Application of On-line C30 RP-HPLC-NMR forthe Analysis of Flavonoids from Leaf Extract ofMaytenus aquifolium

Wagner Vilegas,1* Janete H. Y. Vilegas,2 Markus Dachtler,3 Tobias Glaser3 and Klaus Albert3

1Universidade Estadual Paulista, Instituto de Quı´mica de Araraquara, CP 355, 14801-970, Araraquara, SP, Brazil2Universidade de Sa˜o Paulo, Instituto de Quı´mica de Sa˜o Carlos, CP 780, 13560-970, Sa˜o Carlos, SP, Brazil3Universitat Tubingen, Institut fu¨r Organische Chemie, Auf der Morgenstelle 18, D-72076, Tu¨bingen, Germany

The application of on-line C30-reversed-phase high-pressure liquid chromatography–nuclear magneticresonance spectroscopy is described for the analysis of tetraglycosylated flavonoids in aqueous andhydroalcoholic extracts of the leaves ofMaytenus aquifolium(Celastraceae). Triacontyl stationary phasesshowed adequate separation for on-line1H-NMR measurements at 600 MHz and allowed thecharacterisation of these flavonoids by detection of both aromatic and anomeric proton signals. Copyright# 2000 John Wiley & Sons, Ltd.Keywords:On-line HPLC-NMR; C30-stationary phase; flavonoids;Maytenus(Celastraceae).

INTRODUCTION

The application of coupling techniques to the investiga-tion of polar natural products from plant origin isbecoming of increasing importance. The developmentof HPLC coupled with UV and diode array detection wasthe first step for the direct investigation of polarphytocompounds, including aqueous infusions, but itwas obviously limited to UV-absorbing substances. Morerecently, several methods of coupling between HPLC andMS have evolved, allowing the detection and identifica-tion of a broader range of natural compounds in plantmaterial and foods (Wolfenderet al., 1992, 1994;Verpoorte and Niessen, 1994; Careriet al., 1998),including glycosides present in teas (Fingeret al.,1992). However, complete information about chemicalcomposition is usually not possible by HPLC-MS owingto the possible occurrence of sugar and aglycone isomersthat cannot be easily differentiated by their MS. More-over, many natural products can undergo severe degrada-tion when submitted to the elevated temperaturesrequired for some MS interfaces, and sometimes themolecular ions are in low abundance or are even notdetectable (Niessen and van der Greef, 1992).

High-resolution NMR is the most important methodfor the structural elucidation of complex compounds. Inparticular, for unstable compounds such as carotenoidstereoisomers, on-line HPLC-NMR is the method ofchoice since both light and oxygen may be excluded(Dachtleret al., 1999), whereas LC-MS is not suitable for

this kind of situation. The direct coupling of HPLC withNMR gives, in many cases, more complete structuralinformation about complex plant compounds such asglycosides. One-dimensional (1D) and 2D NMR experi-ments can provide not only information concerning theaglycone structure, but can also give data for theidentification of the sugars and for the determination ofthe sequence of the saccharide chain in the molecule.Direct HPLC-NMR coupling is one of the mostpromising tools for the fast analysis of plant extracts thathave recently been developed (Wolfenderet al., 1998).

A crucial step to on-line HPLC-NMR experiments isthe chromatographic separation prior to the introductionof the compound of interest into the NMR probe: spectrausually must be taken from the heart of chromatographicpeaks, which corresponds to the maximum concentrationof the available sample. However, usually HPLCcolumns must be overloaded in order to furnish adetectable amount of each desired compound, whichmust also be base-line separated. These requirements foron-line HPLC-NMR led to the search for new stationaryphases with higher resolution and selectivity, such astriacontyl reversed phase (C30-RP) in which the longerchain provides better resolution than conventional C18columns in the separation of low polarity compounds(Purschet al., 1996). Furthermore, owing to their highloading capacity, C30 phases can be used with addedadvantage for on-line HPLC-NMR experiments. Thesenew stationary phases have been utilised successfully forthe HPLC-NMR analysis of a number of samples,including b-carotene isomers (Strohscheinet al., 1997),vitamin A derivatives (Albertet al., 1996), tocopherolisomers (Strohscheinet al., 1998) and lutein andzeaxanthin isomers in bovine retina (Dachtleret al.,1998). However, C30-RP has not been evaluated for theanalysis of polar compounds or for the analysis ofinfusions prepared from medicinal plants.

The infusion from the leaves ofMaytenus aquifolium

PHYTOCHEMICAL ANALYSISPhytochem. Anal.11, 317–321 (2000)

Copyright# 2000 John Wiley & Sons, Ltd.

* Correspondence to: Prof. W. Vilegas, Universidade Estadual Paulista,Instituto de Quı´mica de Araraquara, CP 355, 14801-970, Araraquara, SP,Brazil.E-mail: vilegasw@iq.unesp.brContract/grant sponsor: FAPESP.Contract/grant sponsor: CNPq.

Received 20 April 1999Revised 26 September 1999Accepted 10 October 1999

Martius (Celastraceae),a Brazilian speciesknown as‘espinheirasanta’,is popularlyusedin the treatmentofgastric ulcers and gastritis (Carlini, 1988). Previousphytochemicalwork with theinfusionled to theisolationand structure elucidation of the two main flavonoidtetraglucosides: quercetin 3-O-a-L-rhamnopyranosyl(1→6)-O-[b-D-glucopyranosyl(1→3)-O-a-L-rhamnopyr-anosyl(1→2)-O-b-D-galactopyranoside](1) and kaemp-ferol 3-O-a-L-rhamnopyranosyl(1→6)-O-[b-D-glucopyr-anosyl(1→3)-O-a-L-rhamnopyranosyl(1→2)-O-b-D-galac-topyranoside](2; Sannomiyaet al., 1998;Vilegaset al.,1999).

Flavonoidsareanimportantclassof compoundswith awide range of biological activities, including anti-oxidative, anti-mutagenicand anti-carcinogeniceffects(Harborne, 1994). The chromatographic analysis(HRGC, HPTLC, HPLC, LC-MS, CE) of the leavesand of the infusion of M. aquifolium hasbeensystem-atically performed in order to propose analyticalproceduresfor the standardisationof ‘espinheirasanta’preparations(Vilegas et al., 1994, 1995, 1998a, b),mainly using triterpenoidsor flavonoids as chromato-graphicmarkers.The evaluationof suchcompoundsin‘espinheirasanta’is imperativesincemanydrugsfoundin commerceareadulterated(VilegasandLancas,1997).ThepresentpaperdescribestheHPLC-NMR analysisofaninfusionfrom theleavesof M. aquifoliumusinga C30stationaryphase.

EXPERIMENTAL

Plant material and extraction. AuthenticatedleavesofMaytenusaquifoliumMartiuswerecollectedin RibeiraoPreto by Dr Ana Maria Soares Pereira (UNAERP,Ribeirao Preto,SP, Brazil) from cultivated specimens.A voucherspecimenis maintainedin our laboratory.Theleaveswere dried at 40°C, powderedand sieved;onlyparticlesbetween0.5 and1.0mm wereused.A sample(1.0g) of theplantmaterialwasboiledwith 10mL waterfor 10min, after which the infusion wascooled,filteredand evaporated to dryness. The material was re-suspendedin 3.0mL methanol,centrifugedanddirectlyanalysedby HPLC-NMR.

HPLC analysis.ChromatographywasperformedunderambientconditionsusingaMerck(Darmstadt,Germany)Lichrograph L-6200A gradient pump and a MerckLichrographL-4000/4200UV–VIS absorbancedetector.Separationswerecarriedout on a stainlesssteelcolumn(250� 4.6mm i.d.) containingC30-reversedphase(par-ticle size,3mm; pore diameter,200A; Bischoff, Leon-berg, Germany) eluted with an isocratic mixture ofmethanol:water(50:50,v/v). Theflow-rateof themobilephasewas 0.8mL/min, and elution was monitored at370nm. For the LC-NMR experiments70mL of a 0.1%solution(w/v) of the plant extractwasinjectedonto thecolumn.

On-line LC-NMR coupling. On-line experimentswereconducted using a Bruker (Rheinstetten,Germany)model AMX 600 spectrometerequippedwith an LCinverseprobewith a detectionvolume of 120mL. Thechromatographicequipmentandthe peaksamplingunit

(BPSU-12,Bruker) necessaryfor stopped-flowexperi-mentswerecontrolledby Chromstarsoftware(Bruker).

To record the 1H-NMR spectra,solvent suppressionwasnecessary.This wasperformedusingshapedpulses(rectanglepulsewith a lengthof 100ms) for low-powerpresaturationof thetwo methanolsignals(d 3.3and4.7)for 1.6 s prior to thestartof theacquisition.For the 1H-NMR stopped-flowmeasurements,2K transientswereaccumulatedin a total acquisitiontimeof about1 h usinga time domainof 16K and a sweepwidth of 9600Hz.Processingwasperformedwith 1D WINNMR software(Bruker). For all spectra,zero filling up to 32K datapointsandanexponentialmultiplicationof theFID with aline broadeningof 1 Hz wereperformedbeforeFouriertransformation.

RESULTS AND DISCUSSION

Previousanalyticalstudies,usingC8 andC18 columns,ofthe flavonoidsfrom Maytenusindicatedaqueousformicacid:acetonitrilemixturesas the bestmobile phasesforthe separation of these compounds. However, theconcentrationof the buffer is often higher than theconcentrationof the analyte and a large number ofsolvent signals are visible in the NMR spectrum.Therefore,this buffer systemis not suitablefor on-lineHPLC-NMR experiments.In the presentwork severalmethanol:deuteriumoxide mixturesweretested,andthe1:1 (v/v) mixture gave satisfactoryresultsconsideringchromatographicseparationvs.HPLC-NMRparameters.

Methanolshowssignalsat d 3.3and4.7,andcanleadto distorted NMR peaks.A challengein the on-lineHPLC-NMR analysisof glycosylatedcompoundsis tosuppress the solvent signals without affecting thecharacterisationof the sugar signals, mainly thosecorresponding to the anomeric protons. The costassociatedwith theuseof deuteriumoxidein themobilephase is acceptable;most other solvents commonlyemployedin LC arevery expensivein deuteratedform.

A typical extractof the leavesof M. aquifoliumhasasimple chromatographicprofile with two main peakscorrespondingto compounds1 and2 (Fig. 1). Underthe

Structures 1 and 2

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conditionsemployedhere,1 elutesat 7.5min while thelesspolar2 elutesat10.0min: thus,thechromatographicconditionsaffordedabaselineresolutionandallowedtheseparationof thesetwo tetraglucosylatedflavonoidsin aconvenienttime.

Despitethe largeamountof sampleinjectedonto theC30 column,which was necessaryto producesufficientsampleconcentrationin the NMR flow cell, no over-loadingeffectsarevisible. This high-loadingcapacityoftheC30 phasesmakesthempreferablyusablefor on-lineHPLC-NMR experimentsdealingwith naturalproducts,becauseit allows the separationand identification ofminor compounds,which can be responsiblefor the

biologicalactivity. Moreover,theselectivityis quitehighandalmostno peaktailing is observed.

Thestopped-flow1H-NMR spectraldatafor thepeakswith retentiontimes7.5 and10.0min, correspondingtocompounds1 and 2, respectively,fully correlatewiththose reported previously as obtained by traditionalphytochemicalstudies(Sannomiyaet al., 1998;Vilegaset al., 1999).Thedifferencesin thechemicalshift valuesaredueto thechangeof solventnecessaryfor LC-NMRandarein therangeof 0.05ppmfor thearomaticprotonsandlessthan0.2ppmfor theanomericprotons.The 1H-NMR spectra (600MHz; aromatic region) of theaglyconesof 1 and 2 are shownin Fig. 2. The protonsof the aromaticportion of the spectraof 1 [Fig. 2(a)]clearlyshowsignalscorrespondingto threeABX systemsat d 7.67 (1H, d, J = 1.5Hz), at d 7.53 (1H, dd, J = 8.5,1.5Hz) andat d 6.92(1H, d, J = 8.5Hz) dueto theH-2',H-6' and H-5', respectively, from the B-ring of theflavonoidnucleus,aswell assignalsfor two AB systemsat d 6.38 (1H, d, J = 1.5Hz) and at d 6.19 (1H, d,J = 1.5Hz), correspondingto the H-8 andH-6 from theA-ring. Thesesignalsallowed the unequivocalrecogni-tion of the aglyconeof 1 asbeingquercetin(Harborne,1994).Compound2 [Fig. 2(b)] showstwo doubletswithJ = 8.0Hz, integratingfor 2H each,atd 8.00and6.94andclearly correspondsto the H-2'/H-6' and H-3'/H-5',respectively,of the B-ring of a flavonoid nucleus,aswell astwo signalsat d 6.21and6.41correspondingtoH-6 and H-8 from a kaempferolderivative (Harborne,1994).

The aliphatic region of the stopped-flow1H-NMRspectraof 1 and2 arealmostidentical, thusrevealingasimilar saccharidechain. As shownfor the kaempferoltetraglycosidein Fig. 3, the four anomericprotonsareclearlydistinguishedatd 5.26(1H, d, J = 7.5Hz, gal-1@),at d5.13 (1H, d, J = 1.5Hz, rha-1@'), at d4.59 (1H, d,J = 7.5Hz, glc-1@@) andat d4.47(1H, d, J = 1.5Hz, rha-1@@'). Thetwo doubletsthatintegratefor 3H eachatd1.10(d, J = 6.0Hz, rha-6@@') andat d1.02(d, J = 6.0Hz, rha-6@') were assignedto the two rhamnosemoieties.Thecoupling constants of J = 7.5Hz indicate that thegalactoseandtheglucosemoietieshaveb-configuration,

Figure 1. HPLC chromatogram with detection at 350 nm of the ¯avonoidfraction from the infusion of leaves of Maytenus aquifolium. Key to peakidentity: 1, quercetin 3-O-a-L-rhamnopyranosyl(1→6)-O-[b-D-glucopyrano-syl(1→3)-O-a-L-rhamnopyranosyl(1→2)-O-b-D-galactopyranoside]; 2, kaemp-ferol 3-O-a-L-rhamnopyranosyl(1→6)-O-[b-D- glucopyranosyl(1→3)-O-a-L-rhamnopyranosyl(1→2)-O-b-D-galactopyranoside]. (For chromatographic pro-tocol see the Experimental section.)

Figure 2. Stopped-¯ow 1H-NMR (600 MHz) of the aglyconeregion of the spectra of (a) quercetin 3-O-a-L-rhamnopyrano-syl(1→6)-O-[b-D-glucopyranosyl(1→3)-O-a-L-rhamnopyrano-syl(1→2)-O-b-D-galactopyranoside] (1); and (b) kaempferol 3-O-a-L-rhamnopyranosyl(1→6)-O-[b-D-glucopyranosyl(1→3)-O-a-L-rhamnopyranosyl(1→2)-O-b-D-galactopyranoside] (2).

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whereastheJ = 1.5Hz couplingsindicatea-configurationfor the rhamnoseunits. Despitethe suppressedsolventsignals,theanomericprotonswereneithersuperimposednor distorted. Thus, on-line LC-NMR measurementsunderthedescribedconditionsallowedthetwo complexflavonoid tetraglycosides from the infusion of M.aquifoliumto be fully recognised.

On-lineLC-NMR couplingusingthenewlydevelopedC30 stationaryphaseis still anunder-exploredtechniquein the field of natural productschemistry.The presentapproachshowedthat LC-NMR coupling is one of themost promising techniqueswith which to investigate

aqueousphytomedicinessince it providesa quick andreliable methodfor the quality control of the complexconstituents.

Acknowledgements

Theauthorswishto thankFAPESPandCNPq(Brazil) for financialaidandfor fellowshipsto W.V. andJ.H.Y.V.Thesupportof theDeutscheForschungsgemeinschaft, der Fonds der ChemischenIndustrie andBischoff AnalysentechnikGmbH (Leonberg, Germany)is gratefullyacknowledged.

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