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journ. Hattori Bot. Lab. No. 67: 377- 382 (Dec. 1989)

PHYTOCHEMICAL STUDIES OF THE SPECIES PLAGIOMNIUM ELATUM AND

1 S. ANHUT , T. SEEGER 1, J. B IEHL 1 , H. D . ZINSMEISTER 1 AND H . GEIGER 2

ABSTRACT. The flavonoid pattern of P/agiomnium cuspidatum and P. e/atum was evaluated. Fifteen dif­ ferent , mainly flavone glycosides were isolated. The new natural compounds Elatin; 5-0H-Amento­ flavone; 2,3-Dihydro-5' -hydroxyamentoflavone, 2,3-Dihydro-5' -hydroxyrobustaflavone and 2,3-Dihydro- 5',3"'-dihydroxyamentoflavone were amongst them. The phytochemical relevance of these results is briefly discussed.

INTRODUCTION The fi rst phytochemical studies on species was done by Kozlowski (1921). He detected in Mnium cu:,pidatum, based on a chemical reaction with iodinepotassium iodide solution. Melchert & Alston (1965) reported on the occurrence of flavon C-glycosides in Mnium affine, McClure and Miller (1967) offlavonoids in Mnium cu.lpidatum. Koponen and Nilsson (1977) compared the flavonoid patterns of Plagiomnium elfipticum, P. medium, P. insigne, P. affine, P. tezukae, P. ciliare, P. ela/um, P. drummondii, P. japonicum, P. acutum and P. cuspidatum. Vandekerkhove (1977) isolated two f1avonoids from Mnium undulatum and characterized them as saponarin and an -6,8 di-C-glycoside. Further C- and C-O-glycosides from the same species were isolated and identified by Osterdahl (1979). Freitag et at. (1986) isolated two new -O-diglycosides from P. affine. According to the "Generic revision of " (Koponen, 1968) all mentioned species were allocated to the genus Plagiomnium. The revised classification of the family Mniaceae resulted in four tribes with ten genera on the basis of morphological and karyological data. The genus Plagiomnium was furthermore divided into five sections: I. Sectio Plagiomnium (e. g. P. cuspidatum) 2. Sectio Venusta 3. Sectio Rosulata (e.g. P. affine, P. e/atum) 4. Sectio Undulata (e.g. P. undulatum) 5. Sectjo Rostrata Recently Koponen (1 988) assembles the genera Plagiomnium, Orthomnion and Pseudobry um to the new family Plagiomniaceae. Al ready earlier (1968) he states the

I Fachbereich Biologie, Botanik, Universitiit des Saarlandes, D - 6600 Saarbriicken, FRG. 2 Institut fUr Chemie, Universitiit Hohenhei m, D - 7000 Stuttgart 70, FRG. 378 Journ. Hattori Bot. Lab. No. 67 I 9 8 9 following" . .. , it seems evident, that the colour substances and their distribution in the genera of Mniaceae are of taxonomic significance ... " . In the context of this statement a thorough study of flavonoid chemistry of the genus Plagiomnium was started. Here some results of the current studies on P. cuspidatum and P. elatum are reported.

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0J , ~ ... , (0 , ~ \_ ... i ,,~ , e ~~, "'IS~',-~' I I , 0 t~ 1 0 I,-, I I' , , \~, ,' " , ® '., I X --2

FIG. I: Type chromatogram of the phenolics obtained from gametophytic material of Plagiomnium elatum, equivalent to 3 mg dry weight. Adsorbent: cell ulose, solvents: J) tert.

BuOH-HOAc-H 20 (3: I : I) , 2) HOAc-H 20 (15: 85). Detection: UV (350 nm) with and without NA, dolled lines indicate minor components, hatched spots show blue ijuorescence. Spot No. Compound field (mg) I 8-C-fJ-D-glucopyranosyl- () 20 2 6-C-fJ-D-glucopyranosyl-luteolin (lsooricntin) 38 3 6-C-fJ-D-glucopyranosyl-8-C-a- L-rhamopyranosyl-luteolin 70 (proposed name: Elatin) 4 6-C-hexosyl-S-C-rhamnopyranosyl-chrysoeriol (tentatively) 3 5 6,8-di-C-fJ-D-glucopyranosyl-luteolin (Lucenin-2) 60 6 6-C-xylosyl-8-C-hexosyl-Iuteolin (tentatively) to 7 Apigenin-7-0 -neohesperidoside 1,5 8 5'-OH- 20 S. ANHUT et al.: Phytochemical studies of Plagiomnium elatum and P. cuspidatum 379

RESULTS I. Plagiomnium elatum The 2-D TLC chromatogram of a 80% methanolic extract shows a complex pattern, especiaJly in the f1avone-C-/O-glycoside region. Up to now seven flavone glycosides (1~7) and one biflavone (8) could be isolated (Fig. I). Compounds 3 and 8 are new natural products. The definite structure elucidation ) ., 4.2 4. 3 @4.4 ~ ,. , • . .;' ,,: o • I \_~,'

\,--, , G " .. -, '-' , I 1 , I \ - - ~,' x CS 1 I --- 2 FIG. 2: Type chromatogram of the phenolics obtained from gametophytic material of Piagiomnium cuspidatum , equivalent to 3 mg d ry weight. Adsorbent: cellulose, solvents: 1) tert.

BuOH-HOAc-H 20 (3: 1: 1),2) HOAc-H 2 0 (15:85). Detection: UV (350nm) with and wi thout NA, dotted lines indicate minor components, hatched spots show blue flu orescence. Spot No. Compound Yield (mg) 1 6-C-fJ-D-Glucopyranosyl-lu teolin-7-0-fJ-D-glucopyranoside 85 ( = Lutonarin) 2 6-C-fJ- D-Glucopyranosy l-ch rysoeriol-7-O-fJ- D­ 160 gl ucopyranoside ( = Isoscoparin-7-0-glucoside) 3 6-C-fJ- D-Glucopyranosyl-apigenin-7 -O-fJ- D-gI ucopyranoside > 1000 ( = Saponarin) 4.1 2,3-Dihydro-5'· hydroxyamentoflavone } 4.2 2,3-Dihydro-5'-hydroxyrobustaflavone 85 4.3 2,3- Dih ydro-5',3'"-dihydr oxyamen tofla vone 35 4.4 5',3'''-Dihydroxyrobustaflavone 20 380 Journ. Hattori Bot. Lab. No. 67 I 989 of compounds 4 and 6 are still in work, 4 seems to be a new f1avone-di-C-glycoside, too. The chemical data of 8 are published by Geiger et al. (1988), those of the glycosides will be published elsewhere. 5-0H-ametoflavone is the first biflavone found in M niaceae. Additionally large amounts (> 2000 mg) of the hydantoine derivative allantoin crystallized during the whole isolation procedure. This compound is also present in other bryophytes (Touffet & Villert 1958). 2. Plagiomnium cuspidatum As seen from figure 2 a type chromatogram of a 80% methanolic extract contains numerous spots of UV absorbing and blue fluorescing substances. So far the structure of seven f1avonoids (1-4.4) could be elucidated. Compounds 4.1, 4.2 and 4.3 are new natural products. Their analytical data are published elsewhere (Anhut et aI., 1989). Compound 4.4 is so far known from six moss families (Geiger et al. 1988, Becker et a1.1986, Markham et al. 1988, Geiger & Bockel 1989). Lutonarin was first time isolated from a moss species, isoscoparin 7-0-glucoside is a new bryophytic substance. The occurrence of saponarin III Plagiomnium cuspidatum could be finally established by NMR spectroscopy.

DISCUSSION The spot pattern of each of both species shows to be fairly constant which is evident by the comparison of the main f1avonoids from specimen of different habitats (6 of P. cuspidatum and 13 of P. elatum). The flavonoid pattern so far known (Table I, Fig. I & 2) of P. cuspidatum and

TABLE I: Chemical features of some Plagiomllium species.

Sectio: Rosulata Pla!:iomllium Undulata

Species: P. affine P. elatum P. cuspidatum P. ulldulatllm

Aglycone type: Apigenin + + ++ Chrysoeriol + + + Luteolin + + ++ +

Glycosidation type: Mono-C-glycosides + + Di-C-glycosides ++ ++ Di-O-glycosides + Mono-C-mono-O-glycosides + + + Mono-C-di-O-glycosides ++ Biflavonoids + ++

Number of isolated substances 5 8 7 7

+ + > 3 compounds isolated. + 1- 2 compounds isolated. - not yet detected. S. ANHUT et al.: Phytochemical studies of Plagiornniurn eta/urn and P. cuspidaLUrn 381

P. elatum however are quite different. While in P. elatum and P. cuspidatum glycosides of the apigenin, luteolin and chrysoeriol type are found, there is a clear predominance of the luteolin type in P. elatum. The identified glycosides of P. cuspidatum are all 6-C-fJ-D-glucopyranosyl-7-0-fJ-D-glucopyranosides. This glycosidation type is not yet found in P. elatum, where mainly di-C-glycosides occur. Lastly the quality and quantity of biflavonoids is clearly distinct. From the occurrence of biflavonoids in P. affine and P. undulatum is up to now nothing reported, but by own 2D-TLC studies there is some evidence that they may occur. For proving the existence of this compound type the components of the corresponding TLC-region must be isolated and studied by MS and NMR techniques. Comparing the flavonoids of P. elatum and P. affine (Freitag et al. 1986) both have glycosides of the common aglycone luteolin, but differ in their glycosidation pattern (see table I). P. undulatum produces mainly apigenin di-C-glycosides (OsterdahI1979). Finally it must be emphasized, that some chemical correlations are not yet sufficient for a chemotaxonomic evaluation. Also it seems not favourable to rely only on the pattern of a single compound class. Further investigations on the chemistry of the genus Plagiomnium including much more species have to be done.

METHODS Extraction. The cleaned and air-dried gametophytic material [512 g P. elatum (B . S. G .) T. Kop. and 470 g P. cuspida/um (Hedw.) T. Kop.] was ground and repeatedly prccxtracted with CHCI3, followed by extraction with 80% aq. EtOH. Isolation. The flavonoids were separated by columm chromatography on cellulose, polyamide and sephadex LH-20 with different solvent systems as mentioned in Geiger et al. ( 1988). Analytical techniques. The structures were elucidated by spcctroscopical methods (UV, FD-MS, FAB-MS, P]'vfR and 13C-NMR). The identity of known compounds was also proved by cochromatography (TLC, HPLC) with authentic samples.

ACKNOWLEDGEMENTS We thank Mrs. E. Henn, Mrs. Ch. Zchren and Mrs. U. Zeitz for their assistance.

LITERA TU RE CITED

Anhut, S., T. Seeger, H. D . Zinsmeister & H. Geiger. 1989. New dihydrobiflavones from the moss Plagiornl1iurn cuspidaturn. Z. Naturforseh. 44c: 189- 192. Beeker, R., R. Mues, H. D. Zinsmeister, F. Herzog & H. Geiger. 1986. A new biflavone and further flavonoids from the moss Hylocorniurn splel1dens. Z . Naturforseh. 41 c: 507- 510. Freitag, P. , R. Mues, C. Brill-Fess, M. Stoll, H . D . Zinsmeister & K. R. Markham. 1986. lsoorientin 3' -O-sophoroside and 3' -O-neohesperidoside from the moss Plagiornl1iurn affine. Phytochemistry 25: 669--671. Geiger, H ., S. Anhut & H . D. Zinsmeister. 1988. Biflavones from some . Z. aturforsch. 43c: 1-4. -- & M. Bockel. 1989. Die Biflavonoidausstattung von Philono/is Ion/ana (Hedw.) Briol. (Batramiaceae). Z. Naturforsch. 44c: 559- 562. Koponen, T . 1968. Generic revision of Mniaceae Mitt. (Bryophyta). Ann. Bot. Fennici 5: 117- 151. --. 1988. The Phylogeny and Classification of Mniaceae and Rhizogoniaceae (Musci). 1. Hattori 382 Journ. Hattori Bot. Lab. No. 67 I 989

Bot. Lab. 64: 37--46. - -- & E. Nilsson 1977. Flavonoid patterns and species pairs in Plagiomnium and Rhizomnium (Mniaceae). Congres International de Bryologie. Bryophytorum Bibliotheca 13: 411--425. Kozlowski, A. 1921. Sur la saponarine chez le Milium cuspidatum. Acad. Sci . C. R. 173: 429--431. Markham, K. R., O. M. Andersen & E. S. Viotto. 1988. Unique Biflavonoid types from the moss Dicralloloma robuslum. Phytochemistry 27: 1745- 1749. McClure, 1. W. & H . A . Miller. 1967. Moss Chemotaxonomy. A survey for flavonoids and the taxonomic implications. Nova Hedwigia 14: 111 - 125. Melchert, T. E. & R. E. Alston. 1965. Flavonoids from the moss Mnium affine Bland. Science 150: 1170-11 71. Osterdahl. B.-G. 1979. Chemical Studies on Bryophytes. 22. Flavonoid C-Glycosides of Milium undulatum. Acta Chem. Scand. ser. B, 33: 400--404. Touffet, 1. & S. Villeret. 1958. Recherches sur la presence des ureides glyoxyJiques et de leurs enzymes chez les Bryophytes. Soc. Bot. de France 105: 312- 318. Vandekerkhove, O. 1978. Ober die Verbreitung von Flavonoiden bei akrokarpen Laubmoosen. l. Die Flavonoide von Milium ulldulatum (L.) Hedw. Z. Pflanzenphysiol. '86: 135- 139.