Taxane analysis by high performance liquid chromatography–Nuclear magnetic resonance spectroscopy...

Taxane Analysis by High Performance LiquidChromatography–Nuclear Magnetic ResonanceSpectroscopy ofTaxusSpecies§

Bernd Schneider,1‡ Yu Zhao,2 Torsten Blitzke,1 Bettina Schmitt,1 Aslieh Nookandeh,2

Xianfeng Sun2 and Joachim Stockigt2

1Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany2Department of Pharmaceutical Biology, Institute of Pharmacy, Staudinger Weg 5, Johannes Gutenberg-University, D-55099 Mainz,Germany

High performance liquid chromatography–nuclear magnetic resonance spectroscopic analyses of taxanediterpenoids from three Taxus species were carried out employing a stopped-flow technique. Severaltaxanes have been identified from 500 mg leaf samples without prior isolation.# 1998 John Wiley & Sons,Ltd.Keywords:High performance liquid chromatography–nuclear magnetic resonance spectroscopy; taxanes; paclitaxel;Taxol1; diterpenoids;Taxus canadensis; Taxus chinensisvar mairei; Taxus�media; Taxaceae.

INTRODUCTION

Numerous phytochemical investigations concerningTaxusspecies have described the isolation and structuralelucidation of more than 100 naturally occurring taxanediterpenoids (Kingstonet al., 1993; Appendino, 1995).Since paclitaxel (Taxol1; 1) is one of the most powerfulanti-cancer drugs of plant origin, the search for newcompounds of this type is still going on. Taxanederivatives are reported to exist only inTaxus andAustrotaxusspecies (Kingstonet al.,1993), and becauseof this limitation the chance of re-isolation of knowntaxanes is increasing. The rapid progress of analyticalinstrumentation and coupled techniques enables the swiftidentification of natural products from crude plantextracts without prior isolation (Hostettmannet al.,1997). High performance liquid chromatography–nuclearmagnetic resonance spectroscopy (HPLC–NMR) is oneof the most powerful combination techniques for thispurpose (Albert, 1995) and is currently increasinglyutilized to analyse natural products of plant origin, e.g.sesquiterpene lactones from a Compositae species(Springet al.,1995), lupulones from hop extracts (Ho¨tzelet al.,1996) and several ingredients from a Gentianaceaespecies (Wolfenderet al., 1997). In order to develop afast-screening method for the detection of various

taxanes in differentTaxusspecies, we have investigatedthe application of the HPLC–NMR method.

EXPERIMENTAL

Plant material. Leaves ofTaxus chinensis(Pilger) Rehdvarmairei (Lemee et Levl) were collected by S. Y. Hu inNovember, 1995 from Heilong valley, Jiyuan County,Henan Province (central China). The specimen wasidentified by Mr. Z. S. Yue, Senior Engineer of Botany,Kunming Institute of Botany, Chinese Academy ofSciences, where the voucher specimen is deposited.Leaves ofT. canadensisMarsh., andT.�mediaRehd. cvHicksii were collected in February 1996, from theBotanical Garden of the University of Mainz, Germany,and identified by Dr. U. Hecker: the voucher specimensare deposited in the laboratory of the authors at Mainzand in the plant data collection of the Garden.T.canadensis and T.�media are assigned the codenumbers 1905/1 and 1618/1, respectively.

Extraction and pre-purification. The air-dried needles(0.5 g) were frozen with liquid nitrogen and ground to afine powder in a mortar. Methanol (1.5 mL) was added,the mixture ground vigorously, and the slurry transferredwith further portions of methanol to a centrifuge tube andmixed for 10 min. The mixture was cooled with liquidnitrogen for a further 10 min and, after equilibration toroom temperature, centrifuged at 14,000g for 15 min.The supernatant was diluted to ten times its volume withdouble-distilled water. This solution was passed throughan Isolute C-18 solid phase extraction (SPE) column andeluted successively with 35 and 55% methanol to removecompounds having less affinity for the C18 material thanpaclitaxel and its derivatives, which remained on the

PHYTOCHEMICAL ANALYSISPhytochem. Anal.9, 237–244, (1998)

CCC 0958–0344/98/050237–08 $17.50# 1998 John Wiley & Sons, Ltd.

§ Dedicated to Prof. Meinhart H. Zenk on the occasion of his sixty-fifth birth-day.* Correspondence to: B. Schneider. Present address: Max-Planck-Institute ofChemical Ecology, Tatzendpromenade 1a, D-07745 Jena, Germany.E-mail: [email protected]/grant sponsor: Fonds der Chemischen Industrie.Contract/grant sponsor: European Community; Contract/grant number: AIR3-CT94-1979.

Received 28 July 1997Revised 15 November 1997

Accepted 20 November 1997

column (Kaufman et al., 1995). The column was thenwashed with 65% methanol and the eluent wasevaporatedto drynessin a streamof nitrogenat 45°C.Ice-coldmethanol(0.5mL) wasaddedto theresidueandmixed for 5 min at 4°C using a vortex with periodicplacement in an ice bath. The clear solution wasevaporatedto dryness.

HPLC. HPLC analyses were carried out using aLiChrospher100 RP-18column(250� 4 mm i.d.). ThemobilephasesweresolventA — deuteratedwater(D2O)containing0.1% trifluoroaceticacid, and solvent B —acetonitrile containing 0.1% trifluoroacetic acid. Twogradientprofileswereemployed:profile 1 was— 0 min80%A–20%B, 15min 50%A–50%B, 20min 40 % A–60% B, 30min 45% A–55% B (flow-rate1.0mL/min);profile 2 was— 0 min 70% A–30%B, 30min 55% A–45%B (flow-rate0.8mL/min). Detectionwasat230nm.

HPLC–NMR. HPLC–NMRanalyseswerecarriedouton

a Bruker DRX 500 spectrometerat 500.13 MHz. AMerck-HitachiLiChrographL-6200Agradientpumpwasfitted with a 4 mm inversedetectionliquid chromato-graphyprobe-head(detectionvolume120mL). Chroma-tographicconditionsasdescribedabovewereusedin thestopped-flow mode. Proton (1H) NMR spectra weremeasuredwith a spectralwidth of 12,000Hz, and datawereacquiredinto 32 K datapoints.An acquisitiontimeof 1.36 s and a relaxationdelay of 1.80 s were used.1H–1H two-dimensional total correlation (2D-TOCSY)spectrawereacquiredwith 32 transientsperFID andthenumberof experimentswassetto 160.A time domainof2 K and a sweepwidth of 12,000Hz were used.Afterbaseline correction the spectra were symmetrized.Double solvent suppressionof acetonitrile and of theresidualwater in the acetonitrile: D2O gradientswereperformed by presaturationapplying standardBrukerpulsesequences.Forcalibration,thesuppressedsignalofacetonitrilewassetat d 2.0.

Standardone- and two-dimensionalNMR spectraof

238 B. SCHNEIDERET AL.

# 1998JohnWiley & Sons,Ltd. Phytochem.Anal. 9: 237–244(1998)

isolatedcompoundswere measuredon a Bruker DRX500 spectrometerat 500.13MHz (1H) and125.70MHz(13C), respectively.

If not otherwisementioned,ca300mg of a dry samplewere dissolvedin 100mL acetonitrile:water (1:1) forHPLCanalysesandin acetonitrile:D2O (1:1) for HPLC–NMR measurements.ForthetaxineB standard,methanol-d4 wasusedasa solvent.

Mass Spectra. ESI massspectra(electrosprayvoltage4.5 kV) were recordedon a FinniganMAT TSQ 7000instrument.

RESULTS AND DISCUSSION

In order to examinethe variation of the 1H chemicalshifts betweendeuterochloroform,which is generallyusedas the NMR solvent for taxanes,and acetonitrile:D2O which is usedas a solvent for HPLC–NMR, thefollowing authenticreferencecompoundsof the taxanetype were measuredunder HPLC–NMR conditions:paclitaxel (1), taxine B (2), 10-deacetylbaccatinIII (3),5-cinnamoyl-10-acetyltaxicin I (4) and 5-cinnamoyl-9-acetyltaxicinI (5). The 1H NMR spectraobtainedin thesolventsystemacetonitrile:D2O are the first of a moreextensivedatabasebeingestablishedfor the rapid iden-tification of taxanesby HPLC–NMR.A partialspectrumof paclitaxel (1) in acetonitrile: D2O is shown as anexamplein Fig. 1 A. Thesignalsof the1H resonancesof

the standardcompoundswere assignedusing literaturedata measuredin deuterochloroform(Ettouati et al.,1991;Appendinoetal., 1992;Chmurnyetal., 1992).Theacetonitrilesignalwassetatd 2.0.Mostof theHPLC–1HNMR signalsof paclitaxelwereslightly high-field,beingshifted by about 0.10 to 0.25 ppm relative to theresonancesmeasuredin deuterochloroform(Chmurnyetal., 1992).Theresonancesof H(5) andH(10)remainedalmostunchanged(Dd =� 0.04).Therelativelargeshiftwhich wasobservedfor H(3') (Dd =ÿ0.30) from d 5.78in deuterochloroformto d 5.48in acetonitrile:D2O is dueto theinfluenceof thepH valueontheadjacentsecondaryaminogroup:consequently,thesignalsof H(3') andH(2)are interchangedin sequencein the latter solvent.Theassignmentof bothsignalsin theHPLC–NMRspectrumwasconfirmedfrom the couplingconstantsof 6 Hz forH(3') andH(2'): in contrast,2JH(2)–H(3) wasslightly butsignificantlygreater(7 Hz).Theopportunityto determineaccuratelycouplingconstantsindicatesthe high qualityof the HPLC–NMR spectra.In general,the differencesbetween1H NMR signalsobtainedin deuterochloroformand in acetonitrile: D2O, respectively,did not exceed0.20 ppm. This fact enablesthe identificationof knowntaxanesby HPLC–NMR even if referencedata in thelatter solventarenot available.

In spite of the employmentof solvent suppressiontechniques,residualsolventsignalsappearin thespectra.In the HPLC–1H NMR spectrumof paclitaxel (1) (Fig.1A), measuredafterdirectinjectionof astandardsolutionin acetonitrile:D2O (7:3) into the HPLC–NMR probe-head,the narrow signal of HDO doesnot disturb any

Figure 1. HPLC±NMR spectra (500 MHz) of taxanes bearing a N-benzoyl-b-phenylisoserinyl sidechain. A: Directly injected solution of standard paclitaxel (1) in acetonitrile: deuterated water (7:3);B: Paclitaxel (1) from a leaf extract of Taxus canadensis measured in the stopped ¯ow mode; andC: 10-Deacetyltaxol (6) from a leaf extract of Taxus�media measured in the stopped ¯ow mode.

TAXANES BY HPLC–NMR 239


proton resonanceof the sample. However, since thesignalof HDO dependson the acetonitrile:D2O ratio inthe HPLC gradient,it is shifteddownfieldwith decreas-ing ratios,in partsuperimposingthesignalsof H(20)andH(7) of paclitaxel(1) (datanot shown).The HPLC–1HNMR spectrumof 10-deacetylbaccatinIII (3), which ismore hydrophilic and thereforeelutesfrom the RP-18columnwith alowerratioof acetonitrilethan1, exhibitedthe HDO signal at d 4.38. Consequently,the multipletcentredatd 4.17which is formedby H(7) andbothH(20)signalscanbeseenin thespectrum(Table1).

The useof non-deuteratedacetonitrileas the solventinevitablycausesinterferencein theHPLC–NMRspectrabetweend 1.8andd 2.2,maskingtheresonancesof mostacetylprotonsandof somedownfieldmethyl protonsoftaxanes,particularlyof H(18).Forexample,thesingletofthe10-acetylgroupis notvisiblewithin theHPLC–NMRspectrumof paclitaxel,while theprotonsof the 4-acetylgroupresonatefurther downfieldat d 2.29(Table1).

In orderto assessthesuitabilityof HPLC–NMRfor theidentification of taxane diterpenoids in actual plantmaterials,extracts of T. canadensis,T. chinensisvarmairei and T.�media have been investigated.Thesedifferent specieswere selectedto presentthe various

types of taxanestructures,e.g. taxus alkaloids,neutraltaxanesandtheanti-cancerdrugpaclitaxel(Taxol1) (1).Sincethe amountsof taxanesin Taxusleavesare verylow in non-enrichedsamples(in general only 10ÿ3–10ÿ4% of freshweight),a shortwork-up procedurewasemployedto enhancetheir concentration.Chlorophylland other matrix matter were removedfrom the crudemethanolicextractsby passingthem through a RP-18SPEcartridge.A fractionelutingwith 65%methanolwassufficiently enrichedto enableHPLC–NMR analysisoftaxanes.

Initially, an enriched, pre-purified extract of theneedlesof T. canadensiswassubjectedto HPLC–NMRanalysisusing an acetonitrile:D2O gradient(profile 1),and a large number of HPLC peaksappeared.Threepeaksin the regionwherepaclitaxel(1) wasexpectedtobe elutedwere transferredto the NMR probehead.TheHPLC peakat Rt 24.3min showeda 1H NMR spectrum(Fig. 1B) which resembledthe spectrumof standard1(Fig.1A), theslightvariations,e.g.for H(3'), beingduetothe different acetonitrile:D2O ratios.Other compoundscouldnotbeidentifiedin thisextractowingto poorsignalto noiseratio and/orimpurity of thepeaks.

From needlesof T.�media threeHPLC peakswere

Table 1. HPLC–1H NMR data of taxanes1–7.a

H Paclitaxel (1)b Taxine B (2)b10-Deacetyl-

baccatin III (3)b5-cinnamoyl-10-

acetyltaxicin I (4)b5-cinnamoyl-9-

acetyltaxicin I (5)b10-Deacetyltaxol

(6)bDecinnamoyl-taxagi®ne (7)b

1 2.53, dd, 12, 22 5.52, d, 8 4.04, d, 6.5 5.52, d, 7 4.03, d, 6.5 4.10, d, 6.5 5.50, d, 7 5.46, dd, 10, 23 3.68, d, 8 2.94, d, 6.5 3.86, d, 7 3.18, d, 6.5 3.19, d, 6.5 3.72, d, 7 3.31, d, 105 4.98, d, 9 5.02, brs 5.02, d, 10 5.20, brs 5.19, brs 4.97, d, 9 4.26, s6a 2.45, m

�1.63, m 2.46, m NQc NQ NQ *d

6b 1.74, dd, 2, 11 1.75, t, 11 NQ NQ NQ 1.52, m7a 4.21, dd, 11, 7 * 4.17, me NQ NQ NQ 5.18, dd, 11, 67b 1.29, m9 4.24, d, 10 4.23, d, 10 5.66, d, 10 5.08, d, 210 6.31, s 5.74, d, 10 5.28, s 5.78, d, 10 4.97, d, 10 5.18, s 5.23, d, 213 5.98, t, 8 4.84, t 5.99, t, 914a *

�2.67, s * 2.67, d, 19 2.68, d, 19 * 2.59, d, 20

14b * * 2.56, d, 19 2.58, d, 19 * 3.05, dd, 20, 1216 1.06, s 1.46, s 0.99, s 1.47, s 1.57, s 1.00, s 3.78, d, 8

4.00, d, 817 1.09, s 1.15, s 1.01, s 1.14, s 1.23, s 1.08, s 1.44, s18 * * * * * 1.79, sf 1.02, s19 1.56, s 1.02, s 1.59, s 1.10, s 0.92, s 1.58, s 0.95, s20 4.13, s 5.36, s 4.19, d, 8e 5.42, s 5.42, s Sg 5.25, s

5.27, s 4.15, d, 8e 5.36, s 5.35, s Sg

2' 4.68, d, 6 3.10, dd, 17, 6 6.37, d, 16 6.37, d, 16 4.67, d, 63.01, dd, 17, 9

3' 5.48, d, 6 4.61, dd, 9, 6 7.60, d, 16 7.60, d, 16 5.46, d, 62-AcO *4-AcO 2.29, s 2.27, s7-Oac *9-Oac * *10-AcO * * * *a Spectra measured at 500.13 MHz for compounds dissolved in acetonitrile: deuterated water (acetonitrile = d 2.0).b The 1H resonances of the phenyl moieties are as follows: 1 and 6 as shown in Fig. 1; 2 Ð d 7.46, m; d 7.40, m; 3 Ð d 8.07, d,J = 7; d 7.56, d, J = 7; d 7.68, t, J = 7; 4 and 5 Ð d 7.74, m; d 7.47, m.c NQ Ð not quoted owing to poor signal to noise ratio.d * Ð superimposed by the acetonitrile signal.e The resonances of H(7) and H(20) are superimposed.f Visible between the signals of acetonitrile and its satellite.g S Ð superimposed by the residual HDO signal.



analysed.The spectrumobtainedfrom the peak at Rt20.7min (Fig. 1C) showed a close similarity to thespectrumof 1 exceptfor the resonanceof H(10). This

singletwasshiftedfrom d 6.31in 1 to d 5.18in thelattercompound,indicating a lack of the acetyl group at 10-OH: this compoundhas beenassignedas 10-deacetyl-taxol (6). Themostintensiveof thethreeHPLCpeaks(Rt13.2min; profile 1) was identified as taxine B (2) bycomparing the spectrumobtainedduring a measuringtime of 5 h 26min (Fig. 2B) with that of the standardcompound(measuringtime 20min; Fig. 2A). The rele-vantNMR signalsof bothspectraarealmostidenticalandclearly indicatethat trans-esterificationand decomposi-tion processesdid not occurto a significantextentunderthe conditions used. The characteristicsignals of acinnamoyl moiety representedthe most conspicuousfeaturein thespectrumof thethird peak(Rt 24.2min). Incontrastto normalHPLC–NMRspectraof taxaneswhichexhibit the threemethyl resonancesof Me(16), Me(17)and Me(19) (the signal of Me(18) being hiddenby theacetonitrilesignal), a double set of six methyl signalsbetweend 0.92 andd 1.57 was indicative of a mixtureof two taxanes.Furthermore,four doubletsat d 4.23,d 4.97, d 5.66 and d 5.78 each showing a couplingconstantof 6.5 Hz, were presentin the spectrum.Thecorrelationsbetweenthesetwo pairs of doubletswereestablishedby a 2D-TOCSY (total correlationspectro-scopy) spectrum(Fig. 3). The cross signals betweend 4.97 and d 5.66, and betweend 4.23 and d 5.78,respectively,wereconsistentwith connectivitiesbetweenH(9) andH(10) of either5-cinnamoyl-10-acetyltaxicin I(4) or 5-cinnamoyl-9-acetyltaxicin I (5) (Appendinoetal., 1992). Comparing the complete HPLC–1H NMRspectrum of the mixture with standard compoundsconfirmedthesefindings(Fig. 4).

In the HPLC chromatogramof freshextractsfrom T.chinensis var mairei, a major peak appearedat Rt13.5min (profile 2). Preliminary HPLC–1H NMRanalysisshowedcharacteristictaxanesignalsalongwithsignalsof a cinnamicacid moiety (Fig. 5C). However,

Figure 2. Stopped ¯ow HPLC±NMR spectra (500 MHz) of taxine B (2) in acetonitrile: deuterated water. A:Authetical standard; B: Taxine B from a leaf extract of Taxus�media.

Figure 3. Two-dimensional TOCSY spectrum (500 MHz) of amixture of isomeric acetyl derivatives of 5-cinnamoyl-taxicin Ifrom an extract of Taxus�media acquired under stopped¯ow HPLC±NMR conditions in acetonitrile: deuterated water.Cross correlations of 5-cinnamoyl-9-acetyltaxicin I (5) areindicated by a square and those of 5-cinnamoyl-10-acetyl-taxicin I (4) by a circle.



further experimentsusingthe samesampleafter storageat 4°C showedthat the peak at Rt 13.5min was sig-nificantly decreased.The 1H NMR spectrummeasuredunder HPLC–NMR conditions(Fig. 5B) did not showcinnamate signals anymore, but the same taxaneresonancesas before were still visible. Furthermore,apeakcorrespondingto a significantly more hydrophiliccomponent(Rt 3.5 min) appearedin the HPLC, andthiswasidentifiedascinnamicacidby HPLC–NMR(d 7.70,d, 16 Hz; d 7.62,m; d 7.44,m; d 6.50,d, 16 Hz). Thespectrumof thefreshextract(Fig.5C)showedataxaneinadmixturewith cinnamicacid,which is normally elutedmuch earlier from the reversedphasecolumn. Conse-quently, this cinnamic acid must have been formedduring the HPLC–NMR run, indirectly indicating thatdegradationof putativecinnamoyltaxanescanoccur inthis extract.Whilst 5-cinnamoyltaxinesare quite stablecompounds,it appearsthat the rearrangedtaxanein thefresh extract is labile in HPLC solvents containingtrifluoroaceticacid.Lossof the cinnamoylsidechainoftaxaneshas alreadybeenobservedin previousstudies(Hegnauer,1962and literaturecited therein).However,the demonstrationdescribedhere is only possiblebyHPLC–NMR,confirmingthepotentialof this technique.

The HPLC–1H NMR spectrum of the compoundeluting at Rt 13.5min (Fig. 5B) exhibited most of thesignalsof decinnamoyltaxagifine(7), suggestingthat the

detected compound is identical with that formerlyisolatedtaxanefrom T. chinensis(Zhanget al., 1990).1H–1H homonuclearcorrelationsbetweendoubletsofH(16a) (d 4.00)/H(16b)(d 3.78), H(3) (d 3.31)/H(2) (d5.46), and H(1) (d 2.53)/H-14b (d 3.05), respectively,wereestablishedfrom theHPLC–2D-TOCSYspectrum.However, several NMR signals were hidden by thesolvent(Table1) and,particularlyin theregionbetween4.9andd 5.6,areinterchangedin sequencein theHPLC–NMR spectrum,ascomparedwith the1H NMR spectrumobtainedin deuterochloroform(Fig. 6). The 1H NMR(H(1): d 2.37,dd, J= 11.5Hz, 1.9Hz; H(2): 5.52,dd, 9.2,1.9; H(3): 3.54,d, 9.2;H(5): 4.36,t, 3.0; H(6a):2.12,m;H(6b): 1.59, ddd, 14.5, 10.4, 3.6; H(7): 5.38, dd, 10.4,6.1; H(9): 4.93,d, 3.6; H(10): 5.38,d, 3.6; H(14a):2.65,d, 18.4;H(14b):3.03,dd, 18.4,11.6;H(16a):3.69,d, 8.0;H(16b):4.18,d, 8.0;H(20a):4.49,s; H(20b):5.26,s; Me-17: 1.52, s; H(18): 1.17, s; H(19): 1.06, s; acetyl-CH3:1.96,2.03,2.10,2.10),1H–1H 2D-COSYand13C NMR(C(1): d 49.52;C(2): 68.85;C(3): 38.31;C(4): 144.35;C(5): 73.25;C(6): 39.37;C(7): 68.57;C(8): 46.52;C(9):76.12;C(10): 64.16;C(11): 91.74;C(12): 80.27;C(13):205.90;C(14):34.95;C(15):49.34;C(16):82.13;C(17):15.72;C(18):11.87;C(19):13.62;C(20):113.23;acetyl-CO: 172.41,169.84,168.73,168.69;acetyl-CH3: 21.47,21.36,20.75,20.71)spectraaswell asthe ESI–MSdata(m/z 605, relative intensity 16 [M � K]ÿ, m/z 589,

Figure 4. Stopped ¯ow NMR spectra (500 MHz) of isomeric acetyl derivatives of 5-cinnamoyl-taxicin I. A:Standard 5-cinnamoyl-10-acetyltaxicin I (4), B: Standard 5-cinnamoyl-9-acetyltaxicin I (5) Ð this spec-trum was generated by subtraction of spectrum A from a spectrum of standard 4 containing a smallamount of 5 as an impurity (the negative signals are due to subtraction); and C: Spectrum of 4 and 5eluting as a mixture at Rt 24.2 min. The signals tagged by an asterisk are due to a solvent impurity(propionitrile).



relative intensity 100 [M � Na]�) of the isolatedcompound7 confirmedthe suggestedstructure(Zhanget al., 1990),exceptthat the assignmentsof C(1), C(8)and C(14) were revised by careful inspection of theinverse-detectedheterocorrelated2D spectra(HMBC,HMQC).

HPLC–NMRis shownfor thefirst time to bea usefulapproachfor the rapid identificationof varioustypesoftaxanesin partially purifiedplant extracts.It is expectedthat this techniquewill be extensivelyemployedin thephytochemical screening of taxane-containingplantmaterialsandfor otherclassesof naturalproducts.

Acknowledgements

Wearegratefulto Dr. C.PoupatandDr. A. Ahond(Gif-sur-Yvette)forproviding standardtaxanes.Prof. H. Sunand Prof. Z. Gu (KunmingInstituteof Botany)are thankedfor their help in collecting the plantsamplesin China,and Mrs. D. Wurth and Mr. J. Arend (Mainz) forassistingin collecting plant samplesin Germany.Oneof the authors(Y. Z.) also gratefully acknowledgesthe Alexandervon Humboldt-Stiftung for a researchfellowship in Germany.We thankProf.W. E.Court(Mold) for checkingtheEnglishversionof this manuscript.TheDeutscheForschungsgemeinschaft(Bonn)is gratefullyacknowledgedby B. S. for the NMR spectrometerused.This work wasfinanciallysupportedby the Fondsder ChemischenIndustrie (Frankfurt a.M.).Thatpartof thiswork whichwasperformedatHallewasin associationwith a project sponsoredby the EuropeanCommunity(AIR3-CT94-1979).

Figure 5. HPLC±NMR spectra (500 MHz) of decinnamoyltaxagi®ne (7) from Taxus chinensis var mairei. A:Directly injected solution of the isolated compound in acetonitrile: deuterated water (1 : 2): insert shows theHPLC±NMR spectrum of standard cinnamic acid; B: Stopped ¯ow HPLC±NMR spectrum of the peak at Rt

13.5 min from an extract stored several days at 4°C; and C: Stopped ¯ow HPLC±NMR spectrum of the peakat Rt 13.5 min from a fresh extract. The inserts to spectra B and C represent the aromatic regions of thecorresponding spectra. The signals tagged by an asterisk are due to a solvent impurity (propionitrile); thosetagged by squares indicate signals of further impurities.

Figure 6. Selected portions of 1H NMR spectra (500 MHz) ofdecinnamoyltaxagi®ne (7) from Taxus chinensis var mairei.A: Spectrum of the isolated compound in deuterochloroform;and B: Stopped ¯ow HPLC±NMR spectrum of the peak at Rt

13.5 min in acetonitrile: deuterated water. Chemical shiftdifferences are �0.2 ppm.



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