Epicuticular Flavonoids of Some Scrophulariaceae

Eckhard Wollenweber 3 *, Marion Dörra and James N. Roitmanb a Institut für Botanik der Technischen Universität, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany. Fax: 06151/166878. E-mail: [email protected] b Western Regional Research Center, USDA-ARS, 800 Buchanan St., Albany, CA 94710, U. S. A. * Author for correspondance and reprint requests Z. Naturforsch. 55c, 5-9 (2000); received October 14, 1999 Scrophulariaceae, Lipophilic Exudates, Flavonoid Aglycones, Flavone Isobutyrate Twenty-two species of Scophulariaceae have been found to accumulate flavonoid aglycones externally on their leaves and stems. They belong to the genera Anarrhinum, Antirrhinum, Asarina, Calceolaria, Mimulus, and Odontites. Most of the flavonoids are methylated flavones and flavonols, some with 6-0 and/or 8-O-substitution. One of them is the natural isobutyryl ester of a rare flavone.

Introduction the lipophilic exudate material accumulated on leaf and stem surfaces. The concentrated material Scrophulariaceae have so far only rarely been was defatted (MeOH, -10°, centrifugation) and reported to exhibit externally accumulated flavo passed over Sephadex LH-20, eluted with MeOH, noid aglycones. Wollenweber recently (1995) listed to separate the flavonoids from the predominant the genera Diplacus, Mimulus and Odontites. In terpenoids. Comparative TLC of Sephadex frac addition, we have now studied one Anarrhinum tions with authentic substances allowed the identi species, six Antirrhinum, two Asarina, five Cal- fication of most of the flavonoids present. Frac ceolara, and two M imulus species for the occur tions were monitored and comparisons with rence of such products, because their aerial parts markers were done by TLC on silica with tolu are more or less glandulous and sometimes sticky ene - MeCOEt 9:1 v/v and with toluene - diox- to the touch. We found a series of more or less ane - H O A c 18:5:1 v/v/v. For TLC on polyam ide trivial flavones and flavonols, several less abun dant flavonoids, and a novel natural flavone ester. DC-11 we used toluene - petrol 10o -i 4o - M eC O E t - M eO H 12:6:2:1 v/v/v/v, toluene - diox- ane - M eO H 8:1:1 v/v/v and toluene - M eC Material and Methods O E t - M eO H 12:5:3 v/v/v. Chrom atogram s were Anarrhinum forskalii (J. F. Gmel.) Cufod., A n viewed under UV before and after spraying with tirrhinum barrelieri Bor., A. braun-blanquetii “Naturstoffreagenz A”. Terpenoids were visual Rothm ., A. graniticum Rothm., A. hispanicum ized by spraying silica plates with MnCl 2 reagent, Chav., A. majus L. ssp. cirrhigerum (Ficalho) followed by heating (Jork et al., 1989). NMR Franco, A. nutallii Benth., Asarina barklaiana spectra were recorded in DMSO-d 6 or acetone-d 6 (Lindl.) Pennell, A. procumbens Mill. [syn. A ntir at 400 M Hz (!H ) and 100 MHz (13C) on a Bruker rhinum asarina L.], Calceolaria arachnoidea R. ARX-400 instrument. Synthetic samples of 5,6,4'- Grah., C. chelidonioides H. B. K., C. irazuensis trihydroxy-7,8-dimethoxyflavone and 5,8,4'-trihy- J. D.Smith, C. mexicana Benth.-, C. pavonii Benth., droxy-6,7-dimethoxy flavone were obtained from C. scabiosaefolia Sims, C. tenella Poepp. et Endl., T. H orie ( Horie et al., 1995). C. tripartita Ruiz et Pav., Mimulus moschatus In the case of Asarina procumbens , the flavo Dougl. ex Lindl., and M. pilosus (Benth.) S.Wats, noid portion was subjected to column chromatog were cultivated in the Botanischer Garten der TU raphy on polyamide SC- 6 . A minute amount of Darmstadt. Aerial parts were collected from flow the major flavonoid was isolated by subsequent ering plants, or just after the flowers had faded. preparative TLC on silica. This compound (1) ap They were briefly rinsed with acetone to dissolve pears as a dark spot on TLC which remains dark

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 6 E. Wollenweber et al. ■ Flavonoids from Scrophulariaceae on spraying with Naturstoffreagenz A. MS m /z (% 2' 3' 2' 3' H °x 7 8 O^ H0X 7 * rel. int.): 400 (20%, M+), 330 (100, M+- (CH 3)2C= i I 1 1 11' V f v C=0), 315 (45, M+- (CH 3)2C = C =0 - C H 3), 297 J3 p l r [5 fp OH (17). NMR (d 6-acetone) 6 12.90, s (5-OH); Flavones HO 0 HO 0 Flavonols 7.85, d, J = 8 Hz (H-2' and H-6 '); 7.00, d, / = 8 Hz (H-3' and H-5'); 6.67, s (H-3); 4.01, s (7-OMe); Flavones: 3.93, s ( 8 -OMe).; 3.01, pentet, J - 7 Hz (H-2"); 5,7-OH-flavone: chrysin 1.37, d, J = 7 Hz (H-3" and H-4"). 13C NMR ö 5,7,4'-OH-flavone: apigenin 5,6J,4'-OH-flavone: scutellarein 165.5 (C-2); 104.1 (C-3); 183.5 (C-4); 152.4 (C-5); 5,7,8,4'-OH-flavone: isoscutellarein 124.2 (C-6 ); 152.7 (C-7); 137.4 (C-8 ); 145.3 (C-9); 5,7,3',4'-tetraOH-flavone: luteolin 5,6,7,3',4'-OH-flavone: 6-OH-luteolin 107.2 (C-10); 123.0 (C-l'); 129.3 (C-2' and C- 6 '); 5-OH-6,7,8,3',4'-OMe-flavone: 5-desmethylnobiletin 116.9 (C-3' and C-5'); 162.3 (C-4'); 61.5 (7-OMe); 5,4'-OH-7,8-OMe-6-0-isobutyryl-flavone: comp. 1 60.7 (8 -OM e); 174.4 (C-l"); 33.1 (C-2"); 18.9 (C-3" Flavonols: and C-4"). 3.5 J,4'-OH-flavone: kaempferol From Antirrhinum graniticum, small amounts of 3.5 J,8,4'-OH-flavone: herbacetin two crystalline non-polar flavonoids were isolated 3,5,7,3'-4'-OH-flavone: quercetin by prep. TLC of relevant Sephadex fractions on 5-OH-3,6,7,8,4'-OMe-flavone: 5-OH-auranetin silica. The molecular weights of both compounds 3,5,7,8,3',4'-OH-flavone: gossypetin were determined to be 388 by EIMS (M+ with m / z 388), indicating flavones or flavonols each with (loss of dimethyl ketene), suggesting that 1 con 1 OH and 5 OMe groups. By direct comparisons tains a four carbon acyl moiety. The proton NMR with markers, one of them was readily identified spectrum had signals corresponding to a 5-OH fla to be 5-OH-6,7,8,3',4'-pentamethoxy flavone (5- vone with a fully substituted A ring, a para substi desmethyl-nobiletin). The second product (comp. tuted B ring and a 2-propyl moiety (methyl 2) was identified by its NMR spectrum as 5-OH- doublet and methine pentet). The carbon 3,6,7,8,4'-pentamethoxy flavone (5-hydroxy-aura- spectrum displayed B and C ring signals virtually netin) (see Discussion). identical to those of other 4'-OH flavones, con A series of further Scrophulariaceae have been firmed the presence of an isobutyryl moiety and checked for the presence of exudate flavonoids. two sterically compressed OMe’s. The A ring of 1 Some of them exhibit a glandular exudate, but therefore has a 5-OH, two OMe’s and a second seem to be devoid of flavonoids, nonetheless. In OH esterified with isobutyric acid. The carbon sig Alonsoa acutifolia, Antirrhinum orontium, Antir nals can be assigned to accommodate either of two rhinum siculum, Asarina antirrhinifolia, Asarina isomeric flavones: 5 ,4 '-di0 H-6 ,7 -di0 M e-8 -0 -is0 - lophospermum, Calceolaria cana, Collinsia hetero- butyryl flavone or 5,4'-diOH-7,8-diOMe-6-isobu- phylla, and Paulownia tomentosa no external fla- tyryl flavone. Long range H-C coupling experi vonoid aglycones were detected. In Antirrhinum ments revealed interactions between OH-5 and sempervirens, Calceolaria glandulifera, C. pavoni, the C-3'74'’ methyl carbons of the isobutyl group, C. pinnata and others there are only traces of fla which indicates that the isobutyryl substituent is vonoids present. attached to C - 6 and not to C- 8 . Compound 1 was therefore identified as 5,4'-dihydroxy-7,8-dimeth- Results and Discussion oxy- 6 -O-isobutyryl flavone. Core structures of the flavones and flavonols A small sample of 1 was refluxed in acetic acid found in this study are presented in Fig. 1. In the to which a few drops of HC1 had been added; after text, abbreviations OH = hydroxy-group, OMe = 14 hrs a sample removed and analyzed by TLC methoxy group, and Me = methyl ether are used showed that a considerable amount of unchanged throughout. 1 remained and reflux was continued for an addi tional 48 hrs. NMR spectra of the purified product Structure elucidation (LH-20 in CHC13) showed that two isomeric fla The base peak in the MS of compound 1, from vones had been formed: 5,8,4'-triOH-6,7-diOMe Asarina procumbens , is a fragment ion at M+-7 0 flavone and 5,6,4'-triOH-7,8-diOMe flavone, pre- E. Wollenweber et al. ■ Flavonoids from Scrophulariaceae 7 sumably via acid-catalyzed opening and closing of laria sepcies (Wollenweber et al., 1989b) , one Mi- the C ring. The latter product was, indeed, ob m ulus and two Odontites species (Wollenweber, tained by acid treatment of the former by Ferreres 1990). In the literature, the occurrence of flavone et al. (1985). and flavonol aglycones has also been reported for 5,6,4'-triOH-7,8-diOMe-flavone has been species belonging to the genera Adenosma, Digi known previously as “thymusin” (see e.g. Hernan talis, Hebe, Kickxia, Limnophila, Pedicularis, Rhi- dez et al., 1987). Its 6 -O -isobutyrate 1, however, is nanthus, Scoparia, Striga, Verbascum, and Veron a novel natural flavone. Besides chrysin-7-O-ben- ica. However, since in these reports no mention is zoate from Baccharis bigelovii (Arriaga-Giner et made of the flavonoid localization, they must not al., 1986) and the 5'-0-acetate of 2',5'.dihydrox- be considered here. Plant material was “perco yflavone from Primula pulverulenta (Wollenweber lated with ethanol“ (Imre et al., 1984: Digitalis), or et al., 1988), compound 1 is the only other natu “cut and extracted with methanol under reflux“ rally occurring ester of a flavone that has thus far (Nakanishi et al., 1985: Striga). In our lab, Digitalis been found. ferruginea, D. luta and D. purpurea were found de The molecular weight of compound 2 from A n void of external flavonoids. Phuong et al. (1997) tirrhinum graniticum (M+ 388 by APCI-MS) was found methoxylated flavones in the hexane (!) ex consistent with a flavone bearing one hydroxy and tract of dried Adenosma capitatum\ these are five methoxy substituents. MS-MS fragmentation likely, therefore, to be exudate constituents. Bohm of the [MH]+ ion with m /z 389 yielded daughter (1992) reported the external occurrence of some ion fragments with m /z 373 [MH-CH4]+ (9%), 359 flavonol aglycones in the leaf exudate of M imulus [MH-2CH3]+ (100%), 341 [359-H 20 ] + (37% ), 331 lewisii. Only once has a trivial flavanone been [359-CO]+ (25%), 316 [331-CH3]+ (22%), and m /z found in Scrophulariaceae, namely sakuranetin 135 [B-ring]+ (23%). The latter fragment ion (naringenin-7-Me) in Hebe cupressoides (Perry pointed to a B-ring with one methoxy substituent, and Foster, 1994). For Diplacus aurantiacus, the which was confirmed by !H NMR analysis. The fi existence of C10-substituted flavanones and dihi- ring protons gave rise to two doublets (öH 8.07 droflavonols in leaf resin has been reported (Lin and 8 H 7.20 d , both J = 8 .8 Hz, H-276' and H-37 coln and Walla, 1986; Wollenweber et al., 1989c), 5') which were shifted downfield compared to the while for Mimulus clevelandii it can only be as B-ring resonances of kaempferol ( 8 H 8.06 d and sumed that the geranyl flavanones and geranyl di- 8 H 6.94 d), indicating O-methylation of the para hydroflavonols found (Phillips et al., 1996: “whole hydroxyl. The low-field resonance at 8 H 12.43 was plant“) are exudate constituents. taken as evidence for a free hydroxyl at C-5. These Table I comprises 9 species from 3 genera in the assignments left the five methoxy groups ( 6 H 4.03, Scrophularioideae-Antirrhinae ( Anarrhinum, An 3.90, 3.88, 3.83, and 8 H 3.82) to be placed at car tirrhinum and Asarina), 8 species of the genus Cal bons 3, 6 , 7, 8 , and 4'. The compound was there ceolaria in the Scrophularioideae-Calceolariae, 3 fore identified as 5-hydroxy-3,6,7,8,4'-penta- species of M imulus in the Scrophularioideae-Grat- methoxy flavone, trivially known as 5-hydroxy ioleae, and 2 species of Odontites in the Rhinan- auranetin. Its identity was confirmed by direct thoideae-Rhinantheae. Anarrhinum, Asarinum comparison with an authentic sample. This flavo- and Antirrhinum show a rather uniform pattern of nol has previously been known from several taxo- flavones, with the exception of Antirrhinum granit nomically independent plants, including e.g. Pola- icum which exhibits a higly methylated flavone (5- nisia trachysperma (Wollenweber et al., 1989a) and desmethyl-nobiletin) plus a highly methylated fla Rosa centifolia (Wollenweber et al., 1993). vonol (5-hydroxyauranetin), and Asarina procumbens, which produces a flavone ester (comp. 1 ). Flavonols are predominant in Calceolaria-, how Chemotaxonomy ever C. irazuensis shows three flavones. Several 8 - The exudate flavonoid patterns of the Scrophu O-substituted products are found in this genus, lariaceae we studied are presented in Table I. In two of them being flavones. In Mimulus, the flavo addition to the results of the present study, we in noid profiles are more diverse: three species ex cluded earlier published results with three Calceo hibit only trivial flavones, but one also produces al I Faood gyoe itiuin n eetd crophulariaceae. S selected in distribution aglycone Flavonoid I. Table o o o v 'oooooeeeskk ******* a V» ö r r r r rr £T r ~ ~ o s? > > > > o ooooeeeccn>a>n>a>a>a>ww»wptuw as c c c c £ £ 5 g o S S“?“?■?"2 .? 3 ^ g &5 n' v i#-x-s.-.-''i'#i'?x'*iO v ^ sc w w w w 1 1 1 1 1 w W'v)U)W4^snJ!3 I § 6 'J u> -J -J J •jS n o 1*n -■JO'Q v; CD u> ."J U"U viuwoo^® ,' ' T 3 U) V V u 0 ^ 0 c C CT\ — -v £. «! 00 00 CD L*> U> JL aff.OOOOvJ^OOj^ ,'• ■sj Cl> 5 ’ 2 5 ' ^ £.*»■ w ? 2 2 .3 o -1^ 4^ 00 3- .' D. Cu ^3 5 ? C D >-t n D. D. 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Anarrhinum forskalii Antirrhinum barrelieri X Antirrhinum braun-blanquetti X Antirrhinum graniticum x x X X Antirrhinum hispanicum x x x X X A ntirrhinum m ajus ssp. cirrhigerum Antirrhinum nutalli Asarina barkleyana Asarina procumbens X XX x <~v X Calceolaria arachnoidea X X Calceolaria chelidonioides Calceolaria irazuensis Calceolaria mexixana Calceolaria pavonii Calceolaria scabiosifolia Calceolaria tenella X X X Calceolaria tripartita X X X X X X X X Mimulus cardinalis Mimulus moschata o -o Mimulus pilosus 3 “ C Odontites lutea Odontites viscosa E. Wollenweber et al. ■ Flavonoids from Scrophulariaceae 9 a series of flavonols. The flavonoid chemistry of Acknowledgements Odontites (Rhinanthoideae-Rhinantheae) is sim ilar to that of M imulus (Scrophularioideae-Gratio- Thanks are due to Mr Helmut Groh from the leae). Both genera are capable of accumulating Botanischer Garten der TU Darmstadt for his at flavones and flavonols, and they seem to hold an tention and for the plant material, to Prof. Dr. T. intermediate position between the Scrophulari- Horie (Tokushima, Japan) for the gift of reference oideae-Antirrhineae (flavones) and the Scrophu- flavones, and to Dr. F. Stevens and Ms M. Ivancic larioideae-Calceolarieae (flavonols). The flavo (Corvallis, OR, USA) for identification of 5-hy- noid patterns clearly support the recognition of droxy-auranetin by NMR spectroscopy. the tribes Antirrhineae and Calceolarieae (Tahta- jan, 1997).

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