Rev. bras. farmacogn. vol.26 número3

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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

vivo

diabetic

wound

healing

effect

and

HPLC–DAD–ESI–MS/MS

profiling

of

the

methanol

extracts

of

eight

Aloe

species

Abeer

M. El

Sayed

a

_,

_Shahira

_M.

_Ezzat

a,∗

_,

_Moataz

_M.

_El

_Naggar

b

_,

_Seham

_S.

_El

_Hawary

a

a_{Pharmacognosy}_Department,_Faculty_of_Pharmacy,_Cairo_University,_Cairo,_Egypt

b_Department_of_{Pharmacognosy,}_Faculty_of_Pharmacy,_Damanhour_University,_Damanhour_City,_Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12October2015 Accepted12January2016 Availableonline31March2016 Keywords:

Aloe

HPLC–DAD–MS/MS Chemotaxonomic Anti-inflammatory Woundhealing

a

b

s

t

r

a

c

t

GenusAloe,Xanthorrhoeaceae,iswelldistributedalloverEgypt,andmanyspecieshavebeenused

asmedicinalplants;mainlyreportedtopreventcardiovasculardiseases,canceranddiabetes.Thisstudy

attemptstoanalyzethesecondarymetabolitesinthemethanolextractoftheleavesofeightAloespecies;

A.vera(L.)Burm.f.,A.arborescensMill.,A.eruA.Berger,A.grandidentataSalm-Dyck,A.perfoliataL.,A.

brevi-foliaMill.,A.saponariaHaw.andA.feroxMill.growinginEgypt.ForthisaimHPLC–DAD–MS/MSinnegative

ionmodewasused.Althoughbelongingtothesamegenus,thecompositionofeachspeciespresented

dif-ferentparticularities.SeventyonecompoundswereidentifiedintheinvestigatedAloespecies,ofwhich

cis-p-coumaricacidderivaties,3,4-O-(E)caffeoylferuloylquinicacidandcaffeoylquinicacidhexoside

werethemostcommonphenolicacidsidentified.AloeresinEandisoaloeresinD,2′_-_O_{-feruloylaloesin}

werethecommonanthraquinonesidentified.Lucenin II,viceninII,andorientinwerethecommon

identifiedflavonoidsintheinvestigatedAloespecies.6′_{-Malonylnataloin,}_{aloe-emodin-8-}_O_-glucoside,

flavone-6,8-di-C-glucosidescouldbeconsideredaschemotaxonomicmarkersfortheinvestigatedAloe

species.TheeightAloespecieshadsignificantanti-inflammatoryactivity,inadditiontothesignificant

accelerationofdiabeticwoundhealinginratsfollowingtopicalapplicationofthemethanolextractsof

theirleaves.Thisisthefirstsimultaneouscharacterizationandqualitativedeterminationofmultiple

phe-noliccompoundsinAloespeciesfromlocallygrowncultivarsinEgyptusingHPLC–DAD–MS/MS,which

canbeappliedtostandardizethequalityofdifferentAloespeciesandthefuturedesignofnutraceuticals

andcosmeticpreparations.

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

Introduction

ThegenusAloeL.(familyXanthorrhoeaceae,subfamily

Aspho-deloideae) is an old world genus which comprises more than

400species,whichhascentersofdiversityinsouthernandeast AfricaandMadagascar(Newton,2004).Aloeisusedasatraditional medicinefortreatmentofdifferentdiseases,asingredientsoffood productsandcosmetics(EshunandHe,2004).Phenoliccompounds includingchromones,anthraquinones,andpyronesconstitutethe majorsecondarymetabolitesofAloe(Conneretal.,1990;Okamura etal.,1998;Durìetal.,2004).Mostofthesecompoundshavebeen reportedtopossessseveralbiologicalactivitiessuchasantitumor, antidiabeticandantityrosinase,inadditiontotheantiulcer activ-ity(Imanishiet al.,1981; Yagiet al.,1987; Beppu etal., 1993; Teradairaetal.,1993).SomecomponentsisolatedfromAloespecies werereportedtopossessanti-inflammatoryeffectforexample;an

∗ _{Corresponding}_author.

E-mail:shahira.ezzat@pharma.cu.edu.org(S.M.Ezzat).

effectiveanti-inflammatorycinnamoyl-C-glucosyl-chromonehas beenisolatedfromAloebarbadensis(Hutteretal.,1996).Moreover, thewound-healingpropertiesofA.barbadensiswasalsoreported (Davisetal.,1989).

ThephytochemicalconstituentsandbioactivityofAloespp.have attractedresearchinterestsincethetradein‘drugaloes’,prepared fromtheleafexudate,expandedrapidlyinthenineteenthcentury (Yeats,1870).Today,theprincipalsourcesofthesenaturalproducts arewildpopulationsofAloeferoxinSouthAfrica,andAloe scabrifo-lia,AloesecundifloraandAloeturkanensisineastAfrica(Oldfield, 2004).Moreover, Aloevera, the sourceof the leaf parenchyma knownas‘aloegel’,iswidelycultivated.

Aloeleavesinadditionofbeingofpharmacologicalimportance, theirchemistryhave systematic significance,particularlyatthe infragenericrank.Secondarymetabolitesprofilewasusedinthe evaluationofinfragenericgroupssuchasseriesLongistylae,section Pachydendron,sectionAnguialoeandseriesPurpurascentes Salm-Dyck(VanHeerdenetal.,1996;Reynolds,1997;ViljoenandVan

Wyk,2001). Phytochemical data mayoffer a tool to studythe

complexityofmaculatespecies,whichcompriseabout40species

http://dx.doi.org/10.1016/j.bjp.2016.01.009

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so-namedbecauseoftheirconspicuousleafmarkings.Although

it is widely regarded as a well-supported group, infrageneric

boundariesandspeciesdelimitationinthemaculatecomplexare problematic(Graceetal.,2008).

Forthispurposewehavestartedadetailedscreeningofeight Aloespecies;A. vera (L.) Burm.f., A. arborescensMill.,A. eru A. Berger, A. grandidentata Salm-Dyck, A. perfoliataL., A. brevifolia Mill.,A.saponariaHaw.andA.feroxMillgrowinginEgypt;using HPLCcoupledtoelectrosprayionizationmassspectrometry

(ESI-MS)[HPLC–DAD–ESI–MS/MS]withtheaimofidentifyingunique

constituentswhichmaybeusedasmarkerswithpossible chemo-taxonomicsignificance.Inaddition,toevaluatethediabeticwound healingandanti-inflammatoryeffectsoftheeightAloespeciesin ratsusingthediabeticwoundmodelandcarrageenan-inducedpaw edematests.

Experimental

Plantmaterial

Theleaves of theeight Aloespecies, A. vera (L.) Burm.f., A. arborescensMill.,A.eruA.Berger,A.grandidentataSalm-Dyck,A. perfoliataL.,A.brevifoliaMill.,A.saponariaHaw.andA.feroxMill. werecollectedinApril2014fromEl-OrmanGarden,Giza,Egypt. TheplantswereauthenticatedbyDr.ReemSamirHamdy,Lecturer ofPlantTaxonomy,BotanyDepartment,FacultyofScience,Cairo University,Giza,Egypt.Vouchersamplesno.2014422oftheplants

weredepositedattheMuseumofthePharmacognosyDepartment,

FacultyofPharmacy,CairoUniversity.

Extraction

Thefresh leavesofthe eightAloespecies (500g each)were

cutintopiecesandextractedwithmethanolusingcoldmethod

ofextraction(percolation)(4×2l)tillexhaustion.Themethanol extractineachcasewasfiltered,distilledandevaporatedunder reducedpressuretogive12.65,12.44,5.21,20.02,6.97,6.94,3.86 and3.96gofA.vera,A.arborescens,A.eru,A.grandidentata,A. per-foliata,A.brevifolia,A.saponariaandA.ferox,respectively.

QualitativedeterminationofthephenoliccontentsofAloe

leavesusingHPLC–DAD–ESI-MS/MS

Samplepreparation

Aloe extracts were separately dissolved in HPLC grade

methanolinaconcentrationof10mg/mlthenfiltratedthrougha syringe-filter-membrane.Aliquotsof 5␮lwereinjectedintothe

LC–DAD/MSDionexUltimate3000HPLC(Germany),usedfor

per-formingtheanalyses.

HPLCconditions

ThemethodofElMaggar(2012)wasemployed.HPLCapparatus

(Hewlett-Packard 1100, Waldbronn, Germany) with a

quater-narypump and on linedegasserwas employed for analysis.A

thermostattedcolumncompartmentwithanautosampler,a

pho-todiodearraydetector(DAD).Theseparation wasperformedon

EclipseXDBC18column(50mm×2.1mm,1.8␮m,Agilent

Com-pany, USA). 1100 ChemStation software was used. The mobile

phase wascomposedof two solvents,A:methanoland B:0.2%

formicacidinwater.Gradientelutionprofilewasperformed:0min, A:B10:90;36min,A:B100:0;40min,A:B100:0.Chromatography wasperformedat30◦_C_with_a_flow-rate_of_0.2_ml/min._UV_traces

weremeasuredat290,254and350nmandUVspectra(DAD)were

recordedbetween190and900nm.

Massspectrometricconditions

Electrospray ionization (ESI) interfaced Bruker Daltonik

Esquire-LC ion trapmass spectrometer(Bremen,Germany) and

anAgilentHP1100HPLCsystemwereusedforHPLC–MSanalysis.

An autosampler and a UV–vis absorbance detector were used.

The ionization parameters were as follows: capillary voltage

4000V,end platevoltage−500V;nebulizing gasofnitrogenat 35.0p.s.i.;dryinggasof10l/minnitrogenat350◦_C._Mass_analyzer

scanned from15 to1000u.The MS–MS spectrawererecorded

in auto-MS–MSmode. The fragmentationamplitudewas setto

1.0V.MS2data.Massspectraweresimultaneouslyacquiredusing electrosprayionizationinthenegativeionizationmode.

Chemicals

Dermazine® _cream _(silver _{sulfadiazine)} _was_used _as _a stan-dardwoundhealingdrug.Indomethacin(Indomethacin)®_,_Eipico, Egyptian Int.PharmaceuticalIndustriesCo. wasusedasa stan-dardanti-inflammatorydrug;CarrageenanandAlloxan(SigmaCo., USA).

Evaluationofthebiologicalactivity

Animals

Adult male rats of Sprague-Dawley strain (130–150g) were

obtained from the laboratory animal facility of the National

ResearchCenter,Dokki,Giza.Animalswerehousedinsteelcages

under standard conditions and fed with standard pellets and

wateradlibitum.Allexperimentalprocedureswereconductedin accordancewithinternationallyacceptedprinciplesforlaboratory animaluseandcare,andwereapprovedbytheEthicsCommittee

(No.9-031)inaccordancewithrecommendationsfortheproper

careand useof laboratoryanimals (NIHPublication No.80-23; revised1978).

Evaluationofanti-inflammatoryactivity

Themethodof Winteret al.(1962)wasadopted,where ten

groupsofrats(eachofsix)wereused,eightgroupsservedasthe testgroups,eachofthemwasadministeredasingleoraldoseof eachofthemethanolextractoftheeighttestedspecies(A1–A8) inadoseof250mg/kgbodyweight.Thedoseoftheextractswas determinedas250mg/kgonthebasisofapreliminaryshort-term pilotstudywitharangeofvariabledoses.Theothertwogroups, oneofthemreceived1mlsalineandservedasanegativecontrol groupandtheotherreceivedIndomethacin,20mg/kgbodyweight asareferencedrug.Drugswereorallyadministered1hpriorto car-rageenaninjection.Edemawasinducedintheratrighthindpawby subcutaneousinjectionof0.1mlof1%carrageenansuspensionin saline.Thicknessoftherighthindpaw(mm)wasmeasured imme-diatelybeforeand1,2,3and4hpostcarrageenaninjectionwith amicrometercaliber.Theresultsareconsideredsignificantwhen theyaredifferentfromzerotimeatp<0.05.

Invivodiabeticwoundhealingstudy

ApreviouslyestablishedchemicallyinducedtypeIIdiabeticfoot ulceranimalmodelwasemployedinthisstudy(Lauetal.,2008). Forthosediabeticrats(plasmaglucoselevels≥250mg/dl,samples collectedfromtailveins),sixtyratsweredividedintotengroups,

a standardizedwound area(2mm×5mmskininfullthickness

removed)wasinducedonthedorsalsurfaceoftherighthindfoot ofratsunderanaesthetizationwithphenobarbitone.Eightgroups weretreatedtopicallywitheachofthemethanolextractofeachof theeighttestedspeciesbytopicalapplicationonthewoundedarea. Theothertwogroupsactasanegative(untreatedgroup)anda posi-tivecontrolgroup(treatedwithDermazine®

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ineachgroupwasstartedatthedayoftheoperationand contin-uedtillthe10thday.Thewoundswerecoveredwithappropriate dressings,whichwerechangedregularly.Onchangingthe

dress-ings,woundswereinspected,measuredandphotographed.The

woundareasweremeasuredwhileanimalswereunderanesthesia

on2nd,6thand10thdayaftersurgery.Theprogressivechanges

inwoundareaweremeasuredbyaplantimeter,wound

contrac-tionwereexpressedaspercentage reductionof originalwound

size[woundareaatday0−woundareaatdayn/woundareaat day0]×100.

Statisticalanalysis

Thestatisticalcomparison of differencebetweenthecontrol

group and the treated groups was carried out using two-way

ANOVAfollowedbyDuncan’smultiplerangetest.

Resultsanddiscussion

The classes of compounds were recognizable from their

characteristic UV spectra, which were identified based on the

HPLC–DAD–ESI–MS/MS data and subsequent confirmation by

comparisonwithliteraturedata.Thechromatographicand spec-troscopicdatasummarizedinTable1andFig.1.

Chromatographicpeaksannotation

Phenolicacids

Hydroxycinnamic acids such as esters of quinic acid were

detectedasabout10compoundsinA.eru,A.grandidentata,A. brevi-folia,A.saponariaandA.feroxextractscondensedeitherwithcaffeic, ferulicorcoumaricacids.Thedetectedmass[M−H]−_at_m_/_z_651,

515,408,353,601,620,529,516,613and483ofthepeaks,1(Rt 118.2s),2(Rt248s),8 (Rt716s),10(Rt728s),11 (Rt796s), 16(Rt875.2s),17(Rt888s),23(Rt942.1s),31(Rt976s),and38 (Rt1025),respectively.Thosepeaksshowedthecharacteristicbase peakofquinicacid,ferulicacid,coumaricacidandcaffeicacidat m/z191,193,163and179,respectively.

Regardingdicaffeoylquinicacids,peak2(Rt248s)showedm/z at515[M−H]−_,_this_was_identified_as_3,4-di-_O_-(_E_{)-caffeoylquinic}

acid(1).Peak17(Rt888s)wasidentifiedas3-O-(E)-caffeoyl-4-O -feruloylquinicacid(2),itshowed[M−H]−_at_m_/_z₅₂₉_(C26H26O12)

inthenegativeionizationmode.Thisparentionwasfragmented inproductionsatm/z367basepeak,producedbythelossofa caffeoylunit,andm/z353,producedbythelossofaferuloylunit. CaffeoylferuloylquinicacidwasdetectedinA.arborescens,A.eru,A. grandidentataandA.perfoliataonly.Peak30(Rt970.2s)showed [M−H]−_at_m_/_z_at₄₉₉_in_the_negative_ionization_mode._Peak₃₀

showeddaughterionsatm/z353,m/z191(basepeak),m/z179and m/z163.Thiscompoundwasidentifiedas3-O-(E)-caffeoyl-5-O-p -coumaroylquinicacid(3)(GouveiaandCastilho,2011),this com-poundwasonlydetectedinA.vera,A.arborescensandA.perfoliata. Peak23(Rt942.1s)wasidentifiedascaffeoylquinichexoside (Gobbo-NetoandLopes,2008)andpeak16(Rt875.2s)waslikely identifiedastransp-coumaricderivative.

Polyketides

Massspectrum ofpeak 3(Rt 599s) showed[M−H]− _at_m_/_z

393whichshowedadaughterionatm/z247duetothelossof 90amuasaresultofacross-ringcleavageinthehexosidic part involvedintheformationoftheionat247.Thisfragmentationwas

previously reportedfor deprotonated and protonated flavonoid

C-glycosides. Common ions were also noted at m/z 41 and 59

andaneutrallossof120amuwasobserved.Thiscompoundwas

identifiedasaloesin(4,aloeresinB),whichwasdetectedinA. gran-didentataandA.perfoliata.Thiscompoundwaspreviouslyreported inleavesofA.castanea(VanHeerdenetal.,2000).AloesinAandB areAloeresinwithnopurgativeaction.AloesinBisa

chromone-C-glucoside, where aloesin A is a p-coumaric acid ester of

aloesinB.

ThenegativemodeESI-MSspectrumof peak13(Rt841.9s)

showedastrong[M−H]−_parent_ion_at_m_/_z₅₉₅_{corresponding}_to

thecaffeoylesterof5-hydroxyaloinA.Fragmentionsatm/z433 and271wereobservedinMS2_consistent_with_the_loss_of_caffeoyl andcaffeoylpluscarbohydratemoietiesfromtheparentmolecules, respectively.Afurtherfragmentionwithm/z314correspondsto thelossofestergrouptogetherwithafourcarbonfragmentofthe carbohydratemoiety.Caffeoylesterof5-hydroxyaloinAwasnoted inA.vera,A.arborescensandA.grandidentata.

AloeresinC(5)wasdetectedaspeak19(Rt915.8s)showing [M−H]−_at_m_/_z_702,₅₄₁_(703-C6_H10O5),_{corresponding}_to_aloeresin

A(6),m/z394equivalenttoaloesinandm/z232correspondingto aloesone.AloeresinCrepresentthefirstexampleofC,O-diglucoside of5-alkylchromoneaglyconedetectedonlyinA.vera,A.arborescens, A.perfoliata,andA.brevifolia.

Mass spectra of peak 29 (Rt 962.6 s) showed [M−H]− _at

m/z 409.1 was identified as aloenin (7, 4-methoxy-6-(2-ˇ-d

-glucopyranosyloxy-4-hydroxy-6-methylphenyl)-2-pyrone). This

compoundwasdetectedonlyinA.eru.AloeninB(8,peak58,Rt 1290s),isabitterglucosidepreviouslyisolatedfromAloeleaves andfoundtoexhibitaninhibitoryactionongastricjuicesecretion ofrats(HirataandSuga,1978).ThiscompoundwasdetectedinA. vera,A.arborescens,A.eruandA.grandidentata.

The negative modeESI-MS spectrum of peak 33 (Rt 998 s)

showed a strong [M−H]− _parent _ion _at _m_/_z _433. _MS2 _of _the compoundyieldeda daughterionatm/z271resultedfromthe

loss of a sugar moiety (C6H11O5) from the parent molecules.

A moderately abundant ion at m/z 313 probably results from

the loss of a (C4H8O4) fragment fromthe [M−H]− parent ion. Compound33wasidentifiedas5-hydroxyaloinA(9)(Holzapfel etal.,1997)andthis wasonly detectedin A.grandidentataand A.perfoliata.

Peak34(Rt1011.9s)showed[M−H]−_at_m_/_z₅₀₅_was

iden-tifiedas6′_{-malonylnataloin}₍₁₀_,_nataloin),_compound_considered

ofgreatimportanceinsystematicdiscriminationofdifferentAloe species.6-Malonylnataloinis aputativemarkerfor Africantaxa inthemaculatespeciescomplex,althoughitwasnotdetectedin Aloemaculata[A. saponaria];commonlyknownastheSoap Aloe orZebraAloe;whichisaSouthernAfricanspeciesofAloe.Onthe contrary,nataloinhasbeenobservedinnon-maculatespeciesas diverseinformandinfragenericpositionasA.vera,A.arborescens, A. eru, A. grandidentata, A. brevifolia and A. ferox; those are relatedeastAfricanspeciesandnataloinmaytherefore,serveasa

phytochemicalmarkerfor them(Wabuyele,2006).Comparative

dataindicatedthattheanthroneC-glycoside,6-malonylnataloin (7-hydroxychrysaloin6-O-malonate)istypicalofmaculatespecies inEastAfrica.

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Table1

PeakassignmentsofmetabolitesidentifiedinLC/MSspectrumofeightdifferentAloespecies.

No. Rt(s) [M−H]− _MSn_ion_m_/_z₍₋₎_ppm _Tentative_{identification} _A

1 A2 A3 A4 A5 A6 A7 A8

1. 118.2 651 591,501,436,349,302, 206/489,325,205,163,119

cis-p-Coumaricacidderivatives + + − − − + + +

2. 248 515 464,382,301 3,4-di-O-(E)-Caffeoylquinicacid − − − − − − + −

3. 599 393 376,304,274,245,219,203, 163

Aloesin(aloeresinB) − − − + + − − −

4. 603 439 380,313,274,135 Unk − + − − + − − −

5. 672 593 431,258,175 Aloeemodin-diglucoside − + − + − − − +

6. 677 456 394,364,333,291,223 Unk − − − + + − − −

7. 698 395 350,274.8,230,202/313,259, 213

1-Hexanol-pentosylhexoside − − − − + − − −

8. 716 408 315,252,147/354,303,269, 233

Caffeoylferulicacidderivatives − − − + − − − −

9. 727 475 405,301,367,286,224,145 Chrysoeriol-7-O-glycuronyl − − − + − − − −

10. 728 353 248,209,191,179,173,161, 135,145

5-O-caffeolyquinicacid − − − − − + − −

11. 796 601 563,537,529,496,429 Malonyl-3,4-O-dicaffeoylquinic acid

+ − − − − − + −

12. 811.8 609 590,518,489 Luteolin-6,8-C-diglucoside (LuceninII)

+ + − + − − + +

13. 841.9 595 533.1,433,415.6,294.4,314, 271,145.1

6′

-O-Caffeoyl-5-hydroxyaloinA + + − + − − − −

14. 857.8 542 145.4,191.8,250.0,300.5, 360.9,415.1,505,396

Unk + − − − − − − +

15. 871.2 593 575,503,473,449,383,353, 287

Apigenin-6,8-C-diglucoside (viceninII)

+ + + + − + − +

16. 875.2 620 583.0,437.0,370.8 trans-p-Coumaricderivatives + + − − − − − −

17. 888 529 475,367,192,145 3-O-(E)-caffeoyl-4-O -feruloylquinic acid

− + + + + − − −

18. 915.2 667 605,513,439,394,285,232 Luteolin-O-xylosylglucoside malonylated

+ + − + + + + −

19. 915.8 702 541,395,233 AloeresinC(isomer) + + − − + + − −

20. 921 739 704,587,577,550,453,310, 289,242

Epi-catechindigalloylrhamnoside − − − − + + − −

21. 923.9 624 584.2,517.0,314 Isorhamnetin-3-O -deoxyhexosyl(1-6) hexoside

+ + − + − + + −

22. 934.8 581 516.2 7-O-Methylkaempferoldimmer + + − − + − − −

23. 942.1 516 483.9,354.9,210.7,186.7 Caffeoylquinicacidhexoside + + − − − + − −

24. 945 411 246.8/249,161,113 Isopentyldihexose − − + − − − − −

25. 948 445 439.6,409,248.8,271,269,153 Apigenin-7-O-glycuronyl − − + − − − − −

26. 948.2 579 495.1,447.4,356.9,287,285 Kaempferol-3-O-hexosyl-O -pentoside

− + + − − − −

27. 948.2 447 357.3/405,327,147 Luteolin-8-C-glucoside(orientin) + + − + + − − +

28. 955.6 447.8 429.8,405,356.9,326.9,271, 147

Luteolin-6-C-glucoside (isoorientin)

+ − − + − − − −

29. 962.6 409.1 247 Aloenin − − + − − − − −

30. 970.2 499 472.0,436.7,312.5 3-O-Caffeoyl-5-O-coumaroylquinic acid

+ + − − + − − −

31. 976 613 570,508,457,391 4-Succinyl-3,4-dicaffeoylquinic acid

− + − − + + + −

32. 998 469 435/395,298,192,149 Unk − − − + − − − +

33. 998 433 396.6,342.9,313,269.8,271, 255

5-HydroxyaloinA − − − + + − − −

34. 1011.9 505 448,343.1,342.9,341.1,172 6′_{-Malonylnataloin}_(nataloin) ₊ ₊ ₊ ₊

− + − +

35. 1013.1 343 316,315,299,287,285,236 5,3′_{-Dihydroxy-6,7,4}′_-trimethoxy flavones(Eupatorin)

− + + + − + − −

36. 1018.1 463 515.8,472.5/301 Isoquercetrin − + + − − − − −

37. 1018.4 563.2 505.5,500,435,406,342.9, 328,290

AloinosideA/B + + + + + − − −

38. 1025 483 446,422,325.9/337,319,173, 163

3,4-Di-O-(E)-p-coumaroylquinic acid

− + − + − − − −

39. 1057 577 451,425,413,305,271,293.4 Epi(afzelechin)–(epi) gallocatechin

− − + + + − − −

40. 1063 543 500,462,361 Unk + + − + − − − −

41. 1081 509 492.6,387.9,345.9,283.8, 212.9

Unk + + − − − − − −

42. 1084.2 506 396.5,343.0,342.9 Unk + + − − − − − −

43. 1096 570 548,510.8,471.9,433,411, 357.5,320,248,203

2′_-_O_{-Feruloylaloesin} ₋ ₊ ₊ ₊ ₋ ₊ ₊ ₋

44. 1114 778 658,570,508,455 Unk − − − + + − − −

45. 1120.5 616 582.7,578.8,516.8,500.1, 393.2,147.1,145.3

7-O-Methylaloesin-pentaacetate + + − − + − + −

46. 1121 583 570,554,477,407,243,272 7-Methyletherof2′_{-feruloylaloesin}

− − + + − − + −

47. 1143.7 604 567.1,500.3,421.0 malonyl-4,5-O-dicaffeoylquinic acid

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Table1(Continued)

No. Rt(s) [M−H]−

MSn_ion_m_/_z₍₋₎_ppm _Tentative_{identification} _A₁ _A₂ _A₃ _A₄ _A₅ _A₆ _A₇ _A₈

48. 1161 629 547,481,208,146/537,425, 378,325,236,178,146

Glucuronides − − + − + − − −

49. 1164 670 616,550,473,274 Unk − − − − − − + +

50. 1167 555 538,512,410,392,366,259, 193,147,119

IsoaloeresinD − − + + + + − −

51. 1198 554 – Unk − − − + + − − −

52. 1224.4 556 494,423,282,207 Aloeresin − − + + − + − −

53. 1226 671 615,581,481 Unk − − − + − − − +

54. 1256.4 418 417.6,343.2 Nataloin + + − + − − − −

55. 1258 297 – VeracylglucanA − + − − − − − −

56. 1265 885 431,307,284 Kaempferoldi

deoxyhexosylhexoside

− − + − − − − +

57. 1281.6 833 636.2,713.5,775.6 Unk + + − + − − − −

58. 1290 713 635.9,564.3,516.5,343.4 AloeninB + + + + − − − −

59. 1292.0 747 482.1 Wighteone-O-diglucoside

malonylate

+ + − − − − − −

60. 1302.1 417 396.1,343.1,296.9 AloinA + + − + − − − −

61. 1318 417 145,297,343 AloinB + − − + − − − −

62. 1328 432 361,359,353,340.7,310,283, 269

Isovitexin(6-C-glucosyl-apigenin) − − − − + − − −

63. 1342 539 432,359,312,254,146 6′

-O-Coumaroylaloesin − − − − + − − −

64. 1366 297 248.9,174.9 Hydroxyoctadecenicacid − − − + − − − −

65. 1396 586.9 587,518.1,427,257,147 Unk − − + − − − − −

66. 1407 540 468,420,393,312 AloeresinAisomer − − − − + − − −

67. 1410 329.7 286,214,147 Trihydroxyoctadecenoicacid − − − − − − + −

68. 1424 1016 979,916.9,859.6,834.9,606, 144.8

Unk + + − − − + − −

69. 1430 657.6 620,530,458/533,480,391, 134

Unk − − − + + − − −

70. 1448 869.2 833,717,650,586.3,511.3, 144.7

Unk + + − + − − − −

71. 1460 569 426,355,285,225,161 Caffeoylesterofaloesin − + + − + − − −

72. 1462 496 418,225,397,285,248,145 Unk − + + − − − − −

73. 1507 580 463,326,296,265 Unk − − − + − + − −

74. 1519 605 562,425,317,267,146,92 Unk − − + − − + − −

75. 1590 565 534,488,343,241 Trihydroxycinnamicacid derivatives

− − − + − − − −

76. 1600 599 564,512,443.0 Unk + + − + − − − −

77. 1611 685 623,571,500,523.3,539,521 AloeresinE + + + + + - - +

78. 1612 557 505,444,392,322,251 Acetyldicaffeoylquinicacid − − − + + − − −

79. 1645 831.3 813.2,711.2,669.8,742,686, 553

Unk + + − − − − − +

80. 1659.9 416.4 267.8 Barbaloin(10R)/Isobarbaloin(10S) + + − − − − − −

81. 1666 445 380,295,256,216,145/269 Apigenin-7-O-glycuronyl − − + − − − − −

82. 1725 741.6 416,301,300/609,301 Quercetinpentosylrutinoside − − − − − − − +

83. 2083 689.1 854,603,505 Unk − + − − + + − −

84. 2097 675.9 640,586,534/640,506,497 Unk − − − − + + + −

85. 2117.8 711.9 675.2,550.6,396.9 Quercetin-7-O-hexoside-3-O -malonylhexoside

+ + − − + − + −

86. 2174 431 316.1,269,225 Aloe-emodin-8-O-glucoside + + + − + − + +

87. 2226 553 408,376,341,275,259,256, 233,193,178,161

2′_-_p_{-Methoxycoumaroylaloresin} ₋ ₋ ₊ ₋ ₊ ₋ ₊ ₋

88. 2235 552 448.1,359.8,145.1 Unk − + − − + − − −

89. 2252 715 625.6,553.6,455.1,357.6, 303.8,207,129.2

Unk − + − − − − − −

90. 2293 567 528,313,271,253,169 Chrysophanol-8-O-(6′_-O_-galloyl-) glucoside

− + − − − − − +

91. 2339 637 579,504,359 Unk − − − − − − + −

92. 2450 825 788,669,626,581,514,452, 394

AloeresinHtetra-O-methylether − − − − − − + −

93. 2549 769 730.5,326.1 AloeresinH − + − − + − − −

94. 2574 612 555,492.3,427,260.4 Unk + + + − + − − −

95. 2603 642,643 612,557,513,446,375,269, 135/550,501/580,508,459

Unk − − + − + − + +

96. 2625 809.8 642,556,515,448 Unk + + − − + − + −

97. 2632 444.2 392,203.7 Unk + + − − − − − −

98. 2650 811.8 777.8,672.4,573.3,512.8, 462.3,392,341,269,135

Unk − + − − + − − −

99. 2712 813.4 529.4,462.4 Unk − + − − + − − −

100. 2768 789.6 672.3,612.6,533.9,467,398.7, 308.8,272,135,93

Unk − + − − − − − −

101. 2871 627.8 571.2,505.8,444.8,388.7, 135.6/481,319

Pentahydroxyflavonol-O-hexosyl rhamnoside

− + − − + − − −

102. 2872 532/533 464,388,171/489,285 Kaempferol-3-O-malonylhexoside − − − + + − − −

(6)

0 500 1000 1500 2000 2500 Time [s] 0

1 2 3 4

5

x10 Intens.

MOATI000039.D aloe1-A.vera: BPC 50.0-1300.0 -All MS

0 500 1000 1500 2000 2500 Time [s]

0.0 0.5 1.0 1.5

6

x10 Intens.

MOATI000058.D: BPC 50.0-1300.0 +All MS

0 500 1000 1500 2000 2500 Time [s]

0.0 0.5 1.0 1.5 2.0

6

x10 Intens.

MOATI000041.Daloe3-eru: BPC 50.0-1300.0 -All MS

0 500 1000 1500 2000 2500 Time [s] 0.0

0.2 0.4 0.6 0.8

6

x10 Intens.

MOATI000043.Daloe4-grandidentata: BPC 50.0-1300.0 -All MS

0 500 1000 1500 2000 2500 Time [s]

0 2 4 6

5

x10 Intens.

MOAT1000044.D aloe5-perfoliata: BPC 50.0-1300.0 -All MS

0 500 1000 1500 2000 2500 Time [s]

0 2 4 6

4

x10 Intens.

MOATI000045.D aloe 6-brevifolia: BPC 50-1300 -All

500 1000 1500 2000 2500 Time [s] 0

2 4 6 8

4

x10 Intens.

MOATI000046.D aloe 7-saponaria: BPC 50-1300 -All MSn

0 500 1000 1500 2000 2500 Time [s]

0 2 4 6 8

5

x10 Intens.

MOATI000047.D aloe 8-ferox: BPC 50-1300 -All

A

B

C

D

E

F

G

H

(7)

ESI-MSspectrumofpeak45(Rt1120.5s)showed[M−H]−_at_m/z

616whichonfurtherfragmentationyieldedionsatm/z516,500, 393and147.Thiscompoundwasidentifiedas7-O-methylaloesin pentaacetateanddetectedinA.vera,A.arborescens,A.perfoliata andA.saponaria.

Thenegative ESI-MS of peak 46 (Rt 1121 s) showedstrong peaks at m/z 583 [M−H]− _which _yielded _a _peak _at _m/z ₄₀₇

[M−177]− _due _to _the _loss _of _a _fragment _with _mass _177. _This

couldbeinterpretedasthelossofa(COCH CHC6H3(OCH3)OH)

fragmentfromaferulicacidderivative.Thisfragmentationis fol-lowedbythelossofwaterfromm/z407.Thespectrumshowed nopeakscorrespondingtothesubsequentlossof the carbohy-drate moiety after the loss of the acyl group. In contrast, the negativeESI-MSof aloesinshoweda strong[M−H]− _ion _and _a

basepeakatm/z272correspondingtothelossofcarbohydrate moiety.Thiscompoundwasidentifiedas(E)-2-acetonyl-8-(2′

-O-feruloyl)-␤-d_{-glucopyranosyl-7-methoxy-5-methylchromone}_(11,

7-methyletherof2′_{-feruloylaloesin)}_and_detected_in_A._eru,_A.

gran-didentataandA.saponaria.7-Methyletherof2′_{-feruloylaloesin}_was

previouslyreportedintheleavesofA.broomii,A.AfricanaandA. speciosa(Holzapfeletal.,1997).

Peak 50 (Rt 1167 s) revealed a molecular ion peak at

m/z 555 [M−H]−_, _consistent _with _a _molecular _weight ₅₅₆

which was detected in A. eru, A. grandidentata, A.

perfoli-ata and A. brevifolia. MS/MS analysis revealed the follow-ing fragments at m/z 538 [M−H2O] ([M−18]−), m/z 512 [M−CH3CHO] ([M−44]−_), _m_/_z ₄₁₀ _[M₋_coumaroyl] _([M₋_146]−_), m/z 392 [M−(146+18)]−_,_m_/_z ₃₆₆ _[M₋₍₁₄₆₊_44)]−_, _m_/_z ₂₅₉

[M−(133+146+18)]−_, _m_/_z _193[M

−(133+146+84)−_], _m_/_z ₁₄₇

[coumaroyl]+ _and _m_/_z ₁₁₉ _[HOC6H4CHCH]+_. _This _spectra _was

consistentwiththe spectrum reportedby Lee et al. (2000) for isoaloeresinD (12,8-C-␤-d-[2-O-(E

)-coumaroyl]glucopyranosyl-2-[2-hydroxy]-propyl-7-methoxy-5-methylchromone).

Nataloinwasdetectedaspeak54(Rt1256.4s)with[M−H]−_,

atm/z418,thiscompoundwasobservedinA.vera,A.arborescens andA.grandidentata.AloinA/B(13,10-C-␤-d-glycopyranosideof

aloe-emodin-anthrone),whichinmostcaseswasdetectedasa mix-tureof␣-and␤-stereoisomers(MW418),waspreviouslyisolated fromtheleafexudatesofthenonmaculateKenyanspecies(Conner etal.,1987).AloinAwasobservedintheleafextractofA.vera,A. arborescens,andA.grandidentataaspeak60with[M−H]−_at_m_/_z

417.Peak61(Rt1318s)whichshowed[M−H]−_at_m_/_z₄₁₇_was

identifiedasaloinBandwasdetectedinA.veraandA.grandidentata.

Peak 63 (Rt 1342 s) was identified as a chromone; 6-O

-coumaroylaloesinhaving[M−H]−_at_m_/_z₅₃₉_which_was_detected

onlyinA.perfoliata.Thischromoneisamarkermetaboliterestricted tothesectionAnguialoe,thusprovidingchemotaxonomic corrob-orationforthepresumedmonophylyofthesection(Allspeciesof AloethatbelongtosectionAnguialoeshareasingle,unique apo-morphywhichisthesessileflowers)(Reynolds,1985).

Peak66(Rt1407s)with[M−H]−_at_m_/_z₅₄₀_consistent_with_a

molecularweight541ofaloeresinA(6).Thedaughterfragmentat m/z393withdifference146amuwasinagreementwiththeloss ofap-coumaroylgroup.Inpreviousworkdealingwiththemass spectralcharacterizationofplantglycoconjugates,aneutralloss of120amuduetoacross-ringcleavageinthehexosidicpartfor deprotonatedandprotonatedflavonoidsC-glycosideswasreported (Wangetal.,2003).Thelatterlosshasalsobeenreportedforthe protonatedchromoneC-glycosides,aloesinandaloeresinA. Alo-eresinAwasdetectedonlyinA.perfoliata.

Mass spectra of peak 71 at (Rt 1460s) showed [M−H]− _at m/z 569which was tentatively interpreted as caffeoyl ester of aloesin. The compound was identified as (E)-2-acetonyl-8-(2′ -O-caffeoyl)-␤-d-glucopyranosyl-7-methoxy-5-methyl chromone

and observed in A. arborescens, A. eru and A. perfoliata. Peak

77 (Rt 1611 s) showed [M−H]− _at _m_/_z ₆₈₅ _which _was

tentativelyinterpretedas(E)-2-acetonyl-(2′_,6′_-di-_O_,_O -coumaroyl)-␤-d-glucopyranosyl-7-hydroxy-5-methylchromone(aloeresinE,

14).ThiscompoundwasobservedinA.vera,A.arborescens,A.eru, A.grandidentata,A.perfoliataandA.ferox.MS2 _showed_daughter

fragmentions at m/z 539and 521corresponded totheloss of

acetonylandcoumaroylpluscarbohydratemoietiesfromthe par-entmolecules,respectively.Massspectrumofpeak86 (Rt2174 s)showed[M−H]−_at_m_/_z₄₃₁_which_was_tentatively_interpreted

asaloe-emodin-8-O-glucoside(14).MS2_of_the_compound_showed fragmentionsatm/z316,269,and225.ThiswasdetectedinA.vera, A.arborescens,A.eru,A.perfoliata,A.saponariaandA.ferox.

Peak87(Rt2226s)inthenegativeionmodeshoweda depro-tonatedparentionpeakatm/z553[M−H]−_._A_daughter_ion_was

obtainedinMS2_due_to_loss_of_p_-methoxy_coumaric_acid_at_m_/_z₃₇₆ andthepresenceofthep-methoxycoumaroylfragmentionatm/z 161whichwereconsistentwiththepreviouslyreporteddatafor2′ -p-methoxycoumaroylaloeresininleavesofA.excels(Mebe,1987). ThiscompoundwasdetectedinA.eru,A.perfoliataandA.saponaria. TheESI-MSspectrumofpeak92(Rt2450s)gavedeprotonated molecule[M−H]− _at_m_/_z₈₂₅_which _was_tentatively_interpreted

asaloeresinHtetra-O-methyletherobservedonlyinA.saponaria. Forpeak93(Rt2549s),theESI-MSspectrumgavedeprotonated molecule[M−H]−_at_m_/_z₇₆₉_which_was_tentatively_interpreted_as

(8)

C-glycosylflavonoids

Theoccurrenceofflavonoidssuchasisoorientin(15, luteolin-6-C-glucoside) and isovitexin (16, apigenin 6-C-glucoside) was observedinbiogeographicaltrends.Isoorientinisacommon con-stituent of tropical and sub-tropical species of Aloe, whereas isovitexinisrestrictedtoafewsouthernAfricanspecies.Isoorientin andisovitexincooccurintheSouthernAfricanmaculatespecies; A.parvibracteata,andthedisjunctWestAfricanmaculatespecies, A.macrocarpa.Thepresenceofisoorientinandisovitexinin mac-ulatespeciesofAloewasfirstreportedbyGraceetal.,2010.The presenceofflavonoidsinsectionPictaeisthereforeoftaxonomic interest.

ESI-MSspectrum ofPeak12(Rt811.8s)showed[M−H]− _at m/z609, itsMS/MSspectrum giverisetodaughter ionsatm/z 590[(M−H)-18]−_,₅₁₈_[(M

−H)-90]−_and_a_base_peak_at_m_/_z₄₈₉

[(M−H)-120]−_._This_compound _was_tentatively_{characterized}_as

luteolin-6,8-C-diglycoside(17, lucenin II).The MS/MSspectrum of peak 15 (Rt 871.2 s) in negativeion modewith[M−H]− _at m/z593whichproduceddaughterionsatm/z575[(M−H)-18]−_, m/z503[(M−H)-90]−_and _a_base_peak _at_m_/_z₄₇₃_[M₋_H-120]−_,

exhibitingafragmentationpatternofflavonesdi-C-glycoside.The ionsat m/z 353[(M−H)-(120+120)]− _and _at _m_/_z ₃₈₃_[(M

−

H)-(90+120)]− _indicated _the _presence _of _apigenin _(MW ₂₇₀₎ _as

aglyconeandtwohexosemoieties(glucoses).ComparingwithMS literaturedata(Piccinellietal.,2008),thiscompoundwas charac-terizedas6,8-di-C-glucosylapigeninandalsoknownasviceninII (18).

Peak27(Rt948.2s),showedESI-MSspectrumwith[M−H]−

atm/z447.Tendammassofthispeakyieldedfragmentionsatm/z 357.3[(M−H)-90]−_and_a_base_peak_at₃₂₇_[(M₋_H)-120]−_,_together

withtheabsenceofthelossof18amusuggestingthatthe mono-C-glycosylationisinposition8(Ferreresetal.,2007).Peak27was tentativelycharacterizedasluteolin8-C-glucoside,alsoknownas orientin(19).ThiscompoundwasdetectedinA.vera,A.arborescens, A.grandidentata,A.perfoliataandA.ferox.ESI-MSspectrumofpeak 28(Rt955.6s)showedadeprotonatedmolecule[M−H]−_at_m_/_z

447.8.Thespectrumexhibitedfragmentsatm/z356.9[(M−H)-90]−

and a base peak at326.9 [(M−H)-120]−_._For _flavones _mono-_C

-hexosides,thepositionofthesugarresiduecanbeassignedthrough observationoftheabundanceoffragmention[(M−H)-18].In gen-eral,thefragmentationofthe6-C-isomersismoreextensive,giving anioncorrespondingtom/z429[(M−H)-18]−_,_probably_due_to_the

formationofanadditionalhydrogenbondbetweenthe2′′_-hydroxyl

groupof thesugar and the 5-or 7-hydroxylgroup of the

agly-cone,whichconfersadditionalrigidity(Abad-Garciaetal.,2009). Theabundanceoffragmentionat429.8suggestedthemono-C -glycosylationisinposition6,hence,compound28wasidentifiedas luteolin-6-C-glucoside,alsoknownasisoorientin(15).Isoorientin wasdetectedinA.veraandA.grandidentata.

Peak62 atRt 1328s,inwhich theESI-MSspectrumshowed

[M−H]− _at_m_/_z₄₃₂_was_{characterized}_as_6-C_-glycosyl _apigenin,

alsoknownasisovitexin(16).ItsMS/MSspectrumgavemass frag-mentationatm/z340.7and310.IsovitexinwasobservedonlyinA. perfoliata.Themainproductionsinthenegativeionizationmode wereduetodehydration,cleavageofsugarring,andthelossof glycosidicmethylgroupasformaldehyde.Inaddition,the inten-sityratiosofthesemajorfragmentscouldbeawaytodifferentiate vitexinfromisovitexin(Abad-Garcíaetal.,2008).TheMS/MSinthe negativeionizationmode,theproductionsobtainedwith cleav-ageofsugarringwasproposedasdiagnosticions,sincem/z313 isthebasepeakofapigenin-8-C-glycosideandm/z283isthebase peakofapigenin-6-C-glycoside.Incontrast,anionatm/z361was onlyfoundinapigenin-6-C-glycoside,probablybecauseofthe addi-tionalhydrogenbondthatisrequiredforlossoftheextrawater (Abad-Garcíaetal.,2008).

O-Glycosylflavonoids

Peak9showedprecursorionsatm/z475[M−H]−_and_MS2_at m/z301,correspondingtothelossofaglycuronylunit.Afurther fragmentationoftheprecursorionatm/z301resultedina prod-uctionatm/z286,producedbythelossofmethylgroup(El-Hela etal.,2010).Therefore,peak9wasidentifiedaschrysoeriol-7-O -glycuronyldetectedonlyinA.grandidentata.

MSspectrainterpretationallowedtheassignmentofthe com-poundluteolin-O-xylosylglucosidemalonylatedaspeak18andits molecularionatm/z667andfurtherfragmentationofparention whichgaveaglyconefragmentatm/z285asthemostabundant ioninthefragmentationspectrum(Wojakowskaetal.,2013) indi-cating thepresence of luteolin. Malonylation increase both the affinity and transportation,of anthocyaninsor otherflavonoids andfacilitatetheirtransportintothevacuolebyadefined trans-porter.Ithasbeensuggestedthataromaticacylationenhancesthe colorofanthocyanins,whereasaliphaticacylation(usually mal-onylation)maystabilizeflavonoidsandincreasetheresistanceof flavonoidglucosidemalonatestoenzymaticdegradation(Luoetal., 2007).Malonylationofflavonoidsoccurswidelyinplants,and mal-onyltransferases havebeen characterizedfrom severaldifferent species(Yuetal.,2008).Malonylation,butnotp-coumaroylation, offlavonoidsinducesaconformationalchangethatmight facili-tatetheirtransportandstorageinthevacuole,possiblythrough improvedsolubilityandreactionwithothervacuolarcomponents (Maternet al.,1983).Compound18 wasobserved in allof the investigatedAloespeciesexceptA.eruandA.ferox.Thepresence offlavonoidsinsectionPictaeisoftaxonomicinterest.

Peak21(Rt923.9s)wasidentifiedasisorhamnetinderivative. Isorhamnetinisamethylatedderivativeofquercetinand itwas identifiedonthebasisofthepresenceofaspecificbasepeakat m/z314, inaddition tocharacteristic loss of a methylfromits methoxygroup(−15amu).Theresultswereconsistentwiththe

lossofadeoxyhexosyl-hexosidemoiety(308amu).Compound21

wasidentifiedasisorhamnetin-3-O-deoxyhexosyl-(1-6)hexoside andwasnotedinA.vera,A.arborescens,A.grandidentata,A.brevifolia andA.saponaria.

Themassspectrumofpeak25at(Rt948s)showedprecursorion inthenegativeionizationmodeatm/z445[M−H]−_and_a

daugh-terionpeakatm/z269,correspondingtothelossofaglycuronyl unit. Therefore, this compound wasidentified as apigenin-7-O -glycuronyl(Surowiecetal.,2007;Petroviciuetal.,2010)notedonly inA.eru.

Massspectrumofpeak26(Rt948.2s)showedprecursorions atm/z579[M−H]−_could_be_identified_as_a

kaempferol-hexosyl-pentosidebasedonthefragmentatm/z447[(M−H)-132]− _and

at m/z285 [(M−H)-132-162]−_. _Therefore, _peak ₂₆ _was

(9)

Table2

Anti-inflammatoryactivityofthemethanolextractoftheeightAloespecies(A1–A8).

Time(h) Zero 1h 2h 3h 4h

Group Paw

diameter (mm)

Paw diameter (mm)

Edema thickness (mm)

Paw diameter (mm)

Control 3.30±0.08 4.41±0.1a _1.11 _4.71_±_0.13a _1.41 _4.81_±_0.12a _1.51 _4.85_±_0.07a _1.55

A.vera 3.33±0.08 4.26±0.10a _0.93 _4.10_±_0.13a _0.77 _3.82_±_0.12a _0.49 _3.64_±_0.08a _0.31

A.arborences 3.49±0.09 4.34±0.09a _0.85 _4.21_±_0.08a _0.72 _4.03_±_0.09a _0.55 _3.84_±_0.09a _0.35

A.eru 3.51±0.10 4.38±0.14a _0.87 _4.28_±_0.14a _0.77 _4.12_±_0.14a _0.₆₁ _3.94_±_0.11a _0.43

A.grandidentata 3.56±0.11 4.16±0.20a _0.6 _3.99_±_0.10a _0.43 _3.95_±_0.05a _0.39 _3.90_±_0.7a _0.34

A.perfoliata 3.57±0.1 4.46±0.12a _0.89 _4.27_±_0.14a _0.70 _4.04_±_0.05a _0.47 _3.94_±_0.05a _0.37

A.brevifolia 3.49±0.06 4.37±09a _0.89 _4.30_±_0.06a _0.81 _4.27_±_0.07a _0.78 _4.03_±_0.6a _0.54

A.saponaria 3.61±0.02 4.51±0.07a _0.90 _4.38_±_0.02a _0.87 _4.14_±_0.08a _0.53 _4.08_±_0.06a _0.47

A.ferox 3.60±0.09 4.49±0.09a _0.89 _4.31_±_0.08a _0.71 _4.11_±_0.09a _0.51 _3.98_±_0.09a _0.38

Indomethacin 3.56±0.08 4.12±0.09a _0.6 _3.99_±_0.06a _0.43 _3.92_±_0.01a _0.36 _3.84_±_0.07a _0.28

a_{Significantly}_different_from_zero_time_at_p_<_0.05₍_n₌_6). Thedoseoftheextractswas250mg/kg.

agreementtokaempferol(CuyckensandClaeys,2004; Vallverdú-Queraltetal.,2010).

Massspectrum ofpeak 35 (Rt1013.1 s)showed [M−H]− _at m/z343,itwasidentifiedasdihydroxyltrimethoxyflavone(5,3′

-dihydroxy-6,7,4′_{-trimethoxyflavone)} _Eupatorin _and _noted _in _A. arborescens,A.eru,A.grandidentataandA.brevifolia.MS/MS

spec-trum gave product ions at m/z316, 315, 299 and 287, which

wereinaccordancetothiscompound(JafferandMahmoud,2011; Akowuahetal.,2012).

Peak 36 (Rt 1018.1 s) showed [M−H]− _at _m_/_z ₄₆₃

consis-tentwith themolecularformula (C21H20O12).MS/MSspectrum

ofthiscompoundshowedaproductionatm/z301indicating a quercetinnucleus(Breiteretal.,2011).Thispeakwascompared withauthenticstandardandidentifiedasquercetin-3-O-glucoside (isoquercetrin)andwasnotedonlyinA.arborensisandA.eru.

HPLCanalysisofthemethanolextractoftheleavesoftheeight investigatedAloespeciesrevealedapartfromtheknown metabo-lites;aloesin(4), nataloin (10), aloin A/B(13), aloeresin E(14) andaloeresinH,whichareofparticularimportanceinthe chemo-taxonomicstudyofthegenusAloe.Barbaloin/isobarbaloin,which isC-glucosideofaloeemodinanthrone(10-␤-d

-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9(10H)-anthracenone)are

con-sideredtobethemostspecificsecondaryphytoconstituentinAloe species.Barbaloinhasbeenfoundtohaveastronginhibitoryeffect on histamine release from mast cells (Patel et al., 2012). Alo-eresinH,8-C-␤-d-glucopyranosyl-7-hydroxy-5-methylchromone

portionthatiscommontoallaloeresins(A–G)weredetectedin mostoftheinvestigatedAloespecies.AloeresinHrepresentsthe firstC,C-diglucosylatedmoleculediscoveredinAloespp.extracts. AlthoughcinnamicacidderivativesarepresentinAloemetabolites inthealoinseries,aloeresinE(14)andaloeresinFrepresentthe firstcinnamicacidderivativesinthealoeresinseriesofmetabolites

(VanHeerdenetal.,1996).AloeninB(8),anewbitterglycosidewas detectedinmostoftheleavesoftheinvestigatedAloespecies.6′

-Malonylnataloin(10),amalonylatedderivativeoftherareanthrone nataloin,alsowasidentifiedin allinvestigatedsamplesof Aloe. AnthroneC-glycosidesareamongasuiteofchemicalconstituents ofsystematicimportanceinAloe.

Aloehaslongbeenrecognizedbydifferentpharmacopeiasover theworldasa purgativedrug(Rowsonetal.,1967;Osol etal., 1973).The purgativeeffectsof Aloeleaf(drug aloes)havebeen attributedtoanthraquinoneC-glycosides,notablybarbaloin,aloin A and B (Steenkamp and Stewart, 2007). Furtherstudies were conductedtoinvestigatetheAloe’s constituentsresponsiblefor thisactivity.Anthranol,aloe-emodin,chrysophanicacid (chryso-phanol), aloin (barbaloin)and p-coumaric acidwerepreviously reportedinA.vera.Athinlayerchromatographicstudyof22species ofAloe(Meredovaetal.,1973)showedthattwelvespeciescontain flavonoids,hydroxyanthraquinonesandcoumarin.

Concerningthediabeticwoundhealingeffectofthemethanol extractsoftheeightinvestigatedAloespecies,resultsobtainedin thepresentstudysuggestedthattreatmentofdiabeticratswith themethanolextractofeachoftheeightstudiedAloespeciesmay have a beneficial influenceonwound healing. It is noteworthy tomentionthattheeightinvestigatedspeciesshowedsignificant anti-inflammatoryactivity(Table2)astheiractioninreducingthe thicknessofedemastartedfromthefirsthourandcontinuedfor thefourhours.Theanti-inflammatoryactivityofAloemaybedue

totheinhibition of thecyclooxygenasepathway and reduction

ofprostaglandinE2productionthiscouldbeattributedtoitsC -glucosylchromones(Hutteretal.,1996).Thefindingsofourstudy confirmedthesignificantaccelerationofdiabeticwoundhealing inratsfollowingtopicalapplicationofthemethanolicextractof theleavesofthevariousAloespecies.Thehealingeffectonthe

Table3

EffectofthemethanolextractoftheeightAloespeciesonwoundareaindiabeticrats.

Thetestedsample Woundarea(cm2₎

Zerotime Day2 Day6 Day10

Control 1.53±0.003 1.55±0.002 1.59±0.001a _1.60_±_0.003a

A.vera 1.58±0.001 1.26±0.004a _0.69_±_0.003a _0.36_±_0.001a

A.arborences 1.63±0.002 1.23±0.002a _0.67_±_0.001a _0.46_±_0.001a

A.eru 1.67±0.002 0.89±0.003a _0.46_±_0.001a _0.23_±_0.001a

A.grandidentata 1.71±0.001 1.49±0.002a _1.13_±_0.002a _0.74_±_0.001a

A.perfoliata 1.69±0.003 1.41±0.001a _0.86_±_0.001a _0.53_±_0.001a

A.brevifolia 1.56±0.001 1.45±0.001a _0.81_±_0.002a _0.63_±_0.002a

A.saponaria 1.68±0.002 1.34±0.002a _0.65_±_0.001a _0.31_±_0.001a

A.ferox 1.51±0.001 1.37±0.003a _0.98_±_0.001a _0.47_±_0.002a

Dermazine® _1.65_±_0.004 _0.72_±_0.001a _0.43_±_0.002a _0.13_±_0.001a

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Table4

Percentofchangeinwoundareaindiabeticratstreatedwithmethanolextractof theeightAloespecies.

Thetestedsamples %ofchangeinwoundarea

Day2 Day6 Day10

Control 1.31 3.92 4.6

A.vera 30.30 56.3 77.2

A.arborences 24.5 58.8 71.8

A.eru 46.71 72.9 86.2

A.grandidentata 12.9 33.9 57.9

A.perfoliata 17.2 59.2 68.6

A.brevifolia 7.1 48.1 59.6

A.saponaria 20.2 61.3 81.6

A.ferox 14 35.1 68.9

Dermazine®

56.4 73.9 92.1

diabeticwoundswasobservedforthetopicalapplicationofthe methanolextractsoftheeightstudiedAloespecies(Table3),

how-ever,A. erushowed thehighest effect in reducing thewounds

areawhichreached86.2%byday10(Table4).Itwaspreviously reportedthatthehealingpropertiesofAloecouldbeattributedto itsglucomannantypepolysaccharide,whichstimulatesthe activ-ityoffibroblastsandcausesproliferation,whichinturnincreases collagensynthesis(Chithraetal.,1998).Thehighwoundhealing activityobservedforA.erucouldbemainlyattributabletoitsmajor constituentaloenin,whichwasreportedtosignificantlypromote hairgrowthanddemonstratedrecuperativeeffectsonhumanskin (WolfsonandGutterman,2009).AloeninwasdetectedonlyinA. eru(Table1).

Conclusion

The occurrence of flavones-C-glycosides, heptaketides (e.g.

5-methylchromones) and octaketides (e.g.

1,8-dihydroxy-9-anthrones)in Aloespecies is known.The resultsobtainedhere showedthatthesespeciespossessdifferentchemicalcomposition,

justifying the importance of studies aiming for the chemical

characterizationofdifferentAloespecies.Theantiinflammatory andwoundhealingactivityoftheAloespecieshasbeenattributed topolyphenoliccompounds.A.erushowedthehighesteffectin reducingthewoundsareaintendayswhich isrich inAloenin. Amongthespeciespresentedinthispaper,flavonoidsarefound asmainconstituentsspeciallylucieninII,viceninII,orientinand isovitexininA.vera,A.arborescens,A.grandidentata,A.perfoliata,A. brevifoliaandA.saponaria.Heptaketides(5-methylchromones)are foundasmainconstituentsinallinvestigatedAloespeciesexceptA. vera,A.perfoliataandA.ferox.Polyketidesareofchemotaxonomic interestinallAloespeciesinvestigated.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

AMEcontributedincollectingtheplantmaterial,preparationof someofthemethanolextracts,interpretationoftheHPLC–MS/MS dataandconstructingthemanuscript.SMEcontributedin prepa-ration of some of the methanol extracts, interpretation of the

HPLC–MS/MS data, revising and finalizing the manuscript and

interpretationofthebiologicalresults. MMEcontributedin per-formingtheHPLC–MS/MSanalysisandrevisingtheinterpretation ofHPLC–MS/MSdata.SSEcontributedinsuggestingthepointofthe researchandsupervisingthework.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

Theauthorsare deeplythankful toProf.Dr. WafaaElEraky,

Department of Pharmacology, National Research Center, Giza,

Egypt,forcarryingoutthebiologicalexperiments.

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