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Caffeoylquinic acids from flowers of Arnica montana and Arnica chamissonis
Phytochtmistry, Vol.31, No. 6, pp. 2111 2113, 1992 Printed in Great Britain.
CAFFEOYLQUINIC
0
0031-9422192 SS.OO+O.oO 1992 Pergamon Prcsa Ltd
ACIDS FROM FLOWERS OF ARNICA MON7’&VA AND ARNICA CHAMISSONIS* IRMGARDMERFORT
Institut fiir Pharmazeutische Biologic der Heinrich-Heine-Universitiit Dusseldorf, 4ooo Dtisseldorf, Germany (Received in revised form 12 November 1991)
Key Word Index--Amica montana; A. chamissonis ssp.j&liosa; Asteraceae; catTeoylquinicacids; NMR.
Abstract-Two ctieoylquinic acids were isolated from flowers of Arnica montana and three from A. chamissonis ssp. foliosa var. incana. Their structures were established on the basis of spectral data (UV, ‘H NMR, ‘3C NMR, FABMS) as 1,4,5-tri-0-caffeoylquinic acid, 1,5-di-0-caffeoylquinic acid and 3,4,5-tri-0-caffeoylquinic acid methyl ester. The first compound is a new natural product.
INTRODUCTION Both chlorogenic acid (5O-caffeoylquinic acid) and cynarin (1,3-di-O-caffeoylquinic acid) have been isolated previously from flowers of A. montana, but only chlorogenie acid has been identified in A. c~amissonis ssp. ~~iosa flowers Cl, 21. This paper reports on the isolation and identification of three further caffeoylquinic acids from flowers of A. montana and A. chamissonis ssp. foliosa. One of these is a new natural compound. It should be noted that throughout this article, numbering of the quinic acid ring follows the new IUPAC nomenclature recommendations [3]; for instance chlorogenic acid formerly marked as 3-c~eoylqui~c acid is now labelled as the S-compound. RESULTSANDDISCUSSION Compounds 1 and 2 were isolated from the ethyl acetate soluble portion of an aqueous methanohc extract of the flowers of A. Montana and A. c~a~isso~is ssp.foliosa var. incana. The structure of 1 was established on the basis of spectral data (UV, ‘H NMR, 13C NMR, FAB mass spectrometry) as 1,4,5-tri-0-cafFeoylquinic acid. The UV spectrum of 1 in methanol and after addition of alumimum chloride and alu~nium chlo~de~ydrochloric acid provided preliminary evidence for a cal?eic acid derivative. The ‘HNMR spectrum exhibited signals for three caffeic acid moieties (Table 1). Six doublets with coupling constants of 15.9 Hz appeared for the trans olefinic protons H-7’ and H-8’. The three aromatic protons (H-2, H-S, H-6’) exhibited an ABX system, respectively. The signals of H-3 (equatori~), H-4 (axial) and H-5 (axial) of the quinic acid moiety were assigned according to their multiplicity and their spin-spin coupling constants. The location of caffeoyl substitution on the quinic acid moiety *Part of short lectures, 36 and 37th Annual Congress of the Society for Medicinal Piant Research Freiburg (1988) and Braun~hw~ (1989),respectively, Germany.
i)Rz 1 2 3
R’ R’ H Caffeoyi H Caffeoyl Me H
bR” R3 H H Caffeoyl
R’ CaffeOyi H Caffeoyl
was deduced from the comparative analysis of ‘H NMR chemical shifts of the protons in free quinic acid (see Table 1). Only the signals for the protons H-4 and H-5 were shifted downfield about 1,833 ppm and 1.734 ppm, respectively, thus indicating esterification at C-4 and C-5 141. Therefore the third caffeoyl residue must be attached at C-l. Further evidence for esterification at C-l was provided by the signals for the two protons at C-2. These are isochronous at 62.049 ppm in quinic acid [Sj, but in 1 they become distinct at 2.5173 ppm (H-2,,) and 2.600-2.775 ppm (H-2,, overlapping with H-6, ), as also observed in 1,3-diacyl quinic acids due to deshie?ding [5]. Furthermore the structure of 1 was confirmed by its i3C NMR spectrum (Table 2). The full assignment of all signals in the 13CNMR spectrum was performed by heteronuclear 2D NMR spectroscopy. The molecular mass was determined by positive- and negative- ion FAB mass spectrometry (m/z 679 [M +H]‘, resp. m/z 677 [M-H]-). Comparing these two techniques more information was obtained from the negative-ion FAB mass spectrum. In the last case fragment ions appeared at m/z 5 15 and 353 corresponding to the successive loss of two cafleoyl moieties. The caffeoyl moiety at C-l was not eliminated, as also observed with 1,3-di-O~affeoylquinic acid (cynarin) [6].
2111
2112
I. Table
1. ‘H NMR spectral
data of compounds
1
H
2eq
MERF~RT
1-4 (3OOMHz, *360MHz. Swi~erland)
CD,OD,
4*
3*
2
dd dd m dd td dd dd d, 7.078 d d, 6.801 d dd, 6.968 dd d, 7.596 d d, 6.285 d
2.673 dd 2.588 dd 5.676 m 5.303 dd 5.710 m 2.846 dd 2.146 dd 7.133 d, 7.003 d, 6.998 d 6.805 d, 6.742 d, 6.726 d 7.040 dd, 6.907 dd, 6.907 dd 7.639 d, 7.552 d, 7.536 d 6.422 d, 6.217 d, 6.156 d 3.642s
2.6OtX2.775 m 2.517 dd 4.511 m 5.224 dd 5.731 t 2.600-2.775 m 2.223 dd 7.115 d, 7.042 d, 7.042 d 6.821 d, 6.771 d, 6.742 d 7.002 dd, 6,925 dd, 6.925 dd 7.648 d, 7.640 d, 7.565 d 6.379 d, 6.326 d, 6.230 d
2.513 2.438 4.326 3.820 5.423 2.608 2.091 7.078 6.801 6.968 7.603 6.321
J zeq.3 J la&J J 2eq.2sx
3.4 3 15
3.4 4.4 15.3
3.4 3.2 16.1
13.0
3.2 9 3.7 9.9 13.4 1.8, 1.8, 1.8
3.4 8.1 3.9 9.0 13.5 1.9, 1.9
3.5 10.0 4.0 10.5 13.3 2, 2, 2
8.1, 8.1, 8.1 15.9, 15.9, 15.9
8.1, 8.1 15.9, 15.9
8.2, 8.2, 8.2 15.9, 15.9, 15.9
2ax 3 4 5 6eq 6ax 2’ 5 6 7 8 OMe
J 4.5 J 5,6cq J J.6SX J tq.(isx J f'.6' J 1’,6’ J T.8’
Table
2. 13CNMR
c
spectra
of compounds l-3 (CD,OD MHz)
1
1’ 2’ 3’ 4 5 6 7 8’ 9’ -CO,& *Assignment
80.7 35.9 67.8 75.3 68.6 37.7 174.6 127.8, 127.7, 127.6 115.2, 115.2, 115.2 146.7, 146.6, 146.6 149.6, 149.6, 149.6 116.5, 116.5, 116.5 123.2, 123.2, 123.2 147.7, 147.7, 147.6 115.2, 114.7, 114.6 168.6, 168.3, 168.1
quinic acid Fluka,
20491 4.090 3.391 3.997 2.126 1.856
m dd td dd dd
3.4 3.4
3.2 9.1 4.7 10.9 13.2
*Spin-echo,
2*
75 MHz, 90.52
3
80.9 35.6 69.4 72.8 71.5 36.9 174.9 127.7, 127.7 115.2, 115.2” 146.7, 146.7 149.5, 149.5 116.5, 116.5 123.2, 123.1 147.5, 147.3 115.2”, 115.0 168.7, 168.0
(+) (+I (-) i-1 f-1
c+t
(+) (+) (-) (+) (+f 1-f
t-1 (-) (-) (+)
73.5 32.9 71.3 73.5 68.3 38.6 167.3 127.8, 127.8, 127.7 115.5”. 115.3”. 115.3” 146.9, 146.X, 146.8 149.9, 149.8, 149.8 i16.6, lf6.6, 116.6 123.3, 123.2. 123.2 147.9, 147. 9, 147.9 115.3”, 114.5, 114.4 168.3, 168.1, 167.8 53.1
interchangeable.
Compound 2 was identified as 1,5-di-O-caffeoylquinic acid (UV, specific rotation, FAB mass spectrometry, ‘H NMR and t3CNMR). From the ‘HNMR spectrum (Table 1) two caffeoyl residues esterified at position C-l and C-5 of quinic acid were deduced. Only the signal for the proton at C-5 shifted downfield about 1.426 ppm
compared to free quinic acid indicating that one caffeic acid residue was attached to the hydroxyl at C-5, the other at C-l. The two protons at C-2 lost their magnetic equivalence. They became distinct as in 1,3-diacyl quinic acids, but not in 1,5-d~substituted quinic acid derivatives with a methoxyl group at C-l f5]. The structure was
CaiTeoylquinicacids from Arnica species confirmed by the 13C NMR spectrum (Table 2). The spin echo spectrum showed the downfield shift of the quatemary C- 1 of quinic acid, compared to the corresponding Cl of chlorogenic acid (5-0-caffeoylquinic acid) [7]. A full assignment of the other carbons of quinic acid was only possible after heteronuclear 2D NMR spectroscopy. In the field desorption mass spectrum a molecular ion at m/z 516 with low intensity was observed, after loss of Hz0 a base peak at m/z 498. The molecular mass was therefore determined by positive- and negative-ion FAB mass spectrometry (m/z 517 [M+H]+, resp. m/z 515 [M-H] -). Only one caffeoyl residue was eliminated, resulting in an ion at m/z 353. Compound 3 was isolated only from the flower extract of A. chamissonis ssp. foliosa var. incana. Its structure was established as the methyl ester of 3,4,5-tri-O-caffeoylquinic acid by UV, ‘H NMR and 13C NMR data (Tables 1 and 2). The methyl signal observed at 3.642 ppm in the ‘HNMR and at 53.082 ppm in the 13C NMR spectrum was unequivocally associated with an ester rather than an aromatic ether, because otherwise both signals would be shifted downfield [S]. Furthermore the signal from C-7 shifted upfield about 7 ppm compared to those in 1 and 2 (Table 2). Whereas 2 has been found previously in other Asteraceae species [9], 1 is a new natural product. This is only the second report of a tricaffeoylquinic acid in nature. The first was isolated from Chrysothamnus paniculatus (Asteraceae) and identified as 3,4,5-tri-O-caffeoylquinic acid [lo], the methyl ester of which (3) was obtained from flowers of A. chamissonis ssp. foliosa var. incana. The presence of 1 and 2, but not 3, was confirmed by TLC and HPLC analysis of fresh flowers of both Arnica species. It is supposed that 3 normally occurs in the free form, as in Chrysothamnus paniculatus, and is formed by esterification with methanol during preparation of the extract. Cynarin (1,3-di-O-caffeoylquinic acid), which has been reported from flowers of A. montana [2], could not be detected by TLC and HPLC analysis either in dried or in fresh flowers. In all probability cynarin is an artifact which is formed during isolation by acyl esterification from 1,5-di-0-caffeoylquinic acid. Artifact formation of this kind has been shown already in the artichoke [ 111. EXPERIMENTAL 1 H and I3 C NMR: 300 (360) and 75 (90) MHz, respectively; spin-echo experiment: 100 MHz; TMS (S=O) as int. standard.
Pos. and neg. FABMS: a methanolic solution of the samples were dissolved in a glycerol matrix and placed to bombardment with Xe atoms of energy 8 kV. Plant material See ref. [12]. Isolation. 16.6g of the EtOAc extract from flowers of A. montana(total 25 & preparation see ref. [12]) were separated into 11 fractions by RLCC [descending, solvent system CHCl,-n-
PHYTO 31:6-S
2113
BuOH-MeOH-H,O (10: 1: 10: 6), flow rate 1.8, rotation 5, angle 30-j. CC of fraction 5 on Sephadex LH-20 with MeOH gave 1 (72 mg). From fraction 7 2 (203 mg) was isolated by RLCC (ascending, EtOAc-n-PrOH-H,O (4: 2 : 7)) and CC on Sephadex LH-20 with MeOH. 13 g of the EtOAc flower extract of A. chamissonisssp.foliosa var. incana(total 24 g, preparation see ref. [12]) were chromatographed on Sephadex LH-20 (Pharmacia) with MeOH yielding 16 fractions. Repeated CC of fraction 9 on cellulose with EtOAc-petrol (3:2), saturated with H,O and increasing amounts of EtOAc and on Sephadex LH-20 with MeOH afforded 1 (51 mg), 2 (13 mg) and 3 (10 mg). High pressure liquidchromatography. Hyperchrome SC 125 x 4.6 mm column packed with Shandon ODS Hypersil 5 pm (Bischoff); flow rate: 1.5ml min-‘; UV detector at 325 nm; mobile phase: M&H (A), H,O-MeOH-HOAc (10:39:1) (B) O-7 min: 10% A, isocratic, 7-25 min: lo-46% A, linear gradient; R, (min:sech 1=20:36,2=8:0,3=21.12, cynarin (Roth)=6:0. 1,4,5-Tri-0-ca~~oylqatinic acid (1) UV,Igp nm: 235, 243, 3OOsh, 327;+NaOMe 260, 307, 373; + AlCl, 263, 3lOsh, 363; + AlCl, + HCI, 235&245,3OOsh, 330. Acknowledgements-We are gratetul to Dr A. Steigel (Institut fiir Organ&he und Makromolekulare Chemie, Heinrich-HeineUniversitiit Diisseldorf) for taking the 300 (resp. 75) MHz NMR spectra, Dr D. Wend&h (Zentralbereich Forschung und Entwicklung der Bayer AG, Leverkusen) for the 360 (resp. 90) MHz spectra, Professor Dr S. Beqer (Fachbereich Chemie der Philipps Universitit Marburg) for the 100 MHz spin-echo spectrum and Dr E. Schriider (Finn&n MAT GmbH, Bremen) for the FAB mass spectra.
REFERENa 1. Kotialewski, Z., Skrzypczakowa, L. and Matlawaska, I. (1969) Dbs. Pharm. Pharmacol. 21, 249. 2. Nikonov, G. K. (1961) Tr. Vses. Nauchno. -Med. Inst. Lek. Aromat.Rast. 11, 169. 3. IUPAC (1976) Biochem.J. 153,23. 4. Corse, J., Lundin, R. E., Sandheimer, E. and Waiss, A. C. (1966) Phytochemistry5,767. 5. Haslam, E. and Turner, M. J. (1971) J. Chem Sot. (C) 1496. 6. Sakushima, A., Hisada, S., Nishibe, S. and Brandenberger, H. (1985) Phytochernistry24, 325. 7. Kelly, C. J., Harruff, R. C. and Cannack, M. (1976) J. Org. Chem. 41,449. 8. Calis, I., Gross, G.-A. and Sticher, 0. (1987) Phytochemistry 26,2057. 9. Nichiforescu, E. A. (1970) Plant. Med. Phytother.4, 56. 10. Timmermann, B. N., Hoffmann, J. J., Jolad, S. D., Schram, K. H., Klenck, R. E. and Bates, R. B. (1983) .I. Nat. Prod. 46, 367. 11. Panizzi, L. and Scarpati, M. L. (1965) Gazz. Chim. Ital. 95,71. 12. Merfort, I. (1984) Planta Med., 107.
CAFFEOYLQUINIC
0
0031-9422192 SS.OO+O.oO 1992 Pergamon Prcsa Ltd
ACIDS FROM FLOWERS OF ARNICA MON7’&VA AND ARNICA CHAMISSONIS* IRMGARDMERFORT
Institut fiir Pharmazeutische Biologic der Heinrich-Heine-Universitiit Dusseldorf, 4ooo Dtisseldorf, Germany (Received in revised form 12 November 1991)
Key Word Index--Amica montana; A. chamissonis ssp.j&liosa; Asteraceae; catTeoylquinicacids; NMR.
Abstract-Two ctieoylquinic acids were isolated from flowers of Arnica montana and three from A. chamissonis ssp. foliosa var. incana. Their structures were established on the basis of spectral data (UV, ‘H NMR, ‘3C NMR, FABMS) as 1,4,5-tri-0-caffeoylquinic acid, 1,5-di-0-caffeoylquinic acid and 3,4,5-tri-0-caffeoylquinic acid methyl ester. The first compound is a new natural product.
INTRODUCTION Both chlorogenic acid (5O-caffeoylquinic acid) and cynarin (1,3-di-O-caffeoylquinic acid) have been isolated previously from flowers of A. montana, but only chlorogenie acid has been identified in A. c~amissonis ssp. ~~iosa flowers Cl, 21. This paper reports on the isolation and identification of three further caffeoylquinic acids from flowers of A. montana and A. chamissonis ssp. foliosa. One of these is a new natural compound. It should be noted that throughout this article, numbering of the quinic acid ring follows the new IUPAC nomenclature recommendations [3]; for instance chlorogenic acid formerly marked as 3-c~eoylqui~c acid is now labelled as the S-compound. RESULTSANDDISCUSSION Compounds 1 and 2 were isolated from the ethyl acetate soluble portion of an aqueous methanohc extract of the flowers of A. Montana and A. c~a~isso~is ssp.foliosa var. incana. The structure of 1 was established on the basis of spectral data (UV, ‘H NMR, 13C NMR, FAB mass spectrometry) as 1,4,5-tri-0-cafFeoylquinic acid. The UV spectrum of 1 in methanol and after addition of alumimum chloride and alu~nium chlo~de~ydrochloric acid provided preliminary evidence for a cal?eic acid derivative. The ‘HNMR spectrum exhibited signals for three caffeic acid moieties (Table 1). Six doublets with coupling constants of 15.9 Hz appeared for the trans olefinic protons H-7’ and H-8’. The three aromatic protons (H-2, H-S, H-6’) exhibited an ABX system, respectively. The signals of H-3 (equatori~), H-4 (axial) and H-5 (axial) of the quinic acid moiety were assigned according to their multiplicity and their spin-spin coupling constants. The location of caffeoyl substitution on the quinic acid moiety *Part of short lectures, 36 and 37th Annual Congress of the Society for Medicinal Piant Research Freiburg (1988) and Braun~hw~ (1989),respectively, Germany.
i)Rz 1 2 3
R’ R’ H Caffeoyi H Caffeoyl Me H
bR” R3 H H Caffeoyl
R’ CaffeOyi H Caffeoyl
was deduced from the comparative analysis of ‘H NMR chemical shifts of the protons in free quinic acid (see Table 1). Only the signals for the protons H-4 and H-5 were shifted downfield about 1,833 ppm and 1.734 ppm, respectively, thus indicating esterification at C-4 and C-5 141. Therefore the third caffeoyl residue must be attached at C-l. Further evidence for esterification at C-l was provided by the signals for the two protons at C-2. These are isochronous at 62.049 ppm in quinic acid [Sj, but in 1 they become distinct at 2.5173 ppm (H-2,,) and 2.600-2.775 ppm (H-2,, overlapping with H-6, ), as also observed in 1,3-diacyl quinic acids due to deshie?ding [5]. Furthermore the structure of 1 was confirmed by its i3C NMR spectrum (Table 2). The full assignment of all signals in the 13CNMR spectrum was performed by heteronuclear 2D NMR spectroscopy. The molecular mass was determined by positive- and negative- ion FAB mass spectrometry (m/z 679 [M +H]‘, resp. m/z 677 [M-H]-). Comparing these two techniques more information was obtained from the negative-ion FAB mass spectrum. In the last case fragment ions appeared at m/z 5 15 and 353 corresponding to the successive loss of two cafleoyl moieties. The caffeoyl moiety at C-l was not eliminated, as also observed with 1,3-di-O~affeoylquinic acid (cynarin) [6].
2111
2112
I. Table
1. ‘H NMR spectral
data of compounds
1
H
2eq
MERF~RT
1-4 (3OOMHz, *360MHz. Swi~erland)
CD,OD,
4*
3*
2
dd dd m dd td dd dd d, 7.078 d d, 6.801 d dd, 6.968 dd d, 7.596 d d, 6.285 d
2.673 dd 2.588 dd 5.676 m 5.303 dd 5.710 m 2.846 dd 2.146 dd 7.133 d, 7.003 d, 6.998 d 6.805 d, 6.742 d, 6.726 d 7.040 dd, 6.907 dd, 6.907 dd 7.639 d, 7.552 d, 7.536 d 6.422 d, 6.217 d, 6.156 d 3.642s
2.6OtX2.775 m 2.517 dd 4.511 m 5.224 dd 5.731 t 2.600-2.775 m 2.223 dd 7.115 d, 7.042 d, 7.042 d 6.821 d, 6.771 d, 6.742 d 7.002 dd, 6,925 dd, 6.925 dd 7.648 d, 7.640 d, 7.565 d 6.379 d, 6.326 d, 6.230 d
2.513 2.438 4.326 3.820 5.423 2.608 2.091 7.078 6.801 6.968 7.603 6.321
J zeq.3 J la&J J 2eq.2sx
3.4 3 15
3.4 4.4 15.3
3.4 3.2 16.1
13.0
3.2 9 3.7 9.9 13.4 1.8, 1.8, 1.8
3.4 8.1 3.9 9.0 13.5 1.9, 1.9
3.5 10.0 4.0 10.5 13.3 2, 2, 2
8.1, 8.1, 8.1 15.9, 15.9, 15.9
8.1, 8.1 15.9, 15.9
8.2, 8.2, 8.2 15.9, 15.9, 15.9
2ax 3 4 5 6eq 6ax 2’ 5 6 7 8 OMe
J 4.5 J 5,6cq J J.6SX J tq.(isx J f'.6' J 1’,6’ J T.8’
Table
2. 13CNMR
c
spectra
of compounds l-3 (CD,OD MHz)
1
1’ 2’ 3’ 4 5 6 7 8’ 9’ -CO,& *Assignment
80.7 35.9 67.8 75.3 68.6 37.7 174.6 127.8, 127.7, 127.6 115.2, 115.2, 115.2 146.7, 146.6, 146.6 149.6, 149.6, 149.6 116.5, 116.5, 116.5 123.2, 123.2, 123.2 147.7, 147.7, 147.6 115.2, 114.7, 114.6 168.6, 168.3, 168.1
quinic acid Fluka,
20491 4.090 3.391 3.997 2.126 1.856
m dd td dd dd
3.4 3.4
3.2 9.1 4.7 10.9 13.2
*Spin-echo,
2*
75 MHz, 90.52
3
80.9 35.6 69.4 72.8 71.5 36.9 174.9 127.7, 127.7 115.2, 115.2” 146.7, 146.7 149.5, 149.5 116.5, 116.5 123.2, 123.1 147.5, 147.3 115.2”, 115.0 168.7, 168.0
(+) (+I (-) i-1 f-1
c+t
(+) (+) (-) (+) (+f 1-f
t-1 (-) (-) (+)
73.5 32.9 71.3 73.5 68.3 38.6 167.3 127.8, 127.8, 127.7 115.5”. 115.3”. 115.3” 146.9, 146.X, 146.8 149.9, 149.8, 149.8 i16.6, lf6.6, 116.6 123.3, 123.2. 123.2 147.9, 147. 9, 147.9 115.3”, 114.5, 114.4 168.3, 168.1, 167.8 53.1
interchangeable.
Compound 2 was identified as 1,5-di-O-caffeoylquinic acid (UV, specific rotation, FAB mass spectrometry, ‘H NMR and t3CNMR). From the ‘HNMR spectrum (Table 1) two caffeoyl residues esterified at position C-l and C-5 of quinic acid were deduced. Only the signal for the proton at C-5 shifted downfield about 1.426 ppm
compared to free quinic acid indicating that one caffeic acid residue was attached to the hydroxyl at C-5, the other at C-l. The two protons at C-2 lost their magnetic equivalence. They became distinct as in 1,3-diacyl quinic acids, but not in 1,5-d~substituted quinic acid derivatives with a methoxyl group at C-l f5]. The structure was
CaiTeoylquinicacids from Arnica species confirmed by the 13C NMR spectrum (Table 2). The spin echo spectrum showed the downfield shift of the quatemary C- 1 of quinic acid, compared to the corresponding Cl of chlorogenic acid (5-0-caffeoylquinic acid) [7]. A full assignment of the other carbons of quinic acid was only possible after heteronuclear 2D NMR spectroscopy. In the field desorption mass spectrum a molecular ion at m/z 516 with low intensity was observed, after loss of Hz0 a base peak at m/z 498. The molecular mass was therefore determined by positive- and negative-ion FAB mass spectrometry (m/z 517 [M+H]+, resp. m/z 515 [M-H] -). Only one caffeoyl residue was eliminated, resulting in an ion at m/z 353. Compound 3 was isolated only from the flower extract of A. chamissonis ssp. foliosa var. incana. Its structure was established as the methyl ester of 3,4,5-tri-O-caffeoylquinic acid by UV, ‘H NMR and 13C NMR data (Tables 1 and 2). The methyl signal observed at 3.642 ppm in the ‘HNMR and at 53.082 ppm in the 13C NMR spectrum was unequivocally associated with an ester rather than an aromatic ether, because otherwise both signals would be shifted downfield [S]. Furthermore the signal from C-7 shifted upfield about 7 ppm compared to those in 1 and 2 (Table 2). Whereas 2 has been found previously in other Asteraceae species [9], 1 is a new natural product. This is only the second report of a tricaffeoylquinic acid in nature. The first was isolated from Chrysothamnus paniculatus (Asteraceae) and identified as 3,4,5-tri-O-caffeoylquinic acid [lo], the methyl ester of which (3) was obtained from flowers of A. chamissonis ssp. foliosa var. incana. The presence of 1 and 2, but not 3, was confirmed by TLC and HPLC analysis of fresh flowers of both Arnica species. It is supposed that 3 normally occurs in the free form, as in Chrysothamnus paniculatus, and is formed by esterification with methanol during preparation of the extract. Cynarin (1,3-di-O-caffeoylquinic acid), which has been reported from flowers of A. montana [2], could not be detected by TLC and HPLC analysis either in dried or in fresh flowers. In all probability cynarin is an artifact which is formed during isolation by acyl esterification from 1,5-di-0-caffeoylquinic acid. Artifact formation of this kind has been shown already in the artichoke [ 111. EXPERIMENTAL 1 H and I3 C NMR: 300 (360) and 75 (90) MHz, respectively; spin-echo experiment: 100 MHz; TMS (S=O) as int. standard.
Pos. and neg. FABMS: a methanolic solution of the samples were dissolved in a glycerol matrix and placed to bombardment with Xe atoms of energy 8 kV. Plant material See ref. [12]. Isolation. 16.6g of the EtOAc extract from flowers of A. montana(total 25 & preparation see ref. [12]) were separated into 11 fractions by RLCC [descending, solvent system CHCl,-n-
PHYTO 31:6-S
2113
BuOH-MeOH-H,O (10: 1: 10: 6), flow rate 1.8, rotation 5, angle 30-j. CC of fraction 5 on Sephadex LH-20 with MeOH gave 1 (72 mg). From fraction 7 2 (203 mg) was isolated by RLCC (ascending, EtOAc-n-PrOH-H,O (4: 2 : 7)) and CC on Sephadex LH-20 with MeOH. 13 g of the EtOAc flower extract of A. chamissonisssp.foliosa var. incana(total 24 g, preparation see ref. [12]) were chromatographed on Sephadex LH-20 (Pharmacia) with MeOH yielding 16 fractions. Repeated CC of fraction 9 on cellulose with EtOAc-petrol (3:2), saturated with H,O and increasing amounts of EtOAc and on Sephadex LH-20 with MeOH afforded 1 (51 mg), 2 (13 mg) and 3 (10 mg). High pressure liquidchromatography. Hyperchrome SC 125 x 4.6 mm column packed with Shandon ODS Hypersil 5 pm (Bischoff); flow rate: 1.5ml min-‘; UV detector at 325 nm; mobile phase: M&H (A), H,O-MeOH-HOAc (10:39:1) (B) O-7 min: 10% A, isocratic, 7-25 min: lo-46% A, linear gradient; R, (min:sech 1=20:36,2=8:0,3=21.12, cynarin (Roth)=6:0. 1,4,5-Tri-0-ca~~oylqatinic acid (1) UV,Igp nm: 235, 243, 3OOsh, 327;+NaOMe 260, 307, 373; + AlCl, 263, 3lOsh, 363; + AlCl, + HCI, 235&245,3OOsh, 330. Acknowledgements-We are gratetul to Dr A. Steigel (Institut fiir Organ&he und Makromolekulare Chemie, Heinrich-HeineUniversitiit Diisseldorf) for taking the 300 (resp. 75) MHz NMR spectra, Dr D. Wend&h (Zentralbereich Forschung und Entwicklung der Bayer AG, Leverkusen) for the 360 (resp. 90) MHz spectra, Professor Dr S. Beqer (Fachbereich Chemie der Philipps Universitit Marburg) for the 100 MHz spin-echo spectrum and Dr E. Schriider (Finn&n MAT GmbH, Bremen) for the FAB mass spectra.
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