Morphological and Chemical Studies on the Taxonomy of 14 Frullania Species, Subgenus Chonanthelia

Joum. Hattori Bot. Lab. No. 63: 425-436 (Dec. 1987)

MORPHOLOGICAL AND CHEMICAL STUDIES ON THE TAXONOMY OF 14 FRULLANIA SPECIES, SUBGENUS CHONANTHELIA

1 2 YO'ICHI YUZAWA , RUDIGER MUEs AND SINSKE HATTORl3

INTRODUCTION

The subgenus Chonanthelia in the genus Frullania was first established by Spruce (1884), who divided it into two groups, Cladocarpicae and Acrocarpicae, on the basis of the gynoecium position and the length of the female branch. Stephani (1910) also recognized two groups in this subgenus: (a) lobuli longe coaliti and (b) lobuli breviter coaiiti. Schuster (1985) regarded Spruce's two groups as sections, sect. Cladocarpicae and sect. Chonanthelia (= Acrocarpicae) and added three new sections, sect. Holosti pulae, Spinilobae and Pluriplicatae. In this paper we present the results of new studies concerning morphology and flavonoid chemistry of 14 species of this subgenus.

R ESUL TS AND DISCUSSION The type species of sect. Cladocarpicae is F. brachyclada Spruce (non Lehmann 1844).' We examined the type of F. brachyclada and found it to be closely related to F. arecae, but with the following differences: (1) the stem-underleaf is usually flat or weakly undulate in F. brachyclada, whereas in F. arecae it is usually undulate, (2) in F. brachyclada the sinus of the underleafis somewhat wider than that of F. arecae. Further more the flavonoid patterns of both are clearly different from each other (Table 1). Thus we propose to treat F. brachyclada as a taxon different from F. arecae, though it is sometimes regarded as synonymous with F. arecae. F. hians, the type of sect. Pluriplicatae Schust., is regarded as a synonym of F. arecae (Clark & Svihla 1947; Svihla 1956; Schiffner & Arnell 1964; Haarbrink 1981). This opinion is supported by the flavonoid pattern of sample 22144 (a form of F. hians; Table 4) which is identical to that of other samples of F. arecae. We also examined the type specimen of F. spiniloba Steph., the type species of sect. Spinilobae Schust., and found this species to be also closely related to F. arecae. The only character different

1 Taira Commercial High School, Nakashio, Taira, Iwaki-shi, Fukushima-ken 970, Japan; also Hattori Botanical Laboratory. 2 Fachbereich 16, Botanik, Universitat des Saarlandes, D-6600 Saarbriicken, Fed. Rep. of Germany. 3 Hattori Botanical Laboratory, 3888 Obi-Honmachi, Nichinan-shi, Miyazaki-ken 889-25, Japan. 4 Clark and Frye (1952) published F. tunguranguana Clark & Frye as a nomen novum for F. brachyclada Spruce, a later homonym of F. brachyclada Lehm. 426 Journ. Hattori Bot. Lab. No. 63 1 987

• ...... ~ : .< >.~. •.••• • ).•.' .' . ~ , ,i. : ~ ,~, 11f". "

FIG.!. Frullania arecae (Spreng.) Gott. a: Portion of stem, x 24. b-e: Lobules of stem-leaves, all x 24. f-g: Stem-underleaves, both x 24. h: Perianth, x 12. i: Cross section of perianth, x 24. j: Innermost pair of female bracts and bracteole, x 12. All drawn from type of F. dentilobula Steph. (G 21654). from F. arecae is dentation of the laminal portion of the leaf-lobule, as shown in Fig. 2. However, the dentation is highly variable and often almost undeveloped. We ex amined a number of specimens of F. arecae and often observed dentation which is almost identical with that of F. spiniloba, less developed or with blunt teeth or angles, or finally slightly repand-Iobulate ones. Haarbrink (1981) illustrated Colombian F. arecae and showed 2-3 blunt teeth on the laminal portion of the leaf-Iobules. F. spini loba should be treated as conspecific with and a variety or form of F. arecae, rather than a distinctive species. Accordingly sect. Pluriplicatae and Spinilobae (Schuster 1985) should be reduced to synonyms of sect. Cladocarpicae. Sect. Cladocarpicae is characterized morphologically by the leaf-lobule with a long Y. YUZAWA et at.: Morphological and chemical studies of 14 Frullania species 42 7

FIG. 2. Frullania spiniloba Steph. a: Portion of stem, x 24. b-d: Lobules of stem leaves, all x 24. e-/: Stem-underleaves, both x 24. All drawn from specim. from Co lombia (NY). keel (more than 0.35 mm long) almost parallel with the stem. This section is equivalent to Stephani's "lobuli longe coaliti" (l91O) and the "groupe du Frullania arecae" of Vanden Berghen (1976). Among 9 species of this section, 3 species (F. obscura, F. riojaneirensis and F. confertiloba) are characterized by a 4-plicate perianth (2 lateral and 2 ventral). Thus, for this group we propose the new series Quadriplicatae. 6 Four further species of sect. Cladocarpicae, F. ovistipula, F. africana, F. sphaerocephala and F. arecae, belong to ser. Cladocarpicae, characterized by an 8-12-plicate perianth. The remaining species, F. ecklonii, is morphologically similar to the F. arecae complex. Clark and Svihla (1947) and Svihla (1956) reduced F. ecklonii to a synonym of F. arecae and Vanden Berghen (1976), Haarbrink (1981) and others followed them. However, Demaret and Vanden Berghen (\948) studied F. arecae and F. ecklonii and concluded that they are separate species. We examined many specimens of F. arecae

• Frullania subgen. Chonanthelia sect. Cladocarpicae ser. Quadriplicatae Yuzawa, Mues & Hatt., sec. novo A !ypo (ser. Cladocarpicae) differ! perianthiis quadriplicatis. Typus: Frullania obscura Sw. 428 Journ. Hattori Bot. Lab. No. 63 1 987

FIG. 3. Frllllania ecklonii (Spreng.) Spreng. a-d: Lobules of stem-leaves, all x 24. e: Stylus, x 240. f-g: Stem-underleaves, both x 24. All drawn from type of F. emergells Mitt. (021683).

and F. ecklonii and confirmed that both taxa are separable from each other by the shape of leaf-lobules and underieaves, as shown in the following key : ]) Beak of leaf-lobule well-developed, underleaf usually strongly undulate (Fig. 1) .. F. arecae 2) Beak of leaf-lobule not developed, underleaf flat, not undulate (Fig. 3) .... F. eck/onU As F. ecklonii is the only species of sect. Cladocarpicae without a developed beak of the leaf-lobule, the new ser. Ecklonii is proposed for this species.6 Sect. Chonanthelia is characterized morphologically by the leaf-lobule with a short keel (less than 0.25 mm long) obliquely spreading (at angles of 30- 60° with the stem). This section is divided into 3 series: Chonanthelia, GradsteinW and Pluricari nataes. Three species, F. laxiflora, F. tolimana and F. gibbosa, are included in ser. Cho nanthelia. This series is characterized by (I) the quadriplicate perianth and (2) the rounded leaf-apex. F. gradsteinii and F. pluricarinata are morphologically clearly dis-

6 Frullania subgen. Chonanthelia sect. Cladocarpicae ser. EcklonU Yuzawa, Mues & Halt., ser. novo A typo (ser. C/odocarpicae) differt rostris lobulorum foliorum haud productis. Typus : Frul/ania eck/onii (Spreng.) Spreng. 7 Frullania subgen. Chonanthelia sect. Chonanthelia ser. Gradsteinii Yuzawa, M ues & Halt., ser. novo A typo (ser. Chonanthelia) differt apicibus loborum foliorum subacutis. Typus: Frullania grad steinii Yuzawa et aI., sp;· nov. (Description is given in foot-not 9). 8 Frullania subgen. Chonanthelia sect. Chonanthelia ser. Pluricarinatae Yuzawa, Mues & Hatt., ser. novo A typo (ser. Chonanthelia) differt perianthiis 8-12-carinatis. Typus : Frullania p/uricarinala Gott. Y. YUZAWA et a!.: Morphological and chemical studies of 14 Frullania species 429

tinguished from each other: F. gradsteinii 9 has a 4-keeled perianth and a subacute leaf-lobe, whereas F. pluricarinata has a 8-12-keeled perianth and a rounded leaf-lobe. Thus both are placed in different series: ser. Gradsteinii (F. gradsteinii) and ser. Pluri carinatae (F. pluricarinata). lo The classification of 14 investigated species of subgen. Chonanthelia as based on morphological studies is shown in Table 3. In a search for further characteristic dis tinguishing features to classify the species of subgen. Chonanthelia we analyzed the flavonoid patterns of the 14 species by chromatographic and spectroscopic methods. The results are listed in Tables 1 and 2. Altogether 18 different flavonoid com pounds were isolated from air-dried plants and at least partially identified (Table 2). Details on extraction and isolation methods are given on page 433 (Procedures in Chemical Analysis). From two species, F. arecae and F. riojaneirensis, four and five different samples respectively were available for comparative analysis and proved to be essentially identical with one exception: one sample of F. riojaneirensis (No. 22149) from Colombia (collected in 1972) differed from the other samples in relatively low flavonoid concentration. From all other species only one sample could be studied. Thus for these species no results were obtained regarding the possible variation of flavonoid patterns. The 18 isolated flavonoid compounds are flavones : 3 free aglycones and 15 gJycosides (Table 2). All compounds for which we obtained completely identified structures had been isolated before, their chromatographic, UV and MS data are given elsewhere (Mabry et al. 1970, Harborne et al. 1975, Markham et al. 1978, Mark-

o Frullania gradsteinii Yuzawa, Mues et Hat!., sp. novo Fig. 4. Planta major, flavo- vel rufo-brunnea. Caulis ad 7 cm longus, ca. 0.2 mm in diam., cum foliis ca. 0.9 mm latus, sparsim 1-2-pinnatim ramosus. Lobi foliorum caulinorum imbricati, recte patuli, ovati, 0.8-0.85 mm longi (cum appendiculis ca. 1.2 mm lo ngi), ca. 0.85 mm lati, margine integerrimo, apice subacuto, basi antica distincte appendiculata, appendiculis ± longe ligulatis ; cellulae superae 8.5- 13.5 x 8.5-1 I " m, flavo-brunneae vel -virentes, ipsae medianae 14.5- 22 x 13- 22 I'm, flavo-brunneae, ipsae basales 26-33 x 17-26 I'm, profunde rufo-brunneae, trigonis majusculis, subnodulosis, pari etibus ub ique fexuosis. Lobulus foli i caulem subparallelus, cylindrici-campanulatus (raro evolutus), lamina subtriangulata, carina brevi, rostro parum producto, decurvo. Stylus minutus, anguste tri angulatus. Amphigastria caulina late ovata, caule duplo latiora, ca. l iS-bifida, margine integerrimo, basi haud appendiculata. Monoica: Perianthium oblongo-ovatum, 4-plicatum, ca. 1.5 mm longum, 0.7 mm latum; lobus bracteae intimae oblongo-ovatus, apice subacuto vel anguste obtuso, margine integerrimo, lobulo fere parum brevi ore, canaliculati-lanceolato; bracteola intima alte conjugata cum lobulis bractearum, 0.7 mm lata. Androecium in caule ramisque laterale, capitatum, subsessile. Typus : Ecuador: Galapagos Is. : Santa Cruz, along the old trail to Bella Vist, in tufts on twigs of Zanthoxylum/agara, S. R. Cradstein & W. A. Weber H3-holotype in u; dup!. in NICH. 10 The remaining section (sect. Holostipulae) is represented by a single species, F. holostipula Hatt. & Griffin ][1. for which we were unable to make flavonoid analysis through lack of adequate material for study. F. holostipula is a remarkable species with flat, orbicular, non-bilobed underleaves and cylindric-campanulate leaf-lobules. However, non-bilobed underleaves are observed rarely in F. arecae and F. gibbosa, though ± undulate along margins, and cylindric-campanulate leaf-lobules are also observed in F. gradsteinii. Therefore we consider sect. Holostipulae may be better treated as a series in sect. Chonanthelia. 430 Journ. Hattori Bot. Lab. No. 63 1 987

,; ·~.·"········...... · ·- · , ; . f/·· .:.'. .. p

FIG. 4. Frullania gradsleinii Yuzawa, Mues et Hatt. a-b: Branching, open ellipses representing gynoecia and solid circles, androecia, both x 0.6. c: Portion of stem, ventral view, x 24. d : Do. with perianth, x 24. e-g: Stem-leaves, all x 24. h : Lobule of stem leaf, x 60. i: Stylus, x 240. j-k: Stem-underleaves, both x 24. I: Perianth, x 24. m : Cross-section of perianth, x 24. n: Beak of perianth, x 60. 0: Innermost pair of female bracts + bracteole, x 12. p: Androecium, x 24. q-s: Cells of lobe of stem-leaf, q from margin, r from middle, s from base, all x 480. All drawn from type (u). ham & Porter 1979, Theodor et al. 1983, Mues et al. 1983, 1984, Stein et al. 1985). The structures were generally determined by co-chromatography (TLC, HPLC) with standard samples (whenever possible), by UV-, IR- and mass-spectrometry and in the case of glycosides by hydrolysis and separate structure determination of sugars and aglycones. The structures of flavone C-glycosides were additionally determined by Y. YUZAWA et al. : Morphological and chemical studies of 14 Frullania species 431

TABLE 1. Flavonoid patterns of investigated species.

Numbers of isolated compounds No. Species 5 4 7 10 6 2 1 3 11 8 9 13 14 15 16 12 17 18 1 Frullania ovistipula x x 2 F. obscura x x x 3 F. aJricana x x x x 4 F. sphaerocephala x x x x x 0 5 F. arecae (incl. F. wallichiana) x x x x x 0 6 F. riojaneirensis x x X x 0 0 7 F. conJertiloba x X x x x 0 x x 8 F. brachyclada x x x x 000 x 9 F. ecklonii x x x x 0 0 x ** 10 F. pluricarinata x x ** 11 F. gradsteinii x x x** 12 F. laxiftora x *** 13 F. tolimana x x x ***** + 14 F. gibbosa x x x **** + + Numbering of isolated compounds according to Table 2. x = luteolin-derivatives; 0 = apigenin-derivatives; * = 6-0H-luteolin-derivatives; + = scutellarein derivatives.

TABLE 2. Flavones isolated from 14 Frullania species, Subgenus Chonanthelia.

No. Compound Apigenin 2 Apigenin 7-0-glucoside 3 Apigenin 6, 8-di-C-p-D-glucopyranoside ( = vicenin 2) 4 Luteolin 5 Luteolin 7-0-glucoside 6 Luteolin 7-0-glucosyl-6" -malonylester 7 Luteolin 7-0-glucoside-x" -acylated 8 L uteolin 7-O-gentiobioside 9 Luteolin 7-0-diglucoside, acylated at the sugar moiety 10 Luteolin 6, 8-di-C-p-D-glucopyranoside ( = lucenin 2) 11 Luteolin derivative, unknown 12 6-hydroxyluteolin 13 6-hydroxyluteolin 7-0-glucoside ~~ } three 6-hydroxyluteolin 7-0-glucosides, acylated at the sugar moiety 16 17 Scutellarein 7-0-glucoside-x" -acylated 18 Scutellarein 7-0-glucoside?

MS of their perdeuterio-methylated derivatives. The free aglycones are luteolin, apigenin and 6-hydroxyluteolin. Luteolin was isolated as the free aglycone from all investigated species except F. laxiflora, apigenin from only F. riojaneirensis, F. brachyclada and in traces from F. ecklonii, and 6-hy- 432 Journ. Hattori Bot. Lab. No. 63 1 987

droxyluteolin only from F. tolimana. Luteolin 7-0-glucoside was the only glycoside found in all species; thus luteolin and its 7-0-glucoside are the marker components for the species of subgen. Chonanthe/ia. Both compounds had already been isolated earlier from F. di/atata (Mues et al. 1983), whereas this is the first report of the oc currence of apigenin and 6-hydroxyluteolin as free aglycones in any Frullania spe cies. Further luteolin glycosides isolated from several species are: luteolin 7-0-genti obioside, luteolin 7-0-glucosyl-6" -malonylester and luteolin 7-0-glucoside and 7-0- diglucoside acylated at the sugar moieties. The acyl functions of the latter two gly cosides and of the 6-hydroxyluteolin- and scutellarein-glycosides (Nos. 14-17, Table 2) could not be identified due to the minute amounts of isolated compounds (see page 433 et seq., "Experimental section"). A further unidentified luteolin glycoside was detected in traces in F. confertiloba. One luteolin di-C-glycoside was isolated from eight species and identified as luteolin 6, 8-di-C-j9-D-glucopyranoside (= lucenin 2). Besides the free aglycone apigenin, two apigenin glycosides were isolated from several species : apigenin 7-0-glucoside and apigenin 6, 8-di-C-j9-D-glucopyranoside (= vicenin 2). From species 9- 14 (Table I) 6- hydroxyluteolin 7-0-glucoside and 3 different x" -acylated derivatives of this glycoside were isolated. Sulfated glycosides can be excluded, because none of the glycosides moved in the buffer for electrophoresis described for sui fated flavonoids (Williams et al. 1976) except the standard sample eupatin 3-S0SCa1/2 (Timmermann et al. 1979). A 6" -malonylester can also be excluded, because none of the three acylated derivatives is identical to the 6" -malonylester of 6-hydroxyluteolin-7-0-glucoside from Bryum capi/lare (Stein et al. 1985). As minor compounds two scutellarein 7-0-glycosides were detected in extracts of F. to/imana and F. gibbosa. Acid hydrolysis revealed scutellarein as the aglycone and glucose as the sugar. From its Rrvalue on cellulose TLC with H 20 as solvent before alkaline hydrolysis compound 17 (Table 2) should be an acylated scutellarein 7-0-glucoside with a free carboxyl-group at acyl function. As a result of comparative flavonoid analysis two basic species-groups (1-9 and 10- 14) are distinguished by their flavonoid patterns (Table I). This result is in com plete accordance with their classification into two sections as obtained from mor phological investigations (Table 3). The first species group belonging to sect. Cladocar picae (species 1-9, Table I) is characterized chemically by the presence of luteolin- and apigenin-derivatives and the absence of 6-hydroxyluteolin compounds, except in F. ecklonii. This species synthesizes two 6-hydroxyluteolin glycosides. The flavonoid pat tern of F. eck/onii is intermediate between sect. Cladocarpicae and Chonanthelia. Thus the flavonoid pattern of F. ecklonii supports its position in a separate series, Ecklonii. The second species group belonging to sect. Chonanthelia (species 10- 14, Table I) is characterized chemically by the presence of 6-hydroxyluteolin-, luteolin- and (partly) scutellarein-derivatives, apigenin-type compounds being absent. A further arrange ment of the two sections into series is not clearly obvious from the flavonoid patterns, except for F. ecklonii. Nevertheless with this study it was demonstrated that morpholo- Y. YUZAWA et al.: Morphological and chemical studies of 14 Frullania species 433

TABLE 3. Classification of 14 investigated species. Number in front of the species-name corresponds with that on Table 1. Sect. Cfadocarpicae Ser. Cfadocarpicae 1. F. ovistipufa (laminal portion of (perianth 8-12-plicate ; 3. F. africana lobule with long, vertical lobule with beak) 4. F. sphaerocephafa keel) 5. F. arecae 8. F. brachycfada Ser. Quadriplicatae 2. F. obscura (perianth 4-plicate; lobule 6. F. riojaneirensis with beak) 7 . F. conferti/oba ...... Ser. Eck fOil;; 9. F. eckfoni; (perianth 8-12-plicate; lobule without beak) Sect. Chonanthefia Ser. pfuricarinatae 10 . F. pfuricarinata (lamina I portion of lobule (perianth 8-12-plicate; leaf with short, oblique keel) rounded) Ser. Gradstei"ii 11 . F. gradsteinii (perianth 4-plicate; leaf acute) Ser. Chonanthefia 12. F.laxi/lora (perianth 4-plicate; leaf 13 . F. tofimana rounded) 14 . F. gibbosa gical combined with flavonoid chemical studies may well contribute to clearer insights to distinguish taxonomic categories of different levels. The analysis of flavonoid pat terns is possible with minute amounts of air-dried herbarium specimens (Table 4), new collecting of fresh plants not being necessary as, for instance, for cytological studies or for studies of volatile compounds. This advantage of flavonoid patterns as addition al markers in the classification of bryophytes will certainly be used for further pro blems in the future.

PROCEDURES I CHEMICAL ANALYSIS Plant material Chemical analysis was performed only from herbarium plants. In Table 4 the amount of extracted plant material of each species is indicated. Voucher specimens are deposited in NICH and the private herbarium of Y. Yuzawa. Extraction and Isolation Each sample was ground with mortar and pestle in 80% aqu. MeOH, the extract shaken for two hours, filtered and evaporated to a small volume of 2-5 m!. This procedure was repeat ed until no flavonoids were detected on TLCs of the spotted extract. All extracts were spotted on 2D-PCs, the amount equivalent to 100 mg or less dry weight extracted plant material per paper. The papers (Whatmann 3 MM) were developed two-dimensionally: first dimension with TBA(= tert. BuOH-HOAc-H20, 3: 1: 1), second with 15% HOAc. After development papers were dried under a hood, the flavonoid spots marked under UV (350 nm), cut out and eluted by 80% or pure MeOH. Further purification of compounds was achieved by column 434 Journ. Hattori Bot. Lab. No. 63 198 7

TABLE 4. Extracted plant material: sample numbers and dry weights. mg dry weight Species Sample number extracted Frullania ovistipu/a 22135 55 F. africana 22134 50 F. obscura 22152 50 F. sphaerocepha/a 22142 60 F. arecae 22138; 22144; 330 22145; 22146 F. riojaneirensis 22147; 22148 ; 22149; 915 22150 ; 22151 F. confertiloba 22132 135 F. brachyclada 22141 200 F. eck/onii 22143 95 F. p/uricarinata 22136 70 F. gradsteinii 22133 45 F. /axiflora 22137 30 F. tolimana 22140 320 F. gibbosa 22139 220 chromatography (CC) on Sephadex LH-20 columns with aqu. MeOH from 70 % to pure

MeOH. Luteolin 7-0-glucoside crystallized from MeOH/H 20 mixtures, yielding altogether

7 mgs. Luteolin crystallized from H 20 - MeOH mixture: 1 mg. All other compounds were not crystalline and yielded less than 1 mg. TLC and HPLC procedures. A) TLC. For co- and comparison chromatography with standard samples the following systems were used (all solvent mixtures used are v/v): 1. Sorbent cellulose, microcrystalline; TLC ready plastic sheets F 1440, Schleicher and Schiill.

1.1. TLC solvents for glycosides : 15 % and 40 % HOAc; BEW( = sec. BuOH-HOAc-H20,

14: 1: 5); BAW (= n-BuOH-HOAc-H20, 4: 1: 5, upper layer); PEW (= n-Pentanol

HOAc-H20, 10 : 5: 5); H 20 . 1.2. TLC solvents for aglycones: 40 % HOAc and BAW. 2. Sorbent polyamide 6, Polygram, Macherey and Nagel.

2.1. TLC solvent for glycosides: WE MA ( = H 20-MeCOEt-MeOH-3, 5 pentanedione, 13: 3: 3: 1). 2.2. TLC solvent for aglycones : BMM (=C.H.-MeCOEt-MeOH, 4 : 3: 3). 3. Sorbent Silica Gel, Merck.

3.1. Solvents for glycosides: EtOAc-MeCOEt-HCOOH-H20, 5: 3: 1: 1; EtOAc-HCOOH

H 20, 67: 13: 20.

3.2. Solvents for aglycones: BPA (=C.H,-CoHoN-HCOOH, 36: 9: 5); CAA (= CHCI3-

Me2(CO)-HCOOH, 9: 2: 1).

TLC of PDM-lucenin-2: Silica Gel, Merck; solvents : EtOAc; CHCI3-EtOAc-Me2(CO), 5: 4: 1 and 5: 1: 4 respectively. B) HPLC. Waters M-45; isocratic; column Lichrosorb RP 8, 10 p,; flow-rate 1,5 mll min.; DV-detector 254nm; solvent for flavone-O-glycosides: MeOH-HOAc-H 20, 35: 5: 60; solvent for flavone C-glycosides : MeOH-HOAc-H20, 15: 5: 80; solvent for aglycones: MeOH

HOAc-H20, 45: 5: 50. Y. YUZAWA et al.: Morphological and chemical studies of 14 Frullania species 435

Acid hydrolysis, sugar analysis and preparation of PDM-derivatives are described in Mues et al. (1984); alkaline hydrolysis according to Markham (1982). Thin-layer electro phoresis was performed with a CAMAG 67701 electrophoresis unit; self-prepared TLC plates, cellulose, microcrystalline, Merck, 0.25 mm, were used; buffer: 2,5 % HCOOH-7,5 % HOAc, 1: 1, v/v (Williams et al. 1976); 4 hours. Standard samples: apigenin, luteolin: Roth; apigenin 7-0-glucoside: isolated from Blasia pusilla (unpublished results); vicenin-2, lucenin-2: isolated from Mylia taylorii (un published results); luteolin 7-0-glucoside, Prof. H. Geiger, Stuttgart-Hohenheim; luteolin 7-0-glucosyl-6"-malonylester: isolated from Bryum capillare (Stein et al. 1985); luteolin 7- O-gentiobioside: isolated from Frullania dilatata (Mues et al. 1983); 6-hydroxyluteolin and 6-hydroxyluteolin 7-0-glucoside: isolated from Bryum capil/are (Stein et al. 1985) and Frullania dilatata (Mues et al. 1983); scutellarein: from acid hydrolysis of scutellarin, Roth; glucose : Merck. UV-spectra: Varian Superscan 3, self registrating spectrophotometer; conditions and shift reagents according to Mabry et al. (1970). IR-spectra : 1 mg glycoside, 100 mg KBr; Beckman 4210, self registrating spectrophoto meter. MS: Varian MAT 311, spectrosystem 188; 70eV; ion source 150°C; probe temp. 160- 280°C.

ACKNOWLEDGEMENTS. We thank Professor Dr. Hans Geiger, Institut fUr Chemie, Universitat Stuttgart-Hohenheim, for a sample of luteolin 7-0 -glucoside and Dr. Richard Graf, Fachrichtung Organische Chemie, Universitat des Saarlandes, for recording the mass spectra.

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