The esters o f p-coumaric, ferulic and caffeic acid with malic acid were isolated from leaves, the ester o f sinapic acid with malic acid from cotyledons o f Raphanus sativus plants via preparative HPLC. The esters were identified by hydrolyses and following capillary GC, by H-NMR and FAB-MS. Their concentrations in leaves and tubers were determinedby gradient HPLC on reversed phase material. © 1984, Walter de Gruyter. All rights reserved

Brandi, Wilfried

Grotjahn, Lutz

Herrmann, Karl

Institutionelles Repositorium der Leibniz Universität Hannover

Hydroxycinnamoyl Esters of Malic Acid in Small Radish {Raphanus sativus L. var. sativus)W ilfried Brandi and Karl H errm annInstitut für Lebensmittelchemie der Universität Hannover, Wunstorfer Str. 14, D-3000 Hannover andLutz GrotjahnGesellschaft für Biotechnologische Forschung, Mascheroder W eg 1, D-3300 Braunschweig Z. Naturforsch. 39c , 5 1 5 -520  (1984); received March 6 , 1984Hydroxycinnamoylmalic Acids, Isolation, Identification, Quantitative Determ ination,Raphanus sativusThe esters o f /?-coumaric, ferulic and caffeic acid with malic acid were isolated from leaves, the ester o f sinapic acid with malic acid from cotyledons o f Raphanus sativus plants via prepara­tive HPLC. The esters were identified by hydrolyses and following capillary GC, by H -NM R and FAB-MS. Their concentrations in leaves and tubers were determined by gradient HPLC on reversed phase material.IntroductionHydroxycinnamic acid derivatives are present ubiquitously in the plant kingdom  [1]. They have been detected in small radish  by pap e r and th in  layer chrom atographic techniques [2], Some o f these compounds, especially those in small radish  con­taining sinapic acid, could be analyzed by the in ­vestigations o f Strack et al. [3, 4]. Two hydroxycin­namoylmalic acids are know n up to now. G erm s and leaves of beans (Phaseolus vulgaris) and red clover ( Trifolium pratense) are containing caffeoyl- malic acid (phaseolic acid) [5, 6 ] and sinapoylm alic acid has been determ ined in small rad ish  seedlings.In this present study, we report the occurrence of four hydroxycinnam oylm alic acids in sm all radish, the known esters o f caffeic and sinapic acid and also the hithertoo unknown esters o f p -coum aric  and ferulic acid.Materials and MethodsPlant m aterialLeaves o f the small radish  varieties H ilm ar, Riesenbutter and Cherry Bell were used for the iso­lation and determ ination o f />-coumaroyl, caffeoyl and feruloylmalic acid. To ob ta in  sinapoylm alic acid, seed of the variety R ota was sown on cottonReprint requests to Dr. K  Herrmann.0341-0382/84/0600-0515 $ 0 1 .3 0 /0in the laboratory. The cotyledons were harvested and worked up 11 days after sowing [4],Isolation o f  hydroxycinnam oylm alic acidsSinapoylmalic acid: The cotyledons w ere com - munited in m ethanol and hom ogenized using a mixer and an U ltra Turrax. A fter filtering the plant pulp through a G 4  glass frit and w ashing the resi­due with hot m ethanol the extract was evaporated  at 40 °C to about 30 ml in vacuo. Sinapoylm alic acid was separated from the extract by prepara tive  HPLC [7],Apparatus: preparative H PLC system LCX (Philips), injection valve: R heodyne7125 w ith 2 ml sample loop, detection: UV 320 nm , solvent: 17% acetonitrile in 1 % acetic acid, flow: 1 1 .2 m l/m in , column: 250 x16 mm, L iC hrosorb RP 18, 10|am (Chrompack).The fractions containing sinapoylm alic acid (rR =  5.4 min) were collected and lyophylisated. The purity o f the com pound was checked by analytical HPLC.p-Coumaroyl, caffeoyl and feru loylm alic  acidA 300 g am ount o f small radish  leaves was worked up to obtain these esters. They were isolated by preparative HPLC using the solvent 13% aceton i­trile in 1% acetic acid. R etention times: caffeoyl- malic acid: ?r =  15.6 min, /?-courm aroylm alic acid: / r  =  27.3 min, feruloylm alic acid: t r  =  32.7 min. TheBereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46516 W. Brandi et al. • Hydroxycinnamoyl Esters o f Malic Acid in Small Radishfractions collected were lyophylisated and the purity  o f the obtained com pounds was controlled by an a­lytical HPLC. The fractions containing /?-coumaroyl- malic acid and feruloylm alic acid were cleaned up once more by preparative HPLC (solvent: 15% acetonitrile in 1 % acetic acid).Identification o f  the hydroxycinnam oylm alic acid  components by G CFor the identification o f the com ponents of hydroxycinnamoylmalic acids about 1 mg o f each com pound was hydrolyzed by adding 0.5 ml o f 0.5 n  HC1 and refluxing for 7 m inutes in boiling water. The hydrolysate was evaporated to dryness in vacuo and trimethylsilylated by adding 0.5 ml BSA/ 5% TMCS and heating at 70 °C  for 60 m inutes. /?-Coumaric, caffeic, ferulic, sinapic, m alic and pimelic acid (internal standard) were treated  in the same way for reference.Apparatus: capillary G C  (Carlo Erba), capillary: SE 30, 30 m (self m ade), carrier: N 2, detector: FID , tem peratur gradient: 120 °C  — 8  °C /m in  -  220 °C  — 15 °C /m in  -  270 °C.The hydroxycinnamic acid and m alic acid could be detected in about equim olar ratio  in each case.H-NM RH-NM R were recorded on a Bruker W H 270 spectrometer with puls fourier transform ation tech­niques. DMSO d 6 (solvent) was assigned 2.5 ppm  as secondary standard.FAB-MSNegativion mass spectra were recorded on a Kratos MS 50 S spectrom eter with a Kratos FAB source. A Xenon atom  gun was used at 9 KV.Quantitative determ inationsThe quantitative determ inations were perform ed with: HPLC system LCX (Philips), injection valve: Rheodyne 7125 with 20 (a 1 sam ple loop, colum n: 250x4 .6  mm, LiChrosorb RP 18, 5 |im  (M erck), detector: UV 320 nm, integrator: D P 101 C om puting Integrator (Pye U nicam ), solvent: A =  2% acetic acid, B =  methanol, gradient: 5% B in A -  45% B in A in 45 minutes, flow: 1 m l/m in.Results and DiscussionThe isolated com pounds were identified  as trans / 7-coumaroylmalic acid, /rarcs-caffeoylmalic acid, /ra«5 -feruloylmalic acid and /ra«s-sinapoylm alic acid by acid hydrolysis and following iden tification of the components via G C, by H -N M R  and nega- tivion FAB-MS.iR ’ 0COOH5 0 — CH7I2 R" CH2 8COOHCharacteristic proton signals o f the hydroxycin­namic acids and malic acid could be detected by NM R (Table I). The coupling o f the doublets (16 Hz) of the olefinic protons m akes possible the determ ination of the trans com pounds. The shift o f about 5.2 ppm of the C H -proton o f m alic acid allows the identification o f the ester bond with malic acid as alcoholic com ponent (m alic acid: (5 (CH) =  4.27 ppm).Negativion FAB mass spectroscopy affords a very good m ethod for the mass spectrom etric characteri­sation of conjugates, produced by plan t cells, w hen they are bonded by oxygen. As well as intense deprotonated m olecular ions characteristic sequence ions are also found. In the present com pounds, con­jugates of malic acid with /?-coum aric acid, or ferulic acid, or caffeic acid, or sinapic acid show intense molecular ions at 279, 295, 309 and 339 d respectively. Cleavage o f the bond betw een the ester oxygen of malic acid and the carbonyl o f the aromatic acid gives the m alic acid sequence ion at 133 d, while the characteristic arom atic  acid se­quence ions for cleavage between the carbon o f the malic acid and the ester oxygen affords ions at 163 d for / 7-coumaric acid, 179 d for caffeic acid, 193 d for ferulic acid and 223 d for sinapic acid. G lycerol was used as matrix for the m easurem ents and this gives rise to ions at 91, 183, 275 and 367 d (Figs. 1 - 4 ) .For the first tim e every possible ester o f hydroxy­cinnamic acids with m alic acid has been character­ized and identified side by side in one plant. The thin layer characteristics (hR f) o f these esters are differing significantly from o ther hydroxycinnam ic acid esters (Table II), facilitating the ir qualita tive determination beside the frequently  present esters with quinic acid or glucose.Bereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46W. Brandi et al. ■ Hydroxycinnamoyl Esters o f  Malic Acid in Small Radish 517Table I. H-NMR o f hydroxycinnamoylmalic acids in DM SO d6.p-CM ppm CM ppm FM ppm SM ppm1 Ar.-H 7.59 d 15 Hz OH o c h 3 3.82 s o c h 3 3.81 s2 Ar.-H 7.59 d 15 Hz Ar.-H 6.77 d 8  Hz Ar.-H 7.14 d 8  Hz o c h 3 3.81 s3 Ar.-H 6.80 d 15 Hz Ar.-H 7.03 m Ar.-H 6.80 d 8  Hz Ar.-H 7.04 s4 Ar.-H 6.80 d 15 Hz Ar.-H 7.03 m Ar.-H 7.35 s Ar.-H 7.04 s5 olef.-H 7.58 unres. olef.-H 7.46 d 17 Hz olef.-H 7.60 unres. olef.-H 7.54 d 16 Hz6 olef.-H 6.46 d 14 Hz olef.-H 6.27 d 17 Hz olef.-H 6.55 unres. olef.-H 6.54 d 16 Hz7 alk.-H 5.24 unres. alk.-H 5.15 dd 8  Hz alk.-H 5.23 unres. alk.-H 5.08 dd 2 Hz8 alk.-H 3.27 s alk.-H 3.10s alk.-H 3.26 s alk.-H 3.11 s/?-CM, /Mxmmaroylmalic acid; CM, caffeoylm alic acid; FM, feruloylmalic acid; SM, sinapoylm alic acid; d, doublet; dd, double doublet; s, singlet; unres., unresolved.Table II. Thin layer chromatography (h R f) o f  hydroxycinnamoylm alic acids on cellulose plates.BAW BPW KFW EMW UV UV +  n h 3 U V +B en./ 7-coumaroylmalic acid 92 17 97 96 nv blue bluecaffeoylmalic acid 79 1 1 89 96 blue green brownferuloylmalic acid 90 15 96 96 blue green bluesinapoylmalic acid 81 8 93 95 blue green greenBAW, butanol/acetic acid/water =  40 /10 /50  (upper layer); BPW, butanol/pyridine/water =  14/3/3; KFW , m ethyliso- butylketone/formic acid/water =  30/10/20 (upper layer); EMW, ethylacetate/m ethanol/water =  200/35/27; Ben., Bene­dicts reagens; nv, not visible.P - C O U M A R O Y L  M A L I C  A C I D  NEC. F A B - M S279Fig. 1. Negativion FAB mass spectrum of/?-coum aroylm alic acid.Bereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46518 W. Brandi et al. • Hydroxycinnamoyl Esters o f  M alic Acid in Small RadishCAFFEOYL MALIC ACID NEG. FAB-HSFig. 2. Negativion FAB mass spectrum of caffeoylm alic acid.FERULOYL MALIC ACID NEG. FAB-nSFig. 3. Negativion FAB mass spectrum o f feruloylmalic acid.Bereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46W. Brandi et al. • Hydroxycinnamoyl Esters o f  M alic Acid in Small Radish 519SINAPOYL MALIC ACID NEG. FAB-MS133Fig. 4. Negativion FAB mass spectrum o f  sinpoylm alic acid.Fig. 5. HPLC chromatogram of Raphanus sativus leaves (“Hilmar”). 1 , caffeoylmalic acid; 2 , p-coumaroylmalic acid; 3, feruloylmalic acid; 4, sinapoylmalic acid.Bereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46Table III. Concentrations o f hydroxycinnamoylmalic acids in Raphanus sativus in ppm.520 W. Brandi et al. • Hydroxycinnamoyl Esters o f  M alic Acid in Small Radish/?-CM CM FM SM 3CQ 5CQ CG p-C  G FG“Hilmar”leaves 43 80 81 56 1 + 6 3 3tuber peeling 4 19 1 0 8 2 + 2 + 2tuber peeled nd 2 2 2 nd nd + nd nd“Riesenbutter”leaves 44 137 8 8 62 2 2 6 2 3tuber peeling 3 53 33 30 nd 1 2 nd 1tuber peeled nd 4 13 6 nd nd nd nd +“Cherry Bell”leaves 79 255 156 60 2 1 7 2 3tuber peeling 1 0 1 1 7 14 nd 1 1 nd 1tuber peeled nd 1 1 nd nd nd + nd +/?-CM, /?-coumaroylmalic acid; CM, caffeoylmalic acid; FM, feruloylmalic acid; SM, sinapoylm alic acid; 3CQ, 3-caffeoyl- quinic acid; 5CQ, 5-caffeoylquinic acid; CG, caffeoylglucose; p-CG, p-coumaroylglucose; FG, feruloylglucose; nd, not de­tected; + , traces.The hydroxycinnam oylm alic acids are present in relative high concentrations in the leaves o f small radish (Table III, Fig. 5). Lower but still consider­able amounts are located in the red coloured peel­ing. In peeled small radish the concentrations are substantially lower, sim ilar to observations at o ther cultivated plants like potatoes [8 ]. As in o ther culti­vated plants the concentrations of these hydroxy­cinnamoyl derivatives m ight be depending on vari­ety. Hydroxycinnamoylquinic acids and glucose were analyzed in addition  to the hydroxycinnam oylm alic acids only in very low concentrations (Table III).AcknowledgementsWe wish to thank Mr. R. R öm er (M edizinische Hochschule Hannover) for recording the H -N M R  spectra. The work was supported  by a grant from Deutsche Forschungsgem einschaft.[1] K. Herrmann, Progr. Chem. Organ. Nat. Products 35, 73 (1978).[2] J. B. Harbome and J. J. Corner, Biochem. J. 81, 242(1961).[3] N. Tkotz and D. Strack, Z. Naturforsch. 35c, 835 (1980).[4] D. Strack, Z. Pflanzenphysiol. 84,139 (1977).[5] M. L. Scarpati and G. Oriente, Gazz. Chim. Ital. 90, 212(1960).[6 ] T. Yoshihara, H. Yoshikawa, S. Sakamura and T. Saku- ma, Agric. Biol. Chem. (Tokyo) 38,1107 (1974).[7] W. Brandi and K. Herrmann, J. Chromatogr. 260, 447(1983).[8 ] W. Brandi and K. Herrmann, Z. Lebensm.-Unters. Forsch. 178,192 (1984).Bereitgestellt von | Technische Informationsbibliothek HannoverAngemeldetHeruntergeladen am | 23.11.17 13:46

Hydroxycinnamoyl esters of malic acid in small radish (raphanus sativus l. var. sativus)

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Institutionelles Repositorium der Leibniz Universität Hannover