Rev. bras. farmacogn. vol.26 número4

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RevistaBrasileiradeFarmacognosia26(2016)471–473

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

Original

Article

Coumarins

of

Loricaria

ferruginea

Gonzalo

Rodolfo

Malca

Garcia

a,∗

_,

_Lothar

_Hennig

a

_,

_Eric

_F.

_Rodríguez

b

_,

_Rainer

_W.

_Bussmann

c

a_Institut_für_Organische_Chemie,_Fakultät_für_Chemie_und_Mineralogie,_Universität_Leipzig,_Leipzig,_Germany b_Herbarium_Truxillense_(HUT),_National_University_of_Trujillo,_Trujillo,_Peru

c_William_L._Brown_Center,_Missouri_Botanical_Garden,_St._Louis,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14December2015 Accepted23February2016 Availableonline27April2016

Keywords: Asteraceae

5,7-Dimethoxycoumarin 5,7,8-Trimethoxycoumarin 5-Hydroxyobliquine 5-Methoxyobliquine

a

b

s

t

r

a

c

t

InthepresentedresearchweisolatedandcharacterizedcompoundsfromLoricariaferruginea(Ruiz& Pav.)Wedd.,Asteraceae.TothebestofourknowledgenodataonanycompoundsfromL.ferrugineahave beenpublishedtothisday.Asmaincompoundsofthehexaneextractwefoundfourknowncoumarins: 5,7-dimethoxycoumarin;5,7,8-trimethoxycoumarin;5-hydroxyobliquineand5-methoxyobliquine.All thestructuresweredeterminedbyspectroscopicandspectrometricmethods.

Introduction

TheAsteraceaearethesecondmostdiversefamilyinthe Peru-vianflora,withapproximately250generaand1590species(Dillon andHensold,1993;UlloaUlloaetal.,2004).MostPeruvian Aster-aceaeareherbs,shrubsandsubshrubs(Beltránetal.,2006).The genusLoricariacontainscurrently22mostlyrhizomatousperennial andafewannualspecies,withconspicuousflatleavesandlaterally compressedstems,glabrousorpubescentacheneswith glandular-stipitatebiseriatehairs(Hind,2011).MostspeciesofLoricariaare restrictedtohighaltitudegrasslands.

DeFeo(1992)reportedtwospeciesofLoricariabeingusedin thedivination practices in thenorthern Peruvian Andes.Other authorsdescribedthetraditionaluseofLoricariaferruginea(Ruiz& Pav.)Wedd.formenstrualdelay,bloodcirculation,andritual pur-poses(spiritualflowering,protection,goodhealth,goodfortune, goodbusiness,fragrance,success,good travels,becoming socia-bleandgoodrelationswithothers)(BussmannandSharon,2007). However,atoxicologicalbrainshrimpbio-assaysshowedaLC50

15␮g/mlinethanol(Bussmannetal.,2011).

Up to 2009 more than 1300 coumarins were isolated from plants, bacteria, and fungi (Iranshahi et al., 2009). Coumarins consist of a large class of phenolic substances biosynthe-sized by medicinal plants. These type of secondary metabo-lites are known for their pharmacological properties such as

∗ Correspondingauthor.

E-mail:[email protected](G.R.MalcaGarcia).

anti-inflammatory,anticoagulant,antibacterial,antifungal, antivi-ral, anticancer, antihypertensive, antitubercular, anticonvulsant, antiadipogenic,antihyperglycemic,antioxidant,and neuroprotec-tiveproperties(Iranshahietal.,2009;Venugopalaetal.,2013).

In this work, we report the isolation and characteri-zation of four known coumarin derivatives (Basnet et al., 1993;BohlmannandZdero, 1980):5,7-dimethoxycoumarin (1); 5,7,8-trimethoxycoumarin (2); 5-hydroxyobliquine (3) and 5-methoxyobliquine(4).

TothebestofourknowledgenoconstituentsfromL.ferruginea

havebeenreportedsofar.Thestructuresofthesecoumarinswere elucidatedbyspectroscopicmethods.

Materialsandmethods

NMRandMSinfrastructureandmethods

NMRspectra(1_H,13_C,_APT,_NOESY1D,_H,H-COSY,_edited_HSQC,

andHMBC)wererecordedonaVarianMercury400plus(400MHz for1_H,₁₀₀_MHz_for13_C)_and_a_Varian_Mercury₃₀₀_plus₍₃₀₀_MHz

for1_H,₇₅_MHz _for13_C) _{spectrometer,}_{respectively,} _at₂₆◦_C _and

withCDCl3asasolvent.Thechemicalshiftswerereported

rela-tivetotheresidualsolventpeak,usedasaninternalreference(1_H:

7.26ppm,13_C:_77.16_ppm)._Chemical_shifts_are_given_in_ı_values,

couplingconstantsJinHz.Allseparationswerecarriedoutin care-fullypurifiedanddriedsolventsandweremonitoredbythin-layer chromatography(TLC)onplatesofSilufolUV/VIS254nm. Prepara-tivecolumnchromatographywascarriedoutonsilicagel(MERCK 70–230mesh)ingradientregime.

http://dx.doi.org/10.1016/j.bjp.2016.02.006

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472 G.R.MalcaGarciaetal./RevistaBrasileiradeFarmacognosia26(2016)471–473

Plantmaterial

BulkmaterialofLoricariaferruginea(Ruiz&Pav.)Wedd., Aster-aceae,wascollectedinMarch2009fromHuamachuco,Sanchez CarrionProvince-PeruandidentifiedbyBotanistEricF.Rodríguez RodríguezatHerbariumTruxillense(HUT),NationalUniversityof Trujillo,Peru.AvoucherspecimenunderNo.50003(HUT) docu-mentingthe collectionwasdeposited atHerbarium Truxillense (HUT)inPeru.

Preparationofplantextracts

Thecrudeextractofthepowderplantmaterialwasobtained bymacerationusinghexane(200g/3l)forfivedays,followedby filtrationandevaporationunderreducepressure,withafinalyield of5.2g(2.60%).

Isolation

Thehexaneextract(5.2g)waspurifiedbyCCusinganeluent sys-temofhexane/ethylacetate(5:3)andtheresultingfractionswere combinedaccordingtoTLCprofiles.Sixfractionswereobtained; thefirstfraction(468mg)waselutedwithhexane/acetone(10:1) toyield40mgof5-methoxyobliquine(4)(Rf0.71)andthesecond

fraction(1.1g)containing5,7-dimethoxycoumarin(1)waspurified againwithhexane/ethylacetate(2:1)andyielded28.1mgof(1)(Rf

0.61).

Thethirdtothefifthfraction(520.7mg)waselutedwith hex-ane/acetone(2:1) to give 43.1mg of 5,7,8-trimethoxycoumarin (2) (Rf 0.91). The sixth fraction (406.1mg) was eluted with

dichloromethane/ethyl acetate (10:0.5) yielding 61.3mg of 5-hydroxyobliquine(3)(Rf0.5).

Resultsanddiscussion

Spottestswereusedforthequalitativedeterminationof sec-ondary metabolites present in L. ferruginea (Dominguez, 1973; Harborne, 1984).We identified coumarins by NaOH. The filter paperwasthenexaminedunderUVlight,withyellowfluorescence indicatingthepresenceofcoumarins.Steroidsandtriterpenoids (darkgreen)wereidentifiedbytheLiebermann–Burchard’stest, flavonoids(red)bytheShinodatest(bothonlyinsmallamounts), andnoalkaloidsweredetectedusingDragendorff’stest.

Previously,5,7-dimethoxycoumarin(1)wasreportedwith cyto-toxicactivityagainstdifferenttypesofcancercells,andaspotent inhibitorofiNOSexpression(Nakamuraetal.,2009;Riveiroetal., 2009),and5,7,8-trimethoxycoumarin(2)wasreportedasanti-HIV active(Chengetal.,2005).

Thespectraofallcoumarinderivativesareinagreementwith literatureresults(BohlmannandZdero,1980;Jaenschetal.,1989; Osborne,1989;Maesetal.,2008).

5,7-Dimethoxycoumarin(1)whitesolid;28.1mg(0.5%);Rf=0.61

(hexane/EtOAc);1_H_NMR ₍₃₀₀_MHz,_CDCl

3):ı=3.86 (s,3H,

C7-OCH3), 3.89(s,3H, C5-OCH3), 6.16(d,J=9.6Hz, 1H,C3-H),6.29

(d,J=2.29Hz,1H, Ar–H),6.42(d,J=2.29Hz,1H, Ar–H),7.97(d,

J=9.6Hz, 1H, C4-H)ppm; 13_C _NMR ₍₇₅_MHz, _CDCl

3): ı=55.73,

55.91,93.00,94.87,103.87,110.72,138.67,156.69,156.92,161.39, 163.67.

5,7,8-Trimethoxycoumarin (2) white solid; 43.1mg (0.82%);

Rf=0.91(hexane/acetone);1HNMR(300MHz,CDCl3):ı=3.91(s,

3H,C5-OCH3),3.91(s,3H,C7-OCH3),3.96(s,3H,C8-OCH3),6.16(d, J=9.7Hz,1H,C3-H),6.34(s,1H,C6-H),7.99(d,J=9.7Hz,1H, C4-H)ppm;13_C_NMR₍₇₅_MHz,_CDCl

3):ı=56.26,56.71,61.82,91.67,

104.27,111.48,130.52,139.02,148.98,152.58,156.35,161.07.

5-Hydroxyobliquine(3)Yellowsolid;61.3mg(1.17%);Rf=0.50

(DCM/EtOAc);1_H _NMR ₍₃₀₀_MHz, _CDCl

3): ı=1.84(s, br, 13-H),

3.98(dd,J=9.6,1.2Hz,9′_-H),_4.34_(dd,_9.6,_1.2_Hz,_9-H),_4.51_(dd,

br,J=9.6,1.2Hz,10-H),5.12(s,br,12-H),5.17(s,br,12′_-H),_6.17

(d,J=9.6Hz,3-H),6.56(s,8-H),7.88(d,J=9.6Hz,4-H)ppm;13_C

NMR(75MHz,CDCl3):ı=18.94,67.68,75.69,99.69,107.68,112.92,

114.71,133.38,138.70,139.12,144.39,147.60,148.65,161.31.

5-Methoxyobliquine(4)Yellow solid; 40mg (0.77%); Rf=0.71

(hexane/acetone);1_H_NMR₍₄₀₀_MHz,_CDCl

3):ı=1.87(s,br,13-H),

4.00(s,OCH3),4.04(dd,9.6,1.2Hz,9′-H),4.38(dd,9.6,1.2Hz,9-H),

4.54(dd,br,9.6,1.2Hz,10-H),5.13(s,br,12-H),5.19(s,br,12′_-H),

6.22(d,J=9.6Hz,3-H),6.61(s,8-H),7.92(d,J=9.6Hz,4-H)ppm;

13_C _NMR₍₁₀₀_MHz,_CDCl

3):ı=18.94,61.81,67.67,75.69,99.67,

107.67, 112.91, 114.70, 133.36, 138.68, 139.11,144.37, 147.58, 148.63,161.30.

Conclusions

The present paper describes the isolation and characteriza-tionoffourconstituentsofL.ferruginea:5,7-dimethoxycoumarin (1); 5,7,8-trimethoxycoumarin (2); 5-hydroxyobliquine (3) and 5-methoxyobliquine (4).The resultsmay be helpful in further investigationsofthebiologicalactivityofthesenaturalcompounds.

Authors’contributions

GRMG(PhDstudent)contributedrunningthelaboratorywork, anddraftedthepaper;LHdidtheNMRinvestigations;EFRRhas identifiedthespeciesandrevisedthepaperandRWBcontributed incollectingplantsamplesandrevisedthepaper.

Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsinterest.

Acknowledgements

TheauthorsaregratefultoMrs.RamonaOehme(Universityof Leipzig,InstituteofAnalyticalChemistry,Germany)forthe mea-surementoftheMSspectra.WethankDeutscherAkademischer AustauschDienst(DAAD)forfinancialsupport.

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