Gentisic acid conjugates of Medicago truncatula roots

Phytochemistry 70 (2009) 1272–1276

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Gentisic acid conjugates of Medicago truncatula roots Anna Stochmal a,*, Iwona Kowalska a, Bogdan Janda a, Angela Perrone b, Sonia Piacente b, Wiesław Oleszek a a b

Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100 Pulawy, Poland Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (Salerno), Italy

a r t i c l e

i n f o

Article history: Received 28 April 2009 Received in revised form 14 July 2009 Available online 13 August 2009 Keywords: Medicago truncatula Gentisic acid glycosides Vicenin-2 Hovetrichoside C Pterosupin Guaiacylglycerol-feruloyl derivatives

a b s t r a c t Three phenolic glycosides 5-O-{[50 0 -O-E-(40 0 0 -O-threo-guaiacylglycerol)-feruloyl]-b-apiofuranosyl-(1?2)b-xylopyranosyl} gentisic acid, 5-O-[(50 0 -O-vanilloyl)-b-apiofuranosyl-(1?2)-b-xylopyranosyl] gentisic acid and 1-O-[E-(400 0 -O-threo-guaiacylglycerol)-feruloyl]-3-O-b-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5-O-b-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, 1H and 13C NMR spectral data. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Medicago truncatula has been a close relative of agriculturally important pasture crop alfalfa (Medicago sativa). This species due to its small diploid genome (5  108 bp), self-fertilization, easy genetic transformation and prolific nature has been chosen as a model legume for genomic studies of Fabaceae (Cook, 1999). The recognition of the genome is followed by functional genomic approach that includes profiling of gene expression (transcriptome), protein (proteome) and metabolites (metabolome). This approach gives bases for the understanding of biological processes (e.g. symbiosis, insect and pathogen resistance) and provides tools for the improvement of legumes considering agriculture important traits (e.g. yield, protein concentration, secondary metabolites accumulation). Unlike alfalfa, which has been studied for primary and secondary metabolites in depth, not much attention was given to M. truncatula in this respect until recently. It was just last decade when some papers regarding its phytochemical profiles have been published. There were two approaches used for profiling of M. truncatula metabolites, the classical one based on isolation and spectral identification of dominant compounds (Kapusta et al., 2005a,b; Kowalska et al., 2007), and the spectral approach based on multicomponent analysis using GC–MS or LC–MS hyphenated techniques (Huhman and Sumner, 2002; Farag et al., 2007;

* Corresponding author. Address: Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, 24-100 Pulawy, Poland. Tel.: +48 81 886342x205; fax: +48 81 8864547. E-mail address: [email protected] (A. Stochmal). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.07.021

Schliemann et al., 2008; Leitner et al., 2008). Both approaches showed rather high similarity of the composition of triterpene saponins (Kapusta et al., 2005a; Huhman and Sumner, 2002) and flavonoids (Kowalska et al., 2007) in aerial parts of M. sativa and M. truncatula, but some differences were also evident. The roots of M. truncatula have been profiled exclusively with hyphenated techniques. Several groups of secondary metabolites were detected including volatile organic compounds (VOCs) emitted after microbial infection (Leitner et al., 2008), primary polar and nonpolar metabolites, isoflavonoids, apocarotenoids, a cell wall-bounded phenolics (Farag et al., 2007; Schliemann et al., 2008). However, many of the metabolites occurring in LC or GC profiles have been identified only tentatively or remained totally uncharacterized due to the lack of appropriate standards and reference spectra in MS data bases. Thus, there is still a need to further characterize metabolites of M. truncatula by classical spectral techniques. This paper reports for the first time in M. truncatula roots the known glycosides 5-O-b-xylopyranosyl gentisic acid (2-hydroxy-5-xylosyl benzoic acid), vicenin-2 (6,8-di-C-b-glucosylapigenin), hovetrichoside C (maesopsin 4-O-glucoside) and pterosupin (dihydrochalcone) as well as two new guaiacylglycerol-feruloyl derivatives (1,3) and vanillic acid esterified gentisic acid glycoside (2), never characterized previously in any plant species. 2. Results and discussion The purified extract from M. truncatula roots was submitted to a combination of chromatographic steps over reversed phase RP-18 to yield seven compounds. Four known compounds, 5-O-b-xylopyranosyl gentisic acid (Fayos et al., 2006), pterosupin (Manickam

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pound 1 showed three sets of three aromatic protons of a ABX system at d 7.49 (1H, d, J = 3.2 Hz), d 7.14 (1H, dd, J = 3.2, 8.9 Hz), d 7.12 (1H, d, J = 1.6 Hz), d 7.07 (1H, d, J = 1.6 Hz), d 7.04 (1H, dd, J = 1.6, 8.5 Hz), d 6.99 (1H, d, J = 8.5 Hz), d 6.90 (1H, dd, J = 1.6, 8.1 Hz), d 6.79 (1H, d, J = 8.9 Hz), d 6.76 (1H, d, J = 8.1 Hz), two olefinic protons at d 7.53 (1H, d, J = 15.7 Hz) and d 6.27 (1H, d, J = 15.7 Hz) and two signals corresponding to methoxy groups at d 3.86 (3H, s) and d 3.84 (3H, s). Additionally, two methine proton signals at d 4.89 (d, J = 5.7 Hz) and d 4.55 (dd, J = 5.7, 6.1 Hz) indicative of secondary alcoholic functions and signals at d 3.88 (dd, J = 5.7, 10.5 Hz) and d 3.85 (dd, J = 3.2, 10.5 Hz) indicative of

et al., 1997), hovetrichoside C (Yoshikawa et al., 1998) and vicenin2 (Lu and Yeap Foo, 2000) were isolated and their structures confirmed by comparison of the respective spectroscopic data with those reported in literature. However, these four known compounds have not been previously reported in M. truncatula plants. Their concentration in roots determined with UPLC was 0.456, 0.028, 0.194 and 0.435 mg/g of root dry matter for 5-O-b-xylopyranosyl gentisic acid, pterosupin, hovetrichoside C and vicenin-2, respectively. The structures of compounds 1–3 were elucidated based on detailed spectral analyses (Table 1). The 1H NMR spectrum of com-

Table 1 1 H (J in Hz) and

13

C NMR spectroscopic data of compounds 1–3 (600 MHz, CD3OD). 1

2

3

dC

dH

1

Gentisic acid –

115.2

Gentisic acid –

115.0

2 3

– 6.79 d (8.9)

158.6 118.4

– 6.65 d (8.9)

158.3 118.4

4 5 6 7

7.14 dd (3.2, 8.9) – 7.49 d (3.2) –

125.7 150.7 118.2 173.8

7.07 dd (3.2, 8.9) – 7.46 d (3.2) –

125.0 150.6 118.0 173.5

10 20 30 40 50

b-Xylose 4.87 d (7.7) 3.66 dd (7.7, 9.3) 3.59 t (9.3) 3.60 m 3.95 dd (4.8, 11.5) 3.33 t (11.5)

102.0 78.2 78.2 71.0 66.8

b-Xylose 4.86 d (7.7) 3.64 dd (7.7, 9.3) 3.58 t (9.3) 3.59 m 3.93 dd (4.8, 11.5) 3.34 t (11.5)

102.3 78.3 78.0 71.0 66.8

dH

dC

50 0

d d d d

110.2 78.5 79.4 75.0

(9.8) (9.8) (10.8) (10.8)

67.2

5.53 4.05 – 4.30 3.95 4.38 4.32

d (1.6) d (1.6) d d d d

(9.8) (9.8) (10.9) (10.9)

174.0

110.4 78.5 79.2 74.9

168.8 112.2 146.6 129.4 112.1 151.3 151.8

68.0

6.99 d (8.5)

117.3

7.12 dd (1.6, 8.5) 3.86 s

123.5 56.3

– 7.06 – – 6.74 6.87 4.87 4.52 3.87 3.84 3.83

133.8 111.8 148.2 146.8 115.4 121.0 73.9 85.3 62.1

60 0 OMe

a0 0 0 b0 0 0

c0 0 0 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0 70 0 0 80 0 0 90 0 0 OMe 10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0 60 0 0 0 70 0 0 0 80 0 0 0 90 0 0 0 OMe

Guaiacylglycerol-ferulic acid – 6.27 d (15.7) 7.53 d (15.7) – 7.12 d (1.6) – – 6.99 d (8.5) 7.04 dd (1.6, 8.5)

Vanillic acid 168.3 116.0 146.7 129.5 112.0 151.6 151.7 117.3 123.4

– 7.47 d (2.0) – – 6.80 d (8.1) 7.49 dd (2.0, 8.1) –

121.9 113.3 148.6 152.6 115.7 125.0 167.7

– 3.86 – 7.07 – – 6.76 6.90 4.89 4.55 3.88 3.85 3.84

s d (1.6)

d (8.1) dd (1.6, d (6.1) dd (5.7, dd (5.7, dd (3.2, s

8.1) 6.1) 10.5) 10.5)

69.6 70.5

Guaiacylglycerol-ferulic acid – 6.47 d (15.7) 7.67 d (15.7) – 7.22 d (1.6) – –

c0 0 d (1.6) d (1.6)

10.1) 10.1)

67.1

– b-Apiose

a0 0

5.51 4.03 – 4.26 3.91 4.30 4.25

11.3) 11.3)

100.7 74.5 73.4 73.1 72.4

b0 0 10 0 20 0 30 0 40 0

dC

b-Galacturonic acid 4.92 d (8.9) 3.71 t (8.9) 3.55 dd (2.4, 8.9) 3.51 dd (2.4, 10.1) 4.08 d (10.1)

60 b-Apiose

dH Glycerol 4.34 dd (4.4, 4.27 dd (5.7, 4.15 m 3.92 dd (2.8, 3.54 dd (5.3,

56.4 133.9 111.5 148.7 147.2 115.4 120.8 73.9 85.4 62.1 56.3

3.89 s

56.3

d (1.6)

d (8.1) dd (1.6, d (6.1) dd (5.7, dd (5.7, dd (3.2, s

8.1) 6.1) 10.5) 10.5)

56.1

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primary alcoholic function were observed. On the basis of DQF– COSY, HSQC and HMBC spectra, it was determined the presence of a 2,5-substituted benzoic acid identified as gentisic acid, a 4substituted feruloyl moiety, a 3-methoxy-4-hydroxy-phenyl group and a glycerol moiety (Table 1). The HMBC spectrum showed correlations between the proton signal at d 4.89 (H-700000 ) of glycerol moiety and the carbon resonances at d 133.9 (C-10000 ), d 111.5 (C20000 ) and d 120.8 (C-60000 ) of 3-methoxy-4-hydroxy-phenyl group. Coupling constants and chemical shifts values were in good agreement with values reported for threo-isomer of guaiacylglycerol (Sakushima et al., 1995). The HMBC correlation between the proton signal at d 4.55 (H-80000 ) of guaiacylglycerol unit and the carbon resonance at d 151.7 (C-4000 ) of feruloyl unit allowed us to identify a 4000 -O-threo-guaiacylglycerol-feruloyl moiety (see Fig. 1). The 1H NMR spectrum displayed also signals corresponding to two anomeric protons at d 5.51 (1H, d, J = 1.6 Hz) and 4.87 (1H, d, J = 7.7 Hz). Complete assignments of the 1H and 13C NMR signals of the sugar portion were accomplished by 1D-TOCSY, HSQC, HMBC and DQF–COSY experiments which led to the identification of one b-xylopyranosyl unit (d 4.87) and one b-apiofuranosyl unit (d 5.51). The sequence and the linkage sites of the sugar units was determined by the HMBC spectrum: key correlation peaks between the proton signal at d 5.51 (H-100 ) and the carbon resonance at d 78.2 (C-20 ), the proton signals at d 4.30 and 4.25 (H-500 ) and the carbon resonance at d 168.3 (C-a0 00 ) and between the proton signal at d 4.87 (H-10 ) and the carbon resonance at d 150.7 (C-5) were observed. Therefore, the structure of 1

was established as the new 5-O-{[500 -O-E-(400 0 -O-threo-guaiacylglycerol)-feruloyl]-b-apiofuranosyl-(1?2)-b-xylopyranosyl} gentisic acid. A detailed analysis of 1H and 13C NMR spectra of compound 2 in comparison with those of compound 1 showed the absence of signals due to threo-guaiacylglycerol-feruloyl moiety. Moreover, a further set of three aromatic protons at d 7.49 (1H, dd, J = 2.0, 8.1 Hz), d 7.47 (1H, d, J = 2.0 Hz) and d 6.80 (1H, d, J = 8.1 Hz) and a methoxy group at d 3.89 (3H, s) was displayed, along with signals ascribable to the b-apiose, b-xylose and gentisic acid units. The HSQC, HMBC and DQF–COSY experiments allowed us to determine the presence of a 3,4-substituted benzoyl moiety, identified as vanilloyl unit. The HMBC correlation between the proton signals at d 4.38 and 4.32 (H-500 ) and the carbon resonance at d 167.7 (C7000 ) indicated that the position 500 of b-apiose was esterified by the vanilloyl unit. Thus the structure of new compound 2 was identified as 5-O-[(500 -O-vanilloyl)-b-apiofuranosyl-(1?2)-b-xylopyranosyl] gentisic acid. The 1H and 13C NMR spectra of compound 3 showed signals of threo-guaiacylglycerol-feruloyl moiety, similarly to compound 1, along with signals at d 4.34 (1H, dd, J = 4.4, 11.3 Hz) and d 4.27 (1H, dd, J = 5.7, 11.3 Hz) and d 3.92 (1H, dd, J = 2.8, 10.1 Hz) and d 3.54 (1H, dd, J = 5.3, 10.1 Hz) ascribable to two primary alcoholic functions, and a signal at d 4.15 (1H, m) indicative of a secondary alcoholic function. The spin system H-1 (d 4.34 and d 4.27)/H-2 (d 4.15)/H-3 (d 3.92 and d 3.54) confirmed by the COSY spectrum indicated the pres-

Fig. 1. Chemical structures of compounds 1–3.

A. Stochmal et al. / Phytochemistry 70 (2009) 1272–1276

ence of a further glycerol unit. The HMBC spectrum showed key correlation peaks between the proton signals at d 4.34 and d 4.27 (H-1) and the carbon resonance at d 168.8 (C-a00 ) indicating that the C-1 position of additional glycerol unit was esterified by the threo-guaiacylglycerol-feruloyl moiety. The 1H NMR spectrum showed also a signal corresponding to an anomeric proton at d 4.92 (1H, d, J = 8.9 Hz). The sequence of this sugar unit was deduced from the analysis of the DQF–COSY and HMBC spectra which allowed us to identify a b-galacturonopyranosyl unit. The linkage site of sugar unit was established by key correlation peak between the anomeric proton at d 4.92 (H-10 ) and the carbon resonance at d 70.5 (C-3), as shown in the HMBC spectrum. All these evidence indicated that the compound 3 was the new 1-O-[E-(400 -O-threo-guaiacylglycerol)-feruloyl]-3-O-b-galacturonopyranosylglycerol. The concentration of new identified compounds in the root dry matter was 0.893, 0.126 and 0.043 mg/g for 1, 2 and 3, respectively. This is the first report of guaiacylglycerol-feruloyl derivatives from M. truncatula, nevertheless it is worthwhile to note that a considerable number of hydroxybenzoic acids combined with a guaiacylglycerol group have been found to occur as breakdown products of lignin (Katayama et al., 1980, 1981). The guaiacylglycerol-feruloyl moiety of compounds 1 and 3 is a neolignan derivative obtained from the oxidative dimerisation of two coniferyl alcohol units. This dimerisation involves an oxidative linkage through the C-8 of the propenyl side chain of one coniferyl alcohol moiety and the C-40 of the other coniferyl alcohol moiety, as suggested by the biosynthetic pathway proposed for lignan derivatives (Beejmohun et al., 2007). The o-hydroxybenzoic acids, as gentisic acid occurring in 1 and 2, are generally synthesized from phenylalanine and cinnamic acid removing a C2 fragment of the side chain or directly from benzoic acid by hydroxylation, while the p-hydroxybenzoic acids, as vanillic acid occurring in 2, are formed from the corresponding hydroxycinnamic acids removing a C2 fragment of the side chain (El-Basyouni et al., 1964). Thus all the moieties which make up compounds 1–3 seem to be derived from the shikimate pathway. The gentisic acid (2,5-dihydroxybenzoic acid), which is a biosynthetic derivative of salicylic acid (2-hydroxybenzoic acid), was found to be present in three of isolated compounds of M. truncatula roots. This compound was reported to accumulate in plants after non-nectrotizing infection and can also play an important signalling function in activating defence genes in tomato (Bellés et al., 1999). Virus and viroid infection in tomato and cucumber resulted in the accumulation of high levels of gentisic acid xyloside (Fayos et al., 2006). Our finding is the second example next to tomato and cucumber showing that the xyloside of gentisic acid accumulates in plant, and not the glucose derivative as for the salicylic acid. This shows also that compound can be further glycosylated with apiose and additionally acylated with vanillic acid like in 2. What is a meaning of this glycosylation and acylation remains to be explained. Occurrence of derivatives of gentisic acid in M. truncatula roots may suggest that similarly as in tomato and cucumber where progressive accumulation after phatogen inoculation was observed (Bellés et al., 1999; Fayos et al., 2006), these compounds can also play a role in defence response system of this plant. So far number of phenolic acids including vanillic, syringic, 4-E-coumaric, E-ferulic and 4-hydroxybenzoic have been identified in cell wall-bound metabolites of mycorrhizal roots of M. truncatula (Schliemann et al., 2008). The significance of other identified compounds for plant physiology/defence system remains unknown. However, we deeply believe that identification of these unusual molecules, occurring in not-bound forms in M. truncatula roots gives a new touch to the ‘‘tip of the iceberg” of the almost hidden multitude of metabolites, especially signalling molecules involved in molecular dialog between plant and environment (Schliemann et al., 2008).

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3. Experimental 3.1. General The optical rotations were obtained in MeOH at 20 °C on a Jasco P-1020 spectropolarimeter (Jasco Inc., Easton, MD). ESI–MS was performed on a Thermo Finnigan LCQ Advantage Max ion-trap mass spectrometer with an electrospray ion source (Thermo Electron Corp., Bellefonte, PA). Compounds were analyzed by direct injection by a syringe pump at a flow rate of 5 ll/min. The spray voltage was set to 4.2 kV and a capillary offset voltage of 60 V. All spectra were acquired at a capillary temperature of 220 °C. The calibration of the mass range (400–2000 Da) was performed in negative ion mode. Nitrogen was used as sheath gas, and the flow rate was 0.9 l/min. The maximum ion injection time was set to 200 ms. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a Bruker 5 mmTCI CryoProbe at 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma–Aldrich) and standard pulse sequences and phase cycling were used for DQF–COSY, HSQC, HMBC and ROESY spectra. The NMR data were processed using UXNMR software. Liquid chromatography was performed on the Acquity ultra performance liquid chromatograph (UPLC, Waters) consisting of binary solvent manager, sample manager, PDA and TQ mass detectors, and Empower Pro 2.0 software was used. The profiling was performed on a 50 mm  2.1 mm i.d., 1.7 lm, UPLC BEH C18 column (Waters) utilizing a gradient elution profile and a mobile phase consisting of 0.1% acetic acid in water and 40% MeCN. The column was maintained at 50 °C and the flow rate was kept constant at 0.35 ml/min. 3.2. Plant material Seeds of Medicago truncatula Gaertn. var. Jemalong A17 were obtained from Dr. XianZhi He, The Samuel Roberts Noble Foundation, Ardmore, OK, where a voucher specimen is deposited. They were planted in an experimental field of the Institute of Soil Science and Plant Cultivation in Pulawy in 2006. Roots of plants were harvested, lyophilized, finely powdered, and used for the successive extraction. 3.3. Extraction and isolation Powdered roots (200 g) were extracted two times with 70% aqueous MeOH over night at room temperature and sonication for 1 h. The solvent was removed under reduced pressure (45 °C). The dry residue was suspended in water and passed through a short preparative column (6 cm  10 cm, LiChroprep RP-18, 40– 63 lm, Merck) previously preconditioned with water. The column was washed first with water to remove sugars and then with 40% MeOH to elute phenolics. Using analytical HPLC as a check, the fraction washed out with 40% MeOH was used for separation of phenolics according to previously used protocol (Stochmal et al., 2001). Crude phenolic powder (3 g) was suspended in distilled water and loaded onto a 4 cm  50 cm, 40–63 lm LiChroprep RP18 column (Millipore Corp., Bedford, MA). The column was washed with distilled water (300 ml) and then with increasing concentration of MeOH in water (5% increments from 0% to 100% MeOH). Ten milliliter fractions were collected, checked by cellulose TLC (Merck) developed in 15% acetic acid, and observed under UV (366 nm). Fractions showing similar TLC patterns (15 fractions) were further analyzed by ultra performance liquid chromatography (UPLC). Fractions possessing one compound were combined

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and evaporated to dryness. Fractions containing more than one compound were further purified on a 2 cm  50 cm, 25–40 lm RP-18 glass column using an isocratic system (MeCN-1% H3PO4) optimized for each fraction based on the analytical separation. This yielded several individual compounds. 3.4. 5-O-b-Xylopyranosyl gentisic acid

MSn: (negative ion mode) m/z 1279 [2 MH], 639 [MH], 621 [MH18]. For 1H and 13C NMR data see Table 1. Acknowledgements This work was financed by the Polish Committee for Science as 2004–2007 Project 2 P06A 037 27.

A pale brownish amorphous powder (2 mg), Rt 2.35 min, on line UV (MeOH) kmax 313 nm. ESI/MSn: (negative ion mode) m/z 285 [MH], 153 [MH132]. 1H and 13C NMR data were consistent with published before (Fayos et al., 2006).

Appendix A. Supplementary data

3.5. Pterosupin (dihydrochalcone)

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A pale brownish amorphous powder (1.6 mg), Rt 3.21 min, on line UV (MeOH) kmax 216, 281, 317sh. ESI/MSn: (negative ion mode) m/z 435 [MH], 271 [MH164]. 1H and 13C NMR data were consistent with published before (Manickam et al., 1997).

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3.6. Maesopsin-4-O-glucoside (hovetrichoside C) A pale brownish amorphous powder (2.5 mg), Rt 5.57 min, on line UV (MeOH) kmax 224, 301 nm. ESI/MSn: (negative ion mode) m/z 449 [MH], 431 [MH18], 287 [MH162], 269 [MH180]. 1H and 13C NMR data were consistent with published before (Yoshikawa et al., 1998). 3.7. 6,8-Di-C-b-glucosylapigenin (vicenin-2) A pale brownish amorphous powder (2.2 mg), Rt 6.50 min, on line UV (MeOH) kmax 216, 271, 335 nm. ESI/MSn: (negative ion mode) m/z 593 [MH], 575 [MH18]. 1H and 13C NMR data were consistent with published before (Lu and Yeap Foo, 2000). 3.8. 1-O-[E-(400 -O-threo-guaiacylglycerol)-feruloyl]-3-O-bgalacturonopyranosyl glycerol (3) A pale brownish amorphous powder (2.1 mg), Rt 7.61 min, on line UV (MeOH) kmax 326 nm, ½a20 D 54.10° (MeOH, c 0.1). ESI/ MSn: (negative ion mode) m/z 1579 [2 MH], 789 [MH], 771 [MH18], 592 [MH197], 417 [MH372], 285 [MH372132]. For 1H and 13C NMR data see Table 1. 3.9. 5-O-[(500 -O-Vanilloyl)-b-apiofuranosyl-(1?2)-b-xylopyranosyl] gentisic acid (2) A pale brownish amorphous powder (1.5 mg), Rt = 8.07 min, on line UV (MeOH) kmax 264, 296, 325sh nm, ½a20 D 28.97° (MeOH, c 0.1). ESI/MSn: (negative ion mode) m/z 1135 [2 MH], 567 (MH], 417 [MH150], 285 [MH150132], 153 [MH150132132]. For 1H and 13C NMR data see Table 1. 3.10. 5-O-{[500 -O-E-(4000 -O-threo-guaiacylglycerol)-feruloyl]-bapiofuranosyl-(1?2)-b-xylopyranosyl} gentisic acid (1) A pale brownish amorphous powder (1.2 mg), Rt = 10.75 min, on line UV (MeOH) kmax 326 nm, ½a20 D 33.17° (MeOH, c 0.1). ESI/

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2009.07.021.