MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem. 2005; 43: 724–728Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1634

HPLC–NMR for tissue-specific analysisof phenylphenalenone-related compoundsin Xiphidium caeruleum (Haemodoraceae)†

Bernd Schneider,∗ Christian Paetz, Dirk Holscher and Stefan Opitz

Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Knoll Str. 8, 07745 Jena, Germany

Received 27 September 2004; Revised 30 March 2005; Accepted 26 April 2005

HPLC–1H NMR has been used to study the tissue-specific distribution of phenylphenalenones,polyphenolic natural products of Xiphidium caeruleum, a neotropical member of the Haemodoraceaeplant family. The present results provide insight into the occurrence of phenylphenalenone-relatedcompounds in root segments of whole plants and different in vitro culture lines of the same species.Copyright 2005 John Wiley & Sons, Ltd.

KEYWORDS: NMR; 1H NMR; HPLC–NMR coupling; Xiphidium caeruleum; phenylphenalenones;phenylbenzoisochromenones; allophanyl glucosides

INTRODUCTION

Most applications of HPLC–NMR techniques in phyto-chemistry1 – 3 have been devoted to identifying and derepli-cating natural products in order to screen them for bio-logically active components for pharmaceutical and agri-cultural applications. However, HPLC–NMR has been asuitable tool for analyzing natural products in biosynthetic,4

chemotaxonomic,5 and enzymatic studies6 as well. Increas-ing interest in ecological functions of secondary plant prod-ucts has supported the development of techniques enablingthe investigation of organ-, tissue-, and cell-specific dis-tribution of natural products in the producing species. Thesimultaneous accessibility of retention time, absorption prop-erties, and NMR spectral data for identifying the analytes,combined with an efficient sample preparation and an optionfor online or off-line MS analyses make HPLC–NMR themost universally useful technique in this field.

Phenylphenalenones7 and related polyaromatic naturalproducts are involved in plant-defense mechanisms ofMusaceae, Haemodoraceae, and a few other monocotyledonfamilies.8,9 In order to study the occurrence of specificcompounds of the phenylphenalenone type in special roottissue, HPLC–NMR was employed in the present workto analyze various root sections of Xiphidium caeruleumplants, a neotropical member of the Haemodoraceae plantfamily. The results are discussed in comparison with thoseobtained by other methods such as confocal laser scanningmicroscopy (CLSM) and in situ multispectral photometric(MSP) techniques.10 Additional HPLC and HPLC–NMR

†Presented as part of a special issue on Hyphenated NMRTechniques.ŁCorrespondence to: Bernd Schneider, Max-Planck-Institute forChemical Ecology, Beutenberg Campus, Hans-Knoll Str. 8, 07745Jena, Germany. E-mail: schneider@ice.mpg.de

studies were undertaken to examine the occurrence andestimate semiquantitatively the levels of special structuraltypes of phenylphenalenones in root and cell tissues collectedfrom different in vitro culture lines. The occurrence or absenceof phenylphenalenone target compounds, especially novelallophanyl glucosides, was used as a criterion to evaluateplant roots and in vitro cultures with regard to their suitabilityfor biosynthetic studies.

RESULTS AND DISCUSSION

Analysis of roots of Xiphidium caeruleum plantsRed pigmentation of the roots due to the accumulation ofphenylphenalenones and related polyphenolics is commonamong many Haemodoraceae species.7 While the main rootsof Xiphidium caeruleum plants are almost colorless, secondaryroots exhibit an intense orange–red color with even strongerpigmentation in the root tip. However, as shown by phy-tochemical studies of X. caeruleum, in addition to typicalphenylphenalenones with distinctive visible absorptions,phenylphenalenone-related compounds such as phenylben-zoisochromenones and corresponding glucosides are alsofound in this plant.11 Although almost colorless, somephenylbenzoisochromenones and their glucosides exhibit anintense autofluorescence, which, together with in situ MSP,was used to localize these compounds by CLSM specificallyin distinct cells of the plant root.10 However, the very strongquantitative differences in autofluorescence led to selectivedetection of intensely fluorescing compounds such as the6-O-allophanylglucoside of compound 1, while the moreabundant but 150 times less fluorescent aglycone 1 (exitationat 364 nm; autofluorescence emission at 520 nm) was hardlydetected by CLSM. In order to study the distribution of

Copyright 2005 John Wiley & Sons, Ltd.

Tissue-specific analysis of phenylphenalenone-related compounds 725

the major metabolites of X. caeruleum in plant tissue, inde-pendent of their individual absorption and autofluorescenceproperties, HPLC–NMR was used in the present work.

The first series of experiments used pigmented sec-ondary roots of X. caeruleum grown in soil under greenhouseconditions. The roots were divided into a tip section (approx-imately 15 mm), a middle section (approximately 25 mm),and the basal part of the root. After lyophilization, a fewmilligrams of the root sections was extracted with a smallvolume of DMSO-d6 and analyzed by means of HPLC–1HNMR coupling in the stopped-flow mode. The HPLC (UV254 nm) traces (Fig. 1A–C) of all investigated root sectionsexhibited two peaks 1 and 2 of intensity appropriate for NMRanalysis. Comparison of 1H NMR spectra, retention times,and UV absorption of both peaks with those of previouslyisolated compounds11 allowed identification of 6-hydroxy-5-methoxy-7-phenyl-3H-benzo[de]isochromen-1-one (1) and5-methoxy-7-phenyl-3H-benzo[de]isochromen-1,6-dione (2)(Fig. 1). The HPLC–1H NMR spectrum of compound 1(Fig. 1D) shows a characteristic AB spin system of H-9, whichgives the signal at the lowest field at υ 8.16, and adjacent H-8(υ 7.34). The singlet of H-4, integrating for one proton, appearsat υ 7.43, and that of the methylene CH2-3, integrating fortwo protons, at υ 5.78. The unresolved multiplet centered atυ 7.39 can be assigned to the lateral phenyl ring. Finally, themethoxyl resonance is visible in the spectrum at the typicalshift value of υ 3.91. The spectrum of compound 2 (Fig. 1E)resembles that of 1, except that the methylene signal waschanged to a methine resonance (H-3), which, due to theadjacent lactone oxygen, now appears at relatively low fieldat υ 7.64. It changed sequence with the resonance signal of

H-4 (υ 6.84), which has been shifted significantly to higherfield. The doublet of H-8 (υ 7.37) is visible on the slope of theunresolved phenyl multiplet.

Although compounds 1 and 2 were found in allinvestigated root sections, their levels were remarkablydifferent (Fig. 1A), indicating longitudinal concentrationgradients for both compounds. The level of 1 is highestin the 15-mm root tip section (Fig. 1A) and decreases towardthe basal part of the root (Fig. 1C), while the opposite wasobserved for compound 2. Taking into account that the totallevel of phenylphenalenone-related natural products in theplant roots is considerably higher in the root tip than in themature root segment,12 compound 1 is the most abundantphenylphenalenone-derived compound in secondary rootsof Xiphidium caeruleum grown in soil. This confirms previousexperiments,10 which, based on HPLC analyses of eight serialsections of 0.5-mm thickness taken from the tip to the roothair zone, showed an amount of 80 µmol g�1 fresh weightof 1 in the first 0.5-mm tip section (section 1), constantlydeclining to about 20 µmol g�1 in section 8 (between 3.5- to4-mm distance to the tip). Although not studied in detail, theaccumulation of phenylphenalenones, especially compound1, in the root tips indicates a specific role in protecting thegrowing young plant tissue against pathogens and predatorysoil organisms.

Two members of a new type of glucosides, bearingthe rare allophanyl moiety at the primary hydroxyl group6-CH2-OH of the glucose, dropped in concentration evenmore drastically from the first section of the root tip tothe more distant 0.5-mm sections.10 Thus, in soil-grownplant roots, reasonable levels of allophanyl glucosides were

6.06.57.07.58.0ppm

O

HO

H3CO

O1

1'

6

O

O

H3CO

O1

1'

6

H-9

H-9

H-8

H-8

OCH3-5

OCH3-5

H-2′ − H-6′

H-3

H-4

H-3

H-4

D

E

1

2

4.0

H-2′ −H-6′

1

A

1

B

0 10 20

min

C

2

Figure 1. HPLC–NMR of phenylbenzoisochromenones 1 and 2 from root sections of Xiphidium caeruleum plants. UV traces(254 nm) of HPLC runs of DMSO-d6 extracts obtained from root tip (A), middle section (B) and basal root section (C). Samples oflyophilized root material (10 mg) were extracted with DMSO-d6 (400 µl) and injected in the HPLC (injection volume 30 µl; gradient 1).HPLC–1H NMR spectra of compounds 1 (D) and 2 (E) (MeCN-D2O; 500.13 MHz; MeCN D υ 2.00; stopped-flow mode; presaturationwas used for solvent suppression).

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 724–728

726 B. Schneider et al.

detected almost solely in the root tip. Since these amountswere considered insufficient for biosynthetic studies, weinvestigated root sections of hydroponically grown Xiphid-ium caeruleum plants for their allophanyl glucoside con-tents. However, no significant levels of glucosides weredetected in all sections; but aglycones especially compound1, were found to be the major phenylphenalenone-relatedconstituents of these roots.

Analysis of cell and root cultures of XiphidiumcaeruleumIn vitro cell- and tissue cultures are suitable experimentalsystems for phytochemical, biosynthetic and enzymaticstudies.13 For example, they are independent of seasonalgrowth periods, and their cultivation in liquid mediumenables easy administration and rapid uptake of precursorsby the cells. Moreover, especially dedifferentiated callusand cell suspensions in general show a high capacityfor glucosidation of a wide variety of endogeneous andexogeneous substrates. Therefore, both cell suspensions androot cultures of Xiphidium caeruleum were investigated forthe occurrence of phenylphenalenone-type compounds withparticular emphasis on glucosides.

Preliminary HPLC studies of extracts of lyophilized rootsections (root tip and mature root section) and culturedcells showed major peaks of hydrophilic glucosides anddiminishing peaks of nonpolar aglycones in all extracts.

However, significant differences were observed in how effi-ciently the lyophilized tissue was extracted with variouswater-free solvents. Methanol was shown to be the most effi-cient of the tested solvents, while DMSO resulted in lowerlevels of glucosides in the extracts. No glucosides werefound in acetone extracts (data not shown). UV/Vis absorp-tion spectra obtained from PDA detection during the HPLCruns (gradient 2a) indicated two different types of structures,phenylphenalenones and phenylisobenzochromenones, pre-viously identified from Xiphidium caeruleum.11,12 Typi-cal phenylphenalenones were characterized by absorptionbands around 280 nm, 375 nm and 470 nm. In contrast,the oxidized type, namely, phenylisobenzochromenones, islacking the long-wave absorption at 470 nm in the UV/Visspectra. However, absorption spectra did not allow fordiscrimination of glucosides either with or without anallophanyl unit, which was considered one of the targetfeatures for biosynthetic investigations. Moreover, reten-tion time of various allophanyl glucosides differed by onlyless than 0.7 min, and weak relative intensities of molec-ular ions were observed in MS analysis.11,12 Thus, liquidchromatography coupled to 1H NMR was considered thesuperior method for unambiguous identification of thedesired allophanyl glucoside. Using this method, isolationof the target compound in each of the various biosyntheticexperiments, for which the present work is a prerequisite,can be avoided and information on incorporation of labeledprecursors can be easily obtained from the HPLC–1H NMR

H-9

H-2′−

H-6′H-3

H-1″

7.58.08.5ppm

3.03.54.04.5ppm

H-4

H-8

H-6″a

H-6″b

H-3″

H-4″

H-2″

*

A

B

H-9H-8

H-4

H-2′−

H-6′

H-3

* *

**

H-1″H-6″a

H-6″b

H-5″

H-4″H-5″

H-3″

3 HO

OHO

O

HOOH

O

ONH

OO

H2N

OH

1″

1

1'

6

Figure 2. Comparison of 1H NMR spectra (500.13 MHz) of the allophanyl glucoside 3 measured in the stopped-flow HPLC–NMRmode in MeCN-D2O (A) and a previously isolated sample of 3 measured in MeOH-d4 in a 5-mm tube (B). For details of structureassignment and stereochemical aspects, see text. H-200 was not detected in MeCN-D2O due to overlay by the solvent signal (MeCN).* represents solvent signal.

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 724–728

Tissue-specific analysis of phenylphenalenone-related compounds 727

spectrum. For example, position-specific incorporation of 13Clabels from phenylpropanoid precursors and phenylalanineinto aromatic methine carbons of phenylphenalenones andphenylbenzoisochromenones is anticipated in C-3 from [3-13C]phenylpropanoids, C-8 from [2-13C]phenylpropanoids,and C-9 from [1-13C]phenylpropanoids. Since these carbonsare protonated, indirect detection of 13C-labelled positionsis possible by means of their 1H-13C coupling pattern inthe HPLC–1H NMR spectrum.14 In addition, because ofthe variable chemical environment of these respective pro-tons, their resonances are extremely useful as fingerprintsignals to identify various types of phenylphenalenones andrelated structures. For example, because of the differenthybridization status of the respective C-3 and the proximityto variable position 2 of phenylphenalenones and phenyl-benzoisochromenones, H-3 of compounds 1–3 appear at thecharacteristic resonances υ 7.19, 7.64, and 5.78, respectively,in the HPLC–1H NMR spectra (Figs 1 and 2).

In this context, HPLC–1H NMR analysis of the majorpeak of extracts obtained from suspension cultures ofX. caeruleum at Rt26.7 min (gradient 2b) identified 6-O-[(6’’-O-allophanyl)-ˇ-D-glucopyranosyl]-2,5-dihydroxy-7-phenyl-phenalen-1-one (3) (Fig. 2). Comparison with the1H NMR spectrum of the previously isolated compound,11

although recorded in different solvents, showed acceptableagreement between chemical shifts and coupling patterns ofboth samples (Fig. 2). The tricyclic phenalenone nucleus hasa few protons, visible in the low-field part of the spectra.Two singlets of H-3 and H-4, which in MeCN-D2O appear atυ 7.19 and υ 7.57, respectively, and two doublets of the ABspin system of H-8 (υ 7.56) and H-9 (υ 8.47) are attributable tothe phenalenone substructure. The latter singlet, because ofits location in peri position to the carbonyl (C-1), is the mostdownfield signal in the spectrum. The phenyl protons appearas broad signals, indicating rotation hindrance by the closespatial proximity of the lateral phenyl ring to the hexoseunit in peri position. The ˇ-configuration at the anomericcenter (H-1’’, υ 4.80) of the hexose was deduced fromJH-100 – H-200 D 8 Hz. The couplings of approximately 8–9 Hzof the other sugar methine protons proved an axial config-uration of H-1 to H-5 of the hexose, which therefore wasidentified as glucose. In the ˇ-glucose unit, especially H-2’’and to a lesser extent H-4’’, are affected by intramolecularinteraction with the lateral phenyl substituent. The signal ofH-4’’ (υ 3.05 in MeCN-D2O, Fig. 2A) appears approximately0.4 ppm at higher field than expected, and H-2’’ is drasti-cally shifted to a high field (υ 2.36 in MeOH-d4, Fig. 2B).Therefore, in MeCN-D2O the latter signal was overlaid bythe large satellite line of the suppressed MeCN resonance.From the shift data of axial protons H-2’’ and H-4’’, it wasconcluded that the ‘‘upper’’ face of the ˇ-glucose must beoriented toward the lateral phenyl ring. This hypothesis wassupported by the chemical shift values of the axial protonslocated on the ‘‘lower’’ face of the glucose ring, namely, H-1’’(υ 4.80), H-3’’ (υ 3.19), and H-5’’ (υ 3.10), which were foundin the expected chemical shift range.

Finally, the attachment of an allophanyl substituent atthe glucose unit of compound 3 was established fromthe chemical shift of the H-6’’ protons. Glucosides of

phenylphenalenone-type lacking an allophanyl moiety11,15

show shift values of υ 3.24–3.47 and υ 3.45–3.57 for H-6’’proton signals. In contrast, H-6’’ signals in allophanyl glu-cosides are significantly shifted to low field and appear atυ 4.16–4.21 and υ 3.91–4.00, indicating a deshielding effectof a substituent on these methylene protons. The HPLC–1HNMR spectrum of compound 3 displays shifts for H-6’’ pro-tons of υ 4.13 and υ 3.86, which, despite measurement indifferent solvents, clearly agree with those of the allophanylglucosides. Thus, the structure of compound 3 has been con-firmed as 6-O-[(6’’-O-allophanyl)-ˇ-D-glucopyranosyl]-2,5-dihydroxy-7-phenylphenalen-1-one and the correspondingstructure without an allophanyl residue is ruled out. Sincethe same compound occurs in cell suspensions and root cul-tures of Xiphidium caeruleum, both in vitro culture lines seemto be suitable for studying the biosynthesis of allophanylglucosides.

CONCLUSIONS

HPLC–1H NMR was shown to be an excellent toolin phytochemistry-related studies of Xiphidium caeruleum.Extracts of lyophilized tissue of root sections of this plant,prepared directly with deuterated solvents, were sub-jected to HPLC–1H NMR study. This approach resultedin rapid tissue-specific identification of phenylphenalenone-type compounds in plant samples. In contrast to previousconfocal laser scanning microscopy and multispectral pho-tometric methods, 1H NMR analysis was independent of thevery different specific autofluorescence and absorption prop-erties of those compounds. HPLC–1H NMR was also shownto be useful to identify phenylphenalenone allophanyl glyco-sides, which exhibited similar UV spectra and retention timesin HPLC, from cell and root cultures of the same species.Since both the occurrence of an allophanyl substituent at theglucose moiety and discrimination of the various structuraltypes of phenylphenalenone tricyclic nuclei was possible byHPLC–NMR, this method would be used for biosyntheticstudies to indirectly detect specific incorporation of 13C bymeans of 1H NMR. Moreover, it has been demonstrated thateven stereochemical characterization of natural products ispossible with hyphenated HPLC–1H NMR spectroscopy.

EXPERIMENTAL

Plant materialPlant material of Xiphidium caeruleum (Aublet) was grown insoil or hydroponic containers under greenhouse conditionsor held in growth chambers. Cell cultures were initiatedfrom surface-sterilized seeds on solid Murashige and Skoogmedium (MS)16 at 22 °C in the dark. After germination, rootswere sliced and placed on solid agar medium, where callideveloped slowly. Separated callus tissue was then grownon solid agar medium and after several passages transferredto liquid MS medium. Root aggregations spontaneouslydeveloping from callus were separated and further cultivatedin liquid Linsmaier and Skoog medium.17

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728 B. Schneider et al.

Sample preparationPlant root sections. Soil-grown roots were divided into tipsections (15 mm length), middle sections (25 mm length)and basal sections. Lyophilized root segment material(10 mg) was extracted with 400 µl DMSO-d6 at roomtemperature for 2 h. After filtration, a volume of 30 µl of eachextract was subjected to HPLC. Samples for off-line HPLCanalysis of serial root sections were performed as previouslydescribed.10 Hydroponically grown roots were divided intoroot tips (2 mm length), followed by a middle section (10 mmlength) and basal section (up to 30 mm length). The collectedmaterial was then separately lyophilized. Root cultureaggregates were removed from the culture medium andwashed with a small amount of de-ionized water to removeremaining medium. The pale colored tips were cut from theroot aggregates. Subsequently, tips and the remaining rootmaterial were separately lyophilized. Suspended cells wereseparated from the culture medium by filtration, washedwith de-ionized water and lyophilized. Freeze-dried material(1 to 15 mg) from hydroponically grown roots, root culturesand cell suspensions was ground and extracted with drymethanol, dry DMSO and dry acetone, respectively, at roomtemperature for 60 min. After filtration, a volume of 15 µl ofeach extract was subjected to HPLC.

HPLC–NMR analysisHPLC. HP 1100 chromatography system (quaternary pumpG1311A, autosampler G1313A). Column: LiChrospher100RP-18 (5 µm; 250 ð 4 mm). Gradient 1: MeCN-D2O (0.1%TFA), 30%–65% MeCN in 30 min; flow rate 0.8 ml min�1.Gradient 2a: MeCN-H2O (0.1% TFA) 5%–50% MeCN in40 min; flow rate 0.8 ml min�1. Gradient 2b: MeCN-D2O(0.1% TFA) 5%–50% MeCN in 40 min; flow rate 0.8 ml min�1.Detection: J&M photodiode array detector, monitoring wave-length 254 nm).

1H NMR. HPLC–1H NMR analyses were carried out on aBruker DRX 500 NMR spectrometer operating at a protonfrequency of 500.13 MHz. The HP 1100 chromatographysystem and the photodiode array detector were connectedthrough a Bruker BPSU-12 interface to a 4-mm inverse-detection LC probehead (detection volume 120 µl). Spectrawere recorded in the stopped-flow mode and data collectedinto 32 K data points with spectral width of 10 kHz,acquisition time 1.63845 s. The number of accumulated scanstypically was 4 K. Solvent suppression of MeCN and residualHDO signals was performed by presaturation, applying theBruker pulse program lc1pnf2. A line-broadening factor of0.8 was used prior to Fourier transformation. For calibration,the suppressed signal of MeCN was set at υ2.0.

Analytical data of compounds 1–36-Hydroxy-5-methoxy-7-phenyl-3H-benzo[de]isochromen-1-one (1): HPLC: Rt 24.8 min (gradient 1); UV (MeCN-H2O)

�max 214, 268, 388 nm; 1H NMR (500.13 MHz, MeCN-D2O):υ 8.16 (1H, d, JH-9 – H-8 D 8 Hz, H-9), 7.43 (1H, s, H-4), 7.39(5H, m, H-2’–H-6’), 7.34 (1H, d, JH-8 – H-9 D 8 Hz, H-8), 5.78(2H, s, H-3), 3.91 (3H, s, OCH3-5); EIMS: m/z 306 [M]C

(rel. int. 100). 5-Methoxy-7-phenylbenzo[de]isochromene-1,6-dione (2): HPLC: Rt 21.9 min (gradient 1); UV (MeCN-H2O)�max 243, 390 nm; 1H NMR (500.13 MHz, MeCN-D2O): υ 8.23(1H, d, JH-9 – H-8 D 8 Hz, H-9), 7.64 (1H, s, H-3), 7.40 (5H, m,H-2’–H-6’), 7.37 (1H, d, JH-8 – H-9 D 8 Hz, H-8), 6.84 (1H, s,H-4), 3.94 (3H, s, OCH3-5); EIMS: m/z 303 [M C H]C (rel.int. 100). 6-O-[(6’’-O-Allophanyl)-ˇ-D-glucopyranosyl]-2,5-dihydroxy-7-phenyl-phenalen-1-one (3): HPLC: Rt 26.2 min(gradient 2a), Rt 26.7 min (gradient 2b); UV (MeCN-H2O)�max 211, 279, 378, 477 nm; 1H NMR (500.13 MHz, MeCN-D2O): υ 8.47 (1H, d, JH-9 – H-8 D 8 Hz, H-9), 7.57 (1H, s, H-4), 7.56(1H, d, JH-8 – H-9 D 8 Hz, H-8), 7.5–7.2 (5H, H-2’–H-6’), 7.19(1H, s, H-3), 4.80 (1H, d, JH-100 – H-200 D 8 Hz, H-1’’), 4.13 (1H, dd,JH-600a – H-600b D 12 Hz, JH-600a – H-500 D 6 Hz, H-6’’a), 3.86 (1H, brd,JH-600b – H-600a D 12 Hz, H-6’’b), 3.19 (1H, dd, JH-300 – H-200 D 9 Hz,JH-300 – H-400 D 9 Hz, H-3’’), 3.10 (1H, m, H-5’’), 3.05 (1H, dd,JH-400 – H-300 D 9 Hz, JH-400 – H-500 D 9 Hz, H-4’’), H-2’’ was notdetected in MeCN-D2O due to overlay by the solvent signal(MeCN); ESIMS: m/z 553 [M C H]C (rel. int. 4), 305 (100).

AcknowledgementWe thank Emily Wheeler, Jena, for linguistic help in the preparationof this manuscript.

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Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 724–728