Notizen 1289

On the Pigmentation of the Pollen of N o th o fa ­gus an tarctica (Forst.) Oerst. (Fagaceae)

Eckhard W ollenw eber

Institut für Botanik der Technischen Hochschule Darm­stadt, Schnittspahnstr. 3, D-6100 Darmstadt

R olf W ierm ann

Botanisches Institut der Universität MünsterAVestf.

Z. Naturforsch. 34 c, 1289— 1291 (1979); received September 4, 1979

Nothofagus antarctica, Fagaceae, Pollen, Pigmentation, Rare Flavonoids

Extraction o f pollen o f Nothofagus antarctica with acid hydrolysis yields kaempferol, quercetin, the rare flavonol sexangularetin, and naringenin. The tetrahydroxy-chalcone isosalipurpol is shown for the first time to occur in such high amount as free aglycone in mature pollen; it yields na­ringenin in usual extraction procedures. The bright yellow coloration o f the pollen is due to the presence o f isosalipur­pol and sexangularetin.

T he intense yellow colour abundan t in pollen is caused in very m any cases by the accum ulation of carotenoids (see [1] for a survey). C halcones can also bring abou t yellow p igm entation to an as yet un­known extent [2, 3; for antheres; 4]. In the following we will show tha t in add ition rare “ yellow flavonols” [5] may contribu te to such coloration.

Materials and Methods

Pollen o f Nothofagus antarctica was collected in spring 1975 and 1976 from a tree grown in the Bota­nical G arden at M ünster. Sam ples were stored deep- frozen. The m aterial was extracted and hydrolyzed by stirring it in boiling m ethano l/w ater w ith some d iluted sulphuric acid for abou t 1 hour. The solution after cooling was treated w ith diethyl ether and ethyl acetate and the upper layer was taken to dryness. The flavonoid aglycones thus ob ta ined could be se­parated on thick layers (polyam ide D C -1 1; solvent I; to luene/m ethylethyl ketone/m ethano l 6 0 :2 5 :1 5 ) . F ou r bands were scraped off and eluted w ith acetone and ethanol. H ereby a rem arkable am ount o f the ad ­sorbent is dissolved. T he unknow n com pound in band 2 was therefore further purified by preparative TLC on silica. The sam ple was sufficient for running U V -spectra and MS. In addition dem ethylation with

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pyridinium hydrobrom ide [6] could be perform ed. The reaction product, as well as the fiavonoids of bands 1, 3, and 4, was com pared directly w ith au ­thentic markers. A second solvent was used for the m ore polar region (polyam ide, solvent II: to luene/m ethanol/m ethylethy lketone/acety l acetone 40 : 30 : 20 : 10). F o r TLC on silica we used solvent III (toluene/acetone 9 : 1). - A small sam ple o f the pollen m aterial was extracted w ith som e ethylene glycol m onom ethyl ether (/. e. w ithout hydrolysis) at room tem perature and the yellow solution was chro­m atographed on cellulose (solvent IV: BAW; solvent V: isobutyric ac id /am m on ia /w ater 6 6 : 1 : 30; sol­vent VI: 30% acetic acid; solvent VII: 30% ethanol). F o r polyam ide both solvents I and II can be used.— H erbacetin 7-Om ethyl ether was obtained by sapo­nification o f its butyryl ester, isolated from Nothola- ene aschenborniana [7], 6-M ethoxy kaem pferol co­mes from the bud excretion o f sweet cherry tree [8]. A sample o f isosalipurpol was kindly supplied by Prof. H. G risebach (Freiburg); herbacetin was a gift o f Prof. H. W agner (M unich).

Results and Discussion

Extraction and hydrolysis o f the pollen o f the an­tarctic beech, Nothofagus antarctica, yields four identifiable fiavonoids, am ong which there is one rare flavonol. K aem pferol obviously is the m ajor constituent, followed by naringenin, an unknow n fla­vonol (called “N othof. Substanz” in [3]), and traces o f quercetin. N aringenin, kaem pferol and quercetine could be readily identified by direct com parison on polyam ide and on silica w ith authentic substances, w ith which they were identical in R F and in colour properties in U V 366 before and after spraying with “N aturstoffreagenz A ” (/?-amino-ethyl ester o f d i­phenyl boric acid). The unknow n flavonol exhibits the following spectral data. UV: Am« 11 376, 327, 272 nm; + A1C13 438, 360, (310), 275, 265 nm, un­changed on addition o f HC1; + N aO E t dec.; + N aO - Ac 402, 313, 284 nm; + N a 0 A c + H 3B 0 3 385, 313, 275 nm. MS: m /e (rel. int.) 316 (57, M +), 302 (16), 301 (100, M + - C H 3), 287 (6), 273 (27), 181 (10), 167(13), 153 (10), 149 (26), 139 (24), 121 (46). T he pro­duct appears on polyam ide as a brownish-yellow spot (h R f in solvent I: 42; naringenin 63, kaem pferol 28, quercetin 17). A fter spraying w ith “N atu rsto ffre­agenz A ” the spot turns greenish-brow nish. This be­haviour is observed e.g. w ith derivatives o f 6-hydro-

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xy kaem pferol; it points to a flavonol w ith 6- or 8- substitution. T he m olecular w eight 316 shows that it m ust be a flavonol w ith 4 O H groups and 1 O C H 3 group, which is confirm ed by the base-peak a t m /e 301 (M + - 15). H ence it should be a m onom ethyl ether either o f 6-hydroxy kaem pferol (galetin) or o f 8-hydroxy kaem pferol (herbacetin). 6-m ethoxy ka­em pferol shows the sam e R F and very sim ilar, but not identical colour behaviour on polyam ide thin- layer. The position o f band I in the U V -spectrum be­yond 372 nm as well as the small peak at 327 point to substitu tion at C -8 [9], O n addition o f A1C13 an ab­sorption spectrum with 4 m axim a is observed, which strongly recalls the flavonols reported recently to oc­cur as esters [7]. As a m atter o f fact dem ethylation of the substance w ith pyrid in ium hydrobrom ide yields herbacetin , w hich can be identified by com parison with an authentic sam ple. It is characteristic for fla­vonols w ith 8-OH substitu tion tha t the spots after spraying w ith the reagent appear bluish in daylight (see [7]). Thus we now know tha t the substance un­der investigation is a m onom ethyl ether o f herbace­tin. The position o f the m ethoxy group can be deter­m ined according to the rules o f classical UV spec­troscopy. T he reaction w ith A1C13 (A Amax i 62 nm) indicates free O H groups a t C-3 and C-5; because of the reaction w ith N aO A c (A Amaxii 12 nm) there m ust also be a free O H at C-7, and the decom posi­tion w ith N aO E t dem onstrates a free O H at C-4'. H ence our substance m ust be herbacetin 8-Om ethyl ether. This is corroborated finally by com parison w ith literature data [10]. In addition d irect com pari­son with the isom er 7-Om ethyl ether (pollenitin) shows tha t it is not identical w ith this com pound.

It has been m entioned several tim es in the litera­tu re that free, i. e. non-glycosylated, chalcones may occur in pollen or in anthers [4, 5, 11, 12], W e wanted to exam ine w hether such com pounds are accum ula­ted in the pollen o f Nothofagus. Extraction with ethylene glycol m onom ethyl ether indeed yields free 2 ',4 ',6 ',4-tetrahydroxy chalcone. Its identity is proven by direct com parison w ith an authentic sam ple in various TLC and PC systems and by its UV spectral properties.

G lycosides o f kaem pferol and quercetin are fairly widely d istribu ted in pollen [3]. The occurrence of

both flavonols in hydrolysed extract o f Nothofagus pollen thus could be expected. T he accum ulation of the tetrahydroxy chalcone as aglycone, which tends readily to isom erize, however, is a phenom enon which has been reported as yet only for the system “pollen” . It cannot yet be decided w hether the chal­cone observed here is perhaps a non-m etabolized re­sidue o f the chalcone pool tha t exists during certain stages o f the pollen developm ent (described for the contents o f antheres = pollen + tapetum ) o f Tulipa and Lilium in [11] and [12]). A m ong the phenylpro- panoid com pounds occuring in pollen naringenin is encountered often, though m ostly in trace am ounts [13, 3]. In m any cases, perhaps in most, this flavano- ne may have been built by cyclization o f the chalco­ne during extraction procedures as an artefact.

This is the first report on the occurence o f isosali- purpol as a free aglycone in such high am ount in m ature pollen. A lthough its 4 ',4-dim ethyl ether is abundant as a farina constituent o f goldback ferns[14], the tetrahydroxy com pound itself has not been detected as yet. Sim ilarly, the 4/-m onom ethyl ether o f 2 ',4 ',6 '-trihydroxy chalcone is produced by these plants in abundance, w hereas the free trihydroxy com pound has been found only twice in nature [15].

8-Om ethyl herbacetin (sexangularetin) had been found previously only th ree tim es in the plant k ing­dom: in Sedum acre var. sexangularis [16], in Lotus corniculatus [17], and in Dorycnium suffruticosum [18], always as a glycoside in leaves. Like o ther 6- and 8-substituted flavonols, too, this com pound is a “yellow flavonol” . T ogether w ith the deep yellow chalcone it is responsible for the brigh t yellow co­lour o f the Nothofagus pollen. C arotenoids tha t m ight contribute to the p igm entation could not be found in this pollen.

Acknowledgements

Thanks are due to Profs. H. G risebach (Freiburg) and H. W agner (M unich) for sam ples o f authentic flavonoids (E. W.). F inancial support by the D eutsche Forschungsgem einschaft (R. W.) is greatly acknowledged.

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[1] R. G. Stanley and H. F. Linskens. Pollen. Biology, Biochemistry, Management. Springer, Berlin, H eidel­berg, N ew York 1974.

[2] J. B. Harbome, Bull. Nat. Inst. Sc. India 31 ,52 (1965).[3] R. Wiermann, Ber. Dtsch. Bot. Ges. 81 ,3 (1968).[4] P. D e Vlaming and K. F. F. Kho, Phytochemistry 15,

348 (1976).[5] J. B. Harbome, Phytochemistry 8 ,177 (1969).[6] G. Howard and T. J Mabry, Phytochemistry 9, 2413

(1970).[7] E. Wollenweber, J. Favre-Bonvin, and M. Jay, Z. N a ­

turforsch. 33 c, 831 (1978).[8] P. Lebreton, E. Wollenweber, L. Southwick, and T. J.

Mabry, C. R. Acad. Sei. Paris 272,1529 (1971).[9] A. Hasan, Contribution ä l’etude biochem ique de trois

representants de la tribu des lotees: Dorycnium Vill., Bonjeanea Reichenb., Lotus L. (Legumineuses). These (3 ecycle), Lyon 1976.

[10] M. Jay, J.-F. Gonnet, E. Wollenweber, and B. Voirin, Phytochemistry 1 4 ,16005 (1975).

[11] R. Wiermann, Planta 95,133 (1970).[12] L. Quast and R. Wiermann, Experientia 29, 1165

(1973).[13] M. J. Strohl and M. K. Seikel, Phytochemistry 4 , 383

(1964).[14] E. Wollenweber, Am. Fern J. 68,13 (1978).[15] E. Wollenweber, Flora 1 6 8 ,138 (1979).[16] H. Combier, K. Markham, H. Audier, P. Lebreton, T.

J. Mabry, and M. Jay, C. R. Acad. Sei. Paris 266, 2495(1968).

[17] M. Jay, A. Hasan, B. Voirin, and M.-R. Viricel, Phyto­chemistry 17,827 (1978).

[18] M. Jay, A. Hasan, B. Voirin, J. Favre-Bonvin, and M.- R. Viricel, Phytochemistry 1 7 ,1196 (1978).