Journ. Hallori BOI. Lab. No. 69: 195 ~202 (Jan. 1991) THE OCCURRENCE OF FLAVONOIDS IN THE MOSS FAMILY BRYACEAE W. STEIN l AND H. D. ZINSMEISTER l .* ABSTRACT. The flavonoid patterns of Bryum schleicheri and B. pallescens were evaluated. Twenty five different flavones, flavonols and isoflavones, mainly glycosides and some of their malonylesters as well as biflavonoids have been isolated. In further eleven species of the genera Anomobryum, Bryum, Pohlia and Rhodobryum flavonoids could be detected. The phytochemical relevance of these results is discussed. INTRODUCTION Although Herzfelder supposed already in 1921 the occurrence of anthocyanins in Bryum duvalii (= B. weigelii), B. turbinatum and B. pal/ens Bendz and Martensson isolated the first f1avonoids from a Bryum species (B. cryophilum) only 1961. Since that further f1avonoids in the genus Bryum have been described (Bendz et al. 1962, Bendz & Martensson 1963, McClure & Miller 1967, Nilsson 1969, Anhut et al. 1984, Stein et al. 1985, Geiger et al. 1987, Markham & Given 1988, Stein 1988, Stein & Zinsmeister 1990). The flavonoid patterns of Bryum schleicheri and B. pallescens are now reported here. Furthermore the evidence of f1avonoids in some other genera of the moss family Bryaceae is given. RESULTS I. Bryum schleicheri The 2-D TLC of a 80% methanolic extract shows a complex pattern of phenolic compounds (Fig. I). Twenty one different f1avonoids of those could be isolated. They belong to the classes of f1avones, isoflavones and biflavonoids. The f1avones and isoflavones are mainly glycosylated, some of these glycosides are additionally malonylated. Their structures were elucidated by chromatographic, hydrolytic and spectroscopic methods (UV, JR, PMR and 13C NMR-spectroscopy). The analytical data are given elsewhere (Stein et al. 1985, Geiger et al. 1987, Stein 1988, Stein & Zinsmeister 1990). Compounds lOa and lOb are most likely new natural products. The connection between the glucose moiety and the aglycone has still to be proved, as well as the binding of the malonylic residue at the glucose (probably at C-6"). The UV spectra and the green fluorescence after spraying with NA indicate a binding of the glucose on the orobol at C-3' or C-4'. Further studies including the synthesis of these com- 1 Fachbereich Biologie, Botanik, Universitat des Saariandes, 0-6600 Saarbriicken, FRG. • Author to whom correspondence should be addressed. 196 Journ. Haltori Bot. Lab. No. 69 I 9 9 TABLE I. The flavonoid classes and types occuring in some species of the genus Bryum.· :: .;:: :: >.. ~ '" ..., ~ '" ..., "'-' '" ~ Species: '" .g :: ::'" ~ ~ ~ :: >.. :::" .~" .;:.0" ~ §- ;:- ~:: -.: ;;; ..., :: <.) '-' "'- ""'-:::r-'" :: >.. '-' '"$ c:Q c:Q c:Q c:Q ~ .~ c:Q cti'" c:Q Flavonid class: Flavones + + + + + + Flavonols + + 3- Desoxyan thocya nidins + + + Isoftavones + + Biftavones + + Flavone/isoflavone dimers + + Flavonoid type: Methoxylated ftavonoids + + + 6 OH-ftavonoids + + + + 8 OH-ftavonoids + O-glycosides + + + + + + + + C-glycosides + + + Malonylesters + + + + Numbers of flavonoids 8 18 2 6 15 2 21 3 isolated • There are only species considered from which the definitive structure of any compound is elucidated. References: Bendz & Martensson 196 1, 1963, Bendz et al. 1962, Nilsson 1969, Anhut et al. 1984, Stein et al. 1985, Geiger et al. 1987, Markham & Given 1988, and Stein & Zinsmeister 1990. pounds are still in progress. Beside the identified flavonoids two red coloured spots (spot No. 17, 18) are additionally visible on the chromatogram. Because their concentration was very low, these compounds could not be isolated. Due to their chromatographic behavior they might be anthocyanins of the desoxyanthocyanin type. 2. Bryum pallescens The 2-D TLC of a 80% methanolic extract shows also various phenolics mainly flavonoids. The pattern is however not as rich as of B. schleicheri. According to their chromatographical data and cochromatography (TLC, HPLC) with authentic sam­ ples so far six flavonoids could be identified (Fig. 2). Three flavonolglycosides were amongst them. 3. The occurrence of flavono ids in further species of the family Bryaceae Gametophytic and partly sporophytic material of twelve further species belonging to various genera of the family Bryaceae was screened by TLC for flavonoids. The plant material available was in almost every case too li ttle for isolation of sin gle compounds. The comparison with the type chromatograms of well investigated Bryum species permits however some indications on the flavonoid pattern of those W. STEIN & H . D. ZINSMEISTER: The occurrence of ftavonoids in Bryaceae 197 ~ 0) ,-, , \ !11! '... / f;o~ ~ ;~ G '-.' ' (~) 8 ,~, EJY 88 {16J '~ 8 18 ~V CV 1 i x _ 2 FIG. I. The 2-D TLC of the phenolics obtained from airdried gametophytic material (2mg) of Bryum schleicheri. Adsorbent: cellulose, Solvents: I) TBA, tert. BuOH/HOAc/H 2 0 (3: I : I) 2) 15% HOAC, Detection: UV (350 nm) with and without NA, dotted lines indicate minor components. Spot No. Compound Spot No. Compound I 6-0H-luteolin 7-0-glucoside 8b Schaftoside 2 Unidentified phenolic compound 8c Isoschaftoside 3 6-0H-luteolin 7-0-glucoside-6"­ 9 Lucenin 2 malonylester lOa Orobol (3' or 4')-O-glucoside- 4a Luteoli n 7-0 -gl ucoside-6"- 6"-malonylester (tentatively) malonylester lOb Orobol (3' or 4')-glucoside 4b Luteolin 7-0-glucoside 11 Luteoli n 5 Luteolin 7-0-neohesperidoside 12a 5',8"-Biluteoli n 6 Luteolin 7-0-neohesperidoside-6"­ 12b Heterobryoftavone malonylester 13 Orobol 7a OroboI7-0-glueoside-6"- 14 Apigenin 7-0-neohesperidoside malonyleser 15 Vitexin 2" -rhamnoside 7b Orobol 7-0-glucoside 16 OroboI7-0-diglucoside 8a Vieenin 2 17, 18 Unidentified anthocyanin-like compounds specimen. The 2-D TLC of a 80% methanoli c extract of Rhodobryum roseum shows only fo ur main flavonoid containing spots. The single compounds were separated by PC and subjected to an acid hydrolysis which yielded Apometzgerin, Chrysoeriol, Luteolin, Selgin, Tricetin and Glucose in all cases. According to the chromatographic data, the fluorescence and the UV absorption respectively they seem to be 7-0- glycosides. The flavonoid pattern of Bryum flaccidum is quite different from that of 198 Journ. Hattori Bot. Lab. No. 69 199 I ( '-,\ \ . (2) '-~ b G 0 ,-, , -, ( , I ,\ J ... - ~ '-- (2) 0 I'~\ 0'-.1 CD r\ 1 ',I CS> 1 X _2 FIG. 2. The 2-D TLC of the phenolics obtained from airdried gametophytic material (2mg) of Bryum pallescel1.\· (habitat I . sce table 2) . Adsorbent: cellulose. Solvents: I) TBA. 2) 15 % HOAc. Detection: UV (350 nm) with and without NA. dotted lines indicate minor components. Spot No. Compound Spot No. Compound 2a Lucenin 2 9 Kaempferol 3-0-glucosidc 2b Stellarin 2 11 Kaempfcrol 3-0-neohesperidoside 3 Vicenin 2 (tentatively) 6 Kacmpferol 3,4' -di-O-glucoside the closely related B. capillare, it seems to lack bifl avones and all methoxylated and 6-0H-flavonoids for B. capillare characteristic compounds. The chromatograms of gametophytic material of B. caespiticium resemble very much some samples of B. pseudotriquetrum and B. pallescens which can not be distinguished chemically. Samples of these three species originating from different habitats show remarkable variations of the phenolic pattern, this is also true for sporophytes. B. bimum and B. pal/ens have a rich flavonoid pattern. B. turbinatum which is supposed to be closely related to B. schleicheri differs remarkably in the flavonoid pattern. The chromatogram of B. pallens contain additionaly for red spots which might be again anthocyanin like compounds. The phenolic pattern of B. alpinum however is very poor, only few very low concentrated spots representing flavonoids are visible. The same can be observed for all investigated samples of Pohlia species. Pohlia nutans and P. cruda, belonging to the same section, show only one main flavonoid located in the biflavonoid region of the chromatogram. Various samples of P. wahlenbergii differ clearly by not possessing spots in this area. The flavonoid pattern of one sample of Anomobryum julaceum w. STEIN & H. D. ZINSMElSTER: The occurrence of flavonoids in Bryaceae 199 resembles qualitatively and quantitatively more species of the genus Bryum than those of Rhodobryum and Pohlia. DISCUSSION All up to now in vestigated species of various genera (Anomobryum, Bryum, Pohlia, Rhodobryum) of the moss famil y Bryaceae contain flavonoids. From eigth Bryum species almost fifty different flavonoids could be isolated. On the other hand only less than 2 percent of the described Bryum species are until now considered, in terms of the whole family just 0.5 percent. Therefore it is not yet possible to give any chemotaxonomical evaluation which makes sense. Furthermore it has been shown that the flavonoid pattern is sometimes dependent on the physiological stage of de­ velopment, the habitat and the season (Stein et al. 198 5, Siegel et al. 1989). It has to be emphazised, that the specific pattern of any species can not be reliable if these factors are not considered and the pattern of the same species is not checked fo r various habitats (M ues & Zinsmeister 1988). Proceeding with caution there are however already some findings which could be of interest for a later chemotaxonomical evaluation. The variability of the flavonoid pattern in Bryaceae seems to be rather high. Thus flavones, flavonols, isofl avones, biflavonoids, fl avone-isoflavone dimers and anthocyanidins have been detected. Also the glycosidation patterns are highly variable. The malonylation of glycosides seems to be not frequent in bryophytes. Thus it have been observed so far only in some species of the Iiverwort genus Frullania (Yuzawa et al. 1987). Malonylation is how­ ever quite common for higher plants (Barz et al. 1985). 6-hydroxylation, O-methy­ lation and O-glycosidation which was claimed as phyletic markers for higher plants (Harborne 1967, Harborne & Williams 1971) have been found also in Bryaceae. The phytochemical and biochemical characteristics of the genus Bryum are summarized in Table l. The low le vel of flavonoids in Bryum a/pinum and three investigated Pohlia species is striking. It coincides with morphological and anatomical similarity between these species as discussed by Shaw (1984).