt nt nt nt 92.83±0.18mM 37.45±0.16␮M nt nt Kojicacid nt nt nt nt nt nt 98.21±0.18 21.25±0.15 Valuesareexpressedasmeans±S.D.ofthreereplicates.

**_The_IC

50valuewashigherthan500␮g/mL,nt:nottested,AChE:acetylcholinesterase;BChE:butyrylcholinesterase;nt:nottested.

Table6

CytotoxicityofB.papillosaaerialandstemextracts.

Celllines IC50value(␮g/mL)

B.papillosaaerial B.papillosastem

BA-M BA-D BA-H BS-M BS-D BS-H

MCF-7 MDA-MB-231 CaSki DU-145 SW-480 50.34 56.54 85.82 127 154.2 26 14.19 >500** _61.22 _53.24 _225.9 _89.94 82.18 72.46 227.7 116.3 56.39 39.99 164.4 170.2 >500** ₁₈₇ _121.9 _99.17 158.7 78.86 47.29 236.7 94.81 83.04

Valuesareexpressedasmeans±S.D.ofthreereplicates.

**_The_IC50_value_was_higher_than₅₀₀_␮g/mL.

(9)

Fig.5. Relationshipbetweenbiologicalactivitiesandthe4identiﬁedfactors;A:Correlationcircle(belowonfactor1and2);B:Mapofcorrelationbetweenvariablesand factors;C:Mapofsquaredcosinesofthevariables.

Fig.6.Relationshipbetweentotalbioactivecontents(phenolicsandﬂavonoids)anddifferentevaluatedbiologicalactivitiesassessedthroughPearson’slinearcorrelations; **thep-valuewaslowerthan0,01;*thep-valuewaslowerthan0,05.

Hextractshowedmostprominentcytotoxicityagainstthecolon

cancercelllineSW-480(IC50=47.29␮g/mL)andwasalsoactive

againstothertwobreastcelllines,MDA-MB-231andMCF-7(IC50

=61.22and85.82␮g/mL,respectively).IncaseofB.papillosastem

extracts,theBS-Hextractexhibitednotablecytotoxicityagainstthe

MCF-7cellline(IC50=26␮g/mL),andwasalsoconsiderablyactive

againstallotherfourcelllineswithIC50valuesrangingfrom39.99

to99.17␮g/mL.ThecytotoxicitypatternofBS-Mwasquite

sim-ilartothatoftheBA-M,whichalsoshowedthemostsigniﬁcant

cytotoxicityagainstMDA-MB-231cellline(IC50 =53.24␮g/mL).

Similarly,BA-DwasmostactiveagainstCaSki(IC50=56.39␮g/mL)

andSW-480(IC50=94.81␮g/mL).WiththeonlyexceptionofBS-M

andBS-D,allotherextractsshowedthehighestlevelofcytotoxicity

againstMCF-7cellline.Overall,themethanolextractwasthemost

cytotoxicagainstallthetestedcancercells.Cytotoxicactivities

con-cerningBuxusspeciesarescarce, previousstudiesrevealedthat

triterpenoids,alkaloids,tanninsandphenoliccompoundsisolated

fromBuxusspeciesexhibitedcytotoxicactivitiesagainstdifferent

humancancercelllines[7,28,29].Ourcurrentﬁndingsarein

con-cordantwiththeﬁndingsofpreviousstudiesonthecytotoxicity

ofotherBuxusspecies.Forexample,signiﬁcantcytotoxicactivity

isreportedfortriterpenoidalkaloidsisolatedfromB.microphylla

againstHepG2[7].TheobservedhighercytotoxicpotentialofB.

papillosaextractstowardsboththebreastcancercellsissupported

bysomepreviousstudieswhichhadreportedtheB.sempervirens

acetoneextracttoinducedcellcyclearrestinG0/G1phaseand

trig-geredcelldeathbyincreasedsub-G1cellpopulationtestedinﬁve

differentbreastcancercelllines(MCF7,MCF10CA1a,T47D,BT-20

andMDA-MB-435)[29].Similarly anotherstudyreportedtheB.

microphyllaforitscytotoxicityagainstﬁvehumantumorcelllines

(promyelocyticleukemiaHL-60,hepatocellularcarcinoma

SMMC-7721,lungadenocarcinomaA-549,breastcancerMCF-7andcolon

adenocarcinomaSW-480)bythemicro-culturetetrazolium(MTT)

assay[28].Incomparisontoearlierstudies,B.papillosaexhibited

strongtomoderatecytotoxicityagainsttheseselectedcelllines.

Tothebestofourknowledge,thisistheforemostdataregarding

toxicityofB.papillosaaerialextractsagainstanycarcinomacell

(10)

Fig.7.Structuringofthevariabilityexpressedbythesamples.A:IndividualsfactorialmapofPCA,B:DendrogramofAHC.

3.5. Variabilityanalysisofthebiologicalactivity

Toassessthesimilarity ofthebiological activitiesexpressed

bytheextractsandtoanalyzethecorrelationsbetweenthe

mea-suredvariables, a principalcomponent analysis(PCA)hasbeen

performed.17quantitativevariablesrepresentedby17tested

bio-logicalactivitieshaveallowedtoestablishthetypologyof6extracts

derivedfromtheaerialandstempartsofB.papillosa.Accordingto

Kaiser-Guttmanmeasure[30],theﬁrstfourfactorshavingEigen

valuesgreaterthanonehavebeenretained(Fig.4).Thesefactors

accountfor95,76%ofthetotalvariance.Theanalyzeofcorrelation

circle(belowonfactor1and2)completedbythesquaredcosines

andcorrelationmapallowedtocharacterizetheselectedfactors

bythevariables(Fig.5A,B&C).Accordinglytheﬁrstfactor(Eigen

valueof7,94;varianceof49,65%)iscorrelatedpositivelywith

totalbioactivecontents(phenolicsandﬂavonoids),thetests

eval-uatingtheantioxidantcapacity(DPPH,ABTS,CUPRACandFRAP)

and cytotoxicity (SW-480) but negatively with metalchelating

andanti-urease.Thetotalantioxidant(Phosphomolybdenum)and

cytotoxicity(MDA-MB-231)testshighlycontributetothe

forma-tionoffactor 2(Eigenvalueof 3,15;variance of19,7%)while

␣-glucosidasehasalowercontribution.Thefactors3and4were

determinatedbyBChEandMCF-7,respectively.Inadditionalower

negativecorrelationisobservedbetweenthefactor3andtwo

cyto-toxicitytests(CaSkiandDU-145).

ThecorrelationgraphandmapbasedonPearsonlinear

corre-lationshavebeenanalyzedtoestimatethedegreeofassociation

betweenthetotalbioactivecontents(phenolicsandﬂavonoids)and

thedifferentevaluatedbiologicalactivities(Fig.5A&3).Ahighly

signiﬁcantpositivecorrelationhasbeenfoundbetweenTPCand

CUPRAC(r=+0,958;p<0,01),TPCandABTS(r=+0,938;p<0,01),

TFCandSW-480(r=+0,957;p<0,01)whiletherehasbeena

signiﬁ-cantpositivecorrelationforTPCandDPPH(r=+0,885;p<0,05),TPC

andFRAP(r=+0,898;p<0,05),TFCandABTS(r=+0,858;p<0,05),

TFCandFRAP(r=+0,833;p<0,05)(Fig.6).

Toanalyzethevariabilityexpressedbythesamples,the

indi-vidualsgraphofPCAhavebeencompletedbyanagglomerative

hierarchical clusteringbased on thecoordinates of samples on

theﬁrstfourfactorsofPCA.Averyheterogeneousdistributionof

sampleshavebeenobservedinthefactorialplane1–2anda

den-drogramprovided bytheAHCshow3distinctgroupstructured

(11)

4. Conclusion

Thisstudywasaimedtoinvestigatethebiologicaleffectsand

chemicalcompositionofB.papillosaaerialandstemparts.

UHPLC-MSresultsrevealedthepresenceofdepressonolA,antanapeptin

A,lucyosideJ,kaempferol3-lathyroside-7-rhamnoside,robinetin

3-rutinosideandquercetin3-galactoside-7-rhamnosideas

impor-tantsecondary metabolitesin themethanol extracts.Moreover,

thebiologicalscreeningsuggeststhatmethanolextractsexhibited

thestrongestantioxidantabilitiesinlinewiththehigherlevelsof

totalphenolicandﬂavonoidcontents.Bothpartshaveconsiderable

butyrylcholinesterase inhibition and thestem n-hexane extract

hasnotableactivity against␣-glucosidase enzyme. BA-M, BS-H

andBS-Mextractspresentedthemostpotentialcytotoxiceffects.

CurrentﬁndingssuggestthatB.papillosacanbeconsideredasa

sourceforhighvaluepolyphenolsfordevelopingnoveldrugleads.

However,futureresearchintermsofbio-guidedisolationofactive

compoundsisneededtoexploretheunexploitedpharmacological

potentialofthisplanttogetherwithmolecularmechanism.

Conﬂictofinterest

Authorshavenoconﬂictofinterest.

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