Exudate Flavonoids in Three Ambrosia Species

EXUDATE FLAVONOIDS IN THREE AMBROSIA SPECIES

ECKHARD WOLLENWEBER, KARIN MANN, MARION DORR, HANNELORE FRITZa, JAMES J Oftijb and GEORGE YATSKIEVYCHC

alnstitut für Botanik der Technischen Hochschule, D-64287 - Darmstadt; bWestn Regióhal Research Center, USDA-ARSI Albany, CA 94710, U.S.A.; CMissour, Botanical Garden, St. Louis, MO 63166, U.S.A.

(Received 18 July 1995)

Abstract: Three species of Ambrosia (Asteraceae) were studied for leaf and stem exudate flavonoids. Among these, A. bidentata contained 5, 7-dihydroxy-8,4 -dimethoxyflavanone and other rare flavonoids, whereas A. chamissonis and A. trifida were shown to contain a number of trivial flavonoids.

Key Words: Asteraceae, Heliantheae, Ambrosia - Exudate flavonoids, rare flavanone aglycones.

INTRODUCTION

Some years ago, we reported on the flavonoid aglycones present in the leaf and stem exudate of five Ambrosia species from Arizona 1 . In that publication we had stated that further work on this genus should include studies of populations as well as of additional species. Although lack of relevant plant material has thus far precluded population studies, we have

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been able to examine three additional species of Ambrosia in the meantime. Ambrosia bidentata Michx., southern ragweed or lanceleaf ragweed, is an erect annual that grows in prairies and disturbed, open areas from the eastern United States west to Texas and Nebraska. Ambrosia chamissonis (Less.) E. Greene, silver beach bur, is a common perennial herb, found on sand dunes and sandy flats along the western coast of the United States and southern Canada, and also in western South America 2 . Ambrosia trifida L., giant ragweed, is a coarse, erect annual found nearly throughout North America in moist soils along rivers and streams and in disturbed areas.

EXPERIMENTAL I

Aerial parts of flowering Ambrosia bidentata were is collected in St. Genevieve Co., MO (NW of entrance to Hawn

State Park on Highway 144) where it is common on grassy Si roadsides. A voucher (G.Yatskievych 87-20, K. Yatskievych; 6

Sept. 1987) is kept at the Missouri Botanical Garden Herbarium pr

(MO). Fruiting stems of Ambrosia chamissonis var. MI bipinnatisecta (Less.) J. T. Howell were collected in August,

1988 in Humboldt Co., CA (N end of Big Lagoon, just W of US (M

Route 101). A voucher (G. Yatskievych 88-173, K. Yatskievych) m, has been deposited in MO. Aerial parts of flowering Ambrosia 31 trifida were collected in Christian Co., MO (Swan Creek, W of 13 bridge on Highway 125, 3.7 miles north of road UU turnoff) in a 7.4 place where it is common on the rocky open gravel bank of a 5.9 permanent stream. A voucher (G. Yatskievych 87-52, et al.) is S (( kept at MO. (I-I-

Air-dried plant parts were briefly rinsed with acetone (C- to dissolve the epicuticular material. The concentrated

EXUDATE FLAVONOIDS, Ambrosia SPECIES III

solutions were separated into terpenoid fractions and flavonoid fractions by passage through Sephadex LH-20, eluted with MeOH. In the case of Ambrosia bidentata, the combined flavonoid fractions were further chromatographed over a silica gel column, eluted with toluene and increasing amounts of methylethyl ketone and methanol. In the other species, the flavonoids were identified from Sephadex fractions by direct comparisons with authentic substances. Fractions were monitored on TLC and comparisons with markers were performed on silica with toluene/ MeCOEt 9 : 1 and with toluene/ dioxane/ glacial acetic acid 18 : 5 : 1, and on polyamide with toluene/ petroli 00-140/ MeCOEt/ MeOH 12 6: 2 : 1 and toluene/ dioxane/ MeOH 8 : 1 : 1. Chromatograms were viewed under UV366 before and after spraying with "Naturstoffreagenz A" (NA). Some minor flavanones were isolated by preparative TLC on silica with the above solvents.

/ Spectroscopic data of flavanones

) Mass spectra were recorded at 70 eV by direct inlet;

1 proton and carbon NMR spectra were recorded at 200 and 50 MHz, respectively in d6-oMso. 5,7-Dihydroxy-8,4-dimethoxyflavanone (1): UV Xmax (MeOH) 340, 291; + AlC13395, 315 (increase); NaOH 327 nm. MS

) m/z (rel. mt.) 316,0936 (42%, M; calc. for C17H1606:

a 316.0947), 301 (6), 182 (100), 167 (68), 153 (32), 139 (32), 134 (35), 69 (53). 1 H NMR ô: 11.92, s (5-OH); 10.69, s (7-OH);

a 7.46, d, J 9 Hz (H-2and H-6); 6.99, d, J 9 Hz (H-3and H-5);

a 5.97, s (H-6); 5.54, dd, J 12.8, 3.2 Hz (H-2); 3.77, s (OMe); 3.60,

S s (OMe); 3.28, dd, J 18, 12.8 Hz (H3ax); 2.76, dd, J 18, 3.2 Hz (H-3m). 13C NMR ö: 78.5 (C-2); 42.0 (C-3); 196.5 (C-4); 154.3 (C-5); 128.5 (C-8); 160.0 (C-7); 95.8 (C-6); 158.6 (C-9); 101.8 -- w

112 E. WOLLENWEBER etal.

(C-b); 130.7 (C-i ); 128.2 (C-2 and C-6); 114.0 (C-3and C- 5); 159.5 (C-4); 60.4 (8-OMe), 55.2 (4-OMe). S,7,4-Trihydroxy-6- meth oxyflavanone: MS m/z (rel. mt.) 302 (55%, M; C161-11406), 287 (8), 272 (14), 183 (33), 182 (100), 167 (95), 153 (33), 121 (32), 69(91). 5 , 7,4Trihydroxy8methoxyflavanone : MS m/z (rel.int.) 302 (72%, M; C161-11406), 287 (6), 183 (31), 182 (100), 167 (79), 153 (34), 139 (27), 121 (21), 69(42). 5 , 4Dihydroxy7,8cImmethoxyflavanone: MS m/z (rel. mt.) 316 (75%, M, C171-11606), 301 (5), 197 (25), 196 (100), 181 (93), 168 (14), 153 (35), 120 (20), 69(27). S , 7,3,4-Tetrahydroxy-8-methoxy lava none MS m/z (rel. nt.): 318 (77%, M, C161-11407), 303 (6), 183 (79), 182 (89), 167 (90), 153 (36), 139 (30), 136 (35), 69(100).

RESULTS

Most of the flavonoid aglycones present in the lipophilic leaf and stem exudates of the Ambrosia species studied were identified unambiguously by direct comparisons with markers. In the exudate of Ambrosia chamissonis, there are quercetin- 3-methyl ether and quercetagetin-3,6-dimethyl ether (axillarin) as major flavonols, 6-hydroxykaempferol-3,6- dimethyl ether and quercetagetin-3,6,4-trimethylether (centaureidin) as minor flavonols. Ambrosia trifida exhibits 5,7-dihydroxy-6,8,4-trimethoxyfjavone (nevadensin) as the major exudate constituent. 5,7,4-trihydroxy-6,8- dimethoxyflavone (demethoxysudachmtmn), kämpferol-7-methyl ether (rhamnocitrin) and quercetmn-7,3-dimethyl ether e (rhamnazin) are minor constituents, accompanied by traces of eriodictyol-7-methyl ether and eriodictyol-7,3-djmethyl ether. Ambrosia bidentata shows the most interesting exudate EXUDATE FLAVONOIDS, Ambrosia SPECIES 113

flavonoids. Except for a trace of luteolin, they all are flavanones. We identified naringenin (mp 247-2490), eriodictyol (mp 258 - 259 0) and eriodictyol-3-methyl ether (homoeriodictyol), 5,7,4 -tri-hydroxy-6-methoxyflavanone (mp 210-2120), 5,7,4 -tri-hydroxy-8-methoxyflavanone (mp 180- 18 1 0) , 5,7-dihydroxy-8,4 - -dimethoxyflavanone, 5,4- dihydroxy-7,8-dimethoxy-flavanone and 5,7,3 ,4 -tetrahydroxy-8-methoxyflavanone. (Melting points of the latter products were not determined, due to the paucity of material.). The structure of the rare flavanone 1 was elucidated primarily from its proton and carbon NMR spectra. In addition to two methoxyl singlet signals and a low field, sharp singlet indicating the presence of a 5-OH, the proton spectrum displayed two ortho coupled aromatic doublets characteristic of the degenerate pairs of carbons of para substituted benzene rings (4-substituted flavonoid 13-ring) and three one proton double doublets with chemical shifts and coupling constants typical of the H-2 and H-3 protons of flavanones. A one proton singlet in the aromatic region suggested a tn-substituted A- ring. The carbon spectrum revealed 15 signals corresponding to the requisite number of carbons in the proposed structure if allowance is made for the two doubly intense 13-ring signals which represent two degenerate carbons each (C-2/C-6 and C-31C-5). Use of a multiplicity separation pulse sequenz supplied with the instrument (Nicolet NR-WB200 FT) revealed six CH3 and CH signals and nine CH2 and C carbons. Assignment of the B- and C-ring signals was made by comparison with published spectra of naringenin and naringenin 4-methyl ether3 ; our data were in very good agreement with those of naringenin-4-methyl ether (< 0.4 ppm difference), but not with those of naringenin and establish the 4-OMe. The shift of the second OMe indicates it to be sterically crowded; i.e. there J

114 E. WOLLENWEBER etal.

is no adjacent unsubstituted position in the A-ring and thus it cannot be attached at C-7. The A-ring carbon signals are consistent with placing hydroxyl groups at C-S and C-7 and an OMe at either C-6 or C-8. The position of the UV maximum in ethanol at (for a flavanone) relatively high wavelength (340 nm) and in particular the significant shift on addition of Aid3 indicate that the OMe group must be placed at Also the spot colour on polyamide TLC favours C-8-substitution when parallel pairs of flavanones such as 5,6,7-tri0H-flavanone vs. 5,7,8-triOH-flavanone, 5,7,4-tri-OH-6-OMe-flavanone vs. 5,7,4 -trioH-8-OMe-flavanone, 5,4 -diOH-6,7-diOMe-flavanone VS. 5,4 -diOH-7,8-di0Me-f lava none, 5-OH-6,7,4 -triOMe- flavanone vs. 5-OH-7,8,4 -triOMe-flavanone are considered and compared. The spots of 6-0-substituted flavanones are dark in UV354 before and after spraying NA, while the spots of 8-0- substituted flavanones appear brown and turn reddish-brown6. Compound 1 is, therefore, ascribed the structure of 5,7- dihydroxy-8,4 - -dimethoxyflavanone.

DISCUSSION

In an earlier investigation 1 , five Ambrosia species were studied for their exudate flavonoids: A. ambrosiodes, A. artemisiifolia, A. camphorata, A. cordifolia and A. deltoidea. They were found to exhibit flavones and flavonols, many of which are 6- and! or 8-0-methoxylated. Among the three species now studied, there are two that produce similar flavonoid aglycones. The third one, however, is exceptional in that it exhibits only flavanones. Most of these flavanones also bear 6- and/or 8-0-methyl groups. Thus the flavonoid pattern observed for flavones and flavonols is also paralleled by that of the flavanones. EXUDATE FLAVONOIDS, Ambrosia SPECIES 115

To the best of our knowledge, 5,7-dihydroxy-8,4- dimethoxy flavanone (subscandenin) has been reported only once before from a plant source, namely from leaves of Chromolaena subscandens 7. The other flavanones found in this species are also very rare natural products. 5,7,4-Trihydroxy- 6-methoxyflavanone (6-methoxynaringenin) has been found in roots of Scutellaria baicalensis 8 and in flower heads of Tanacetum sibiricum 9 . 5,7,4-Trihydroxy-8 -met hoxyflavanone (8-methoxynaringenin) is known from Scutellaria rivularis 10 and Encelia frutescens 5 only. 5,4-Dihydroxy-7,8- dimethoxyflavanone is present in the farinose frond exudate of the fern Chellanthes argentea 6 and. 5,7,3,4-tetrahydroxy-8- methoxyflavanone (8 -met hoxyeriodictyol) has been reported from Encelia frutescens 5.

REFERENCES

1. E. Wollenweber, D. Hradetzky, K. Mann, J. N. Roitman, G. Yatskievych, M. Proksch and P. Proksch (1987) Exudate flavonoids from aerial parts of five Ambrosia species. J. Plant Physiol. 131, 37 - 43. 2. M. Mc Auley (1985) Wildflowers of the Santa Monica Mountains. Canyon Publishing Co., Canoga Park, CA, p. 193. 3. K. R. Markham, V. M. Chari and T. J. Mabry (1982) Carbon-13 NMR spectroscopy of flavonoids. In "The Flvonoids: Advances in Research", J. B. Harborne and T. J. Mabry, Eds., Chapman and Hall, London, 19-134. 4. P. Proksch, M. Breuer, A. Mitsakos and H. Buezikiewicz (1987) New dihydroflavonols from Enceliopsis and Geraea. Planta Med. 53, 334-337. 116 E. WOLLENWEBER eral.

5. P. Proksch, U. Politt, E. Wollenweber, V. Wray and C. Clark (1988) Epicuticular flavonoids from Encelia. Planta Med. 54, 542 - 546. 6. E. Wollenweber, V. H. Dietz, D. Schillo and G. Schilling (1980) A series of novel flavanones from fern exudates. Z Naturforsch. 35c, 685-690. 7. J. M. Amaro-Luis and P. Delgado-Mendez (1993) Flavonoids from the leaves of Chromo/aena subscandens. J. Nat. Prod. 56, 610-612. 8. S. Takagi, M.Yamaki and K. Inoue (1980) Studies on water- soluble constituents of the roots of Scutellaria baicalensis. Yakugaku Zasshi 100, 1220 - 1224. 9. T.A.Stepanova, V. I. Sheichenko, L. P. Smirnova and V. I. Glyzin (1981) Flavanones of Tanacetum sibiricum. Khimiya Prirodnykh Soedinenii 1981, 721 - 728. 10. T. Tomimori, Y. Miyaichi, V. Imoto and H. Kizu (1986) Studies on the constitutents of Scu tel/aria species (VIII). On the flavonoid constituents of "Ban Zhi Lian", the whole herb of Scute//aria rivu/aris. Shoyakugakv Zasshi40, 432 - 433.