Journ. Hattori Bot. Lab. No. 70: 79- 90 (July 1991)

NEW REFLECTIONS ON THE OF PLEUROZIACEAE SUPPORTED BY FLAVONOID CHEMISTR yl

2 2 3 R. MUES , R. KLEIN AND S. R. GRADSTEIN

ABSTRACT From ten species of Pleuroziaceae (genera and Eopleurozia) the flavonoid patterns have been determined. The results are compared with the morphological classification of the fam il y. Eopleurozia is chemically distinguished from Pleurozia in the diversity of its flavonoid pattern. Within Eopleurozia two sections are recognized: E. sect. Eopleurozia (E. paradoxa) and E. sect. Ampliatae sect. novo (E. simplicis· sima). Both for chemical and morphological reasons Eopleurozia giganteoides is returned to Pleurozia, which is considered to be phylogenetically more advanced than Eopleurozia, because of its more si mpli fied fl avonoid pattern. Based on the presence of "differential" flavonoids the species of Pleurozia fall into three groups, which partly correspond with the sections recognized by Jack (1886). The 3' ·O·glucoside of lucenin·2, detected in P. acinosa, P. caledonica and P. articulata , is a new natural product. All other flavonoids detected in the Pleuroziaceae are rather widespread in li ve rworts.

1. INTRODUCTION The Pleuroziaceae are a small, highly specialized family of , which is sometimes placed in a separate suborder Pleuroziinae (Schuster 1984) because of several unique morphological features. Contrary to all other Jungermanniales, growth of the stem in Pleuroziaceae is by means of an apical cell with only 2 (instead of 3) cutting faces . As a result, only two lateral merophytes with its associated leaves are produced and the ventral merophyte and the underleaves, which in liverworts are derived from the third face of the apical cell, are completely lacking. Another striking feature of the Pleuroziaceae is the presence in most species of complicate leaves divided into two lobes, one of which may become modified to fo rm a watersac with a structurally complex opening. Traditionally the watersac has been interpreted as the ventral or posticallobe ( = "lobule"), but Schuster (1965) demonstrated that the dorsal and ventral faces of the had been confused, the sac actually being dorsal in position. Consequently, the leaves in Pleuroziaceae should be considered succubous rather than incubous. Because of the leaf position as well as other morphological characteristics the Pleuroziaceae are presently considered to be more close to the Jungermanniinae than to the Radulinae and Porellinae, with which they had tradi­ tionally been associated. Sex organs in Pleuroziaceae are always produced on very

I Publication No. 36 from the "Arbeitskreis Chemie und Biologie der Moose," Universitat des Saarlandes. 2 Fachrichtung 13.1 Botanik, Universitat des Saarlandes, D-6600 Saarbriicken, Germany. J Institute of Systematic Botany, Heidelberglaan 2, 3584 CS Utrecht, The Netherlands. 80 Joum. Hattori Bot. Lab. No. 70 199 1 short, abbreviated branches, which are lateral-intercalary in ongm. Unique is the presence in most species of long, hollow, tube-like organs (the "tubi vacui"), which are interpreted as sterile perianths. They are unknown in Eopleurozia simplicissima and Pleurozia purpurea . The taxonomy of the Pleuroziaceae is not very well known. The only monograph available is the treatmently Jack (1886), who recognized 10 species in the single Physotium Nees ( = Pleurozia Dum.). Several new species and one new genus, Eopleurozia Schust. ( = Pleurozia sect. Anotia Jack), have been added since. Most of the species occur in the lndo-Pacific region, which is the centre of diversity of the family. Africa has one species (the widespread palaeotropical P. gigantea), South America has two (P. heterophylla, E. paradoxa) and one species occurs on the Atlantic coast of western (P. purpurea). For a better understanding of the Pleurozia­ ceae taxonomy, information on other characters is needed. Little is known about the occurrence of chemical compounds in the Pleuroziaceae. In the course of a survey on the occurrence of lunularic acid in liverworts, Gorham (1977) detected this natural growth inhibitor in P. purpurea. Terpenoids were reported from P. acinosa and P. gigantea (Table 1). Based on their terpenoid chemistry, Asakawa et al. (1990) regarded P. gigantea as more advanced than P. acinosa. However, only one sample of each species was investigated and the possible variability of the terpenoid patterns therefore remains unknown. As to flavonoids, nothing has been known about their

TABLE I. Terpenoids reported from Pleuroziaceae.

P. acinosa P. gigantea Terpenoids (Wu 1985; Wu & Asakawa 1988) (Asakawa et al. 1990) Monoterpenes ,8-pinene + Sesquiterpenes a-, ,8-, o..elemene + elemol + ( + ) aristol-9-ene + ,8-chamigrene + bicyclogermacrene + ent-spathulenol + Diterpenoids ( - )-3, 14-c lerodadien-13-ol + + 14-labden-8,13-diol + + fusicogigantone A + fusicogigantone B + fusicogigantepoxide + 18-hydroxy-4,8-dola-bell adien + pleuroziol + ent-kaurene + Triterpenoids campesterol + stigmasterol + sitosterol + R. MUES et al.: Taxonomy of Pleuroziaceae by flavonoid chemistry 81 occurrence in the Pleuroziaceae. Therefore a study has been undertaken to determine the flavonoid contents of the species of Pleurozia and Eopleurozia, and to compare the results with morphological data.

2. MORPHOLOGICAL NOTES The species and genera of Pleuroziaceae are mostly distinguished by means of leaf characters and two main groups can be recognized. Species with bifid leaves are placed in Pleurozia, those with simple, undivided leaves in Eopleurozia. a) Eopleurozia Schust. Three species are currently recognized: E. paradoxa (Jack) Schust. (type species) from South America, E. simplicissima (Herz.) Schust. from Borneo and E. giganteoides (Horik.) Inoue from southern Japan and Taiwan. We have checked herbarium material of all three species and found that E. paradoxa and E. simplicissima are genuine Eopleurozia's, characterized by the possession of undivided vegetative leaves only. In E. giganteoides, however, both undivided and bifid leaves occur. The undivided leaves are small and mainly present on sterile shoots and at the bases of female branches, the bifid leaves are much larger and restricted to the central and upper portions of the female branches, gradually turning into bracts. Since female bracts are divided in E. paradoxa, Inoue (1976) argued that P. giganteoides might better be placed in Eopleurozia . However, the bracts in Eopleurozia are only shallowly 2-3-lobed in the upper part and have a smooth lamina, without keel. In E. giganteoides, however, the bifid leaves are clearly complicated and keeled. The presence or absence of divided leaves with a sharp keel, which is usually expanded into a wing (as in Micropterygium. Schistochila and Balantiopsis), seems to be a major morphological character to distin­ guish between Pleurozia and Eopleurozia. On this basis, E. giganteoides is better placed in Pleurozia . Within Pleurozia, P. giganteoides is not the only species which besides ordinary complicate-bifid may produce small undivided leaves. As Jack (1886) has shown, undivided leaves may also be found in P. acinosa . Their presence at shoot bases seems to indicate that the undivided leaf is the juvenile condition in Pleurozia and possibly presents the plesiomorphic condition. Although we have not attempted a phylogenetic analysis of the family, it would seem plausible on the basis of leaf characters that Eopleurozia (as the name suggests) is the more primitive genus of the family, from which Pleurozia has been derived based on elaboration of keeled, bifid leaves. Some species of Pleurozia have apparently retained a few undivided leaves in the adult plant (P. acinosa. P. giganteoides), which could be interpreted as paedamorphosis. In most species of Pleurozia , however, undivided leaves seem to have become lost entirely. The two remaining species of Eopleurozia are very different in general habit and leaf characters, as is shown in the following key: - Leaves erect, clasping the stem, laxly imbricate, broadly ovate; leaf bases without or with small auricles; leaf margins entire, with a conspicuous whitish border. Tubular organs common, gametoecia unknown. South America ...... E. paradoxa (Jack) Schust. 82 Journ. Hattori Bot. Lab. No. 70 I 9 9 I

- Leaves obliquely to widely spreading, densely imbricate, narrow ovate-triangular; leaf bases strongly ampliate-auriculate; leaf margins with a few large indentations, lacking a whitish border. Tubular organs unknown, gametoecia commonly present (dioicous). Borneo ...... E. simplicissima (Herz.) Schust. Based on these differences, the two species may be placed in different sections, as follows: 1. Eopleurozia Schust. sect. Eopleurozia. Type: E. paradoxa (Jack) Schust. Specimens examined: see Table 3. 2. Eopleurozia Schust. sect. Ampliatae Gradst. sect. novo Type: E. simplicissima (Herz.) Schust. A sect. Eopleurozia differt foliis divergentibus, anguste ovato-triangulatis, basi ampliatibus auriculatibusque, sine margine hyalino. Specimens examined: Borneo, Sarawak, Mt. Dulit, K oyana valley, on fallen trees in heath forest, ca. lOOOm , P. W. Richards 1856, Oxford University expedition to Sarawak 1932, female (lE lectotype) . Ibid., Dulit ridge, on tree trunk in open moss forest, ca. 1230m, P. W. Richards 2193. 8.10.1932, female plants (JE). Ibid., on branch of fallen tree in "transition" forest, P. W. R ichards 1725 p.p ., 9.1932, male plants (JE). b) Pleurozia Dum. Jack (1886) recognized nine species, which he grouped in three sections based on lobule shape and gametoecial arrangement: Pleurozia sect. Sphagnoidea Jack ( = sect. Pleurozia), P. sect. Articulata Jack and P. sect. Florida Jack. The present study includes six species recognized by Jack and two further species, P. giganteoides Horik. and P. heterophylla Fulf. Their classification is as follows: Sect. Pleurozia - P. conchifolia , P. gigantea, P. giganteoides, P. heterophylla , P. purpurea Sect. Articulata - P. articulata Sect. Florida - P. acinosa, P. caledonica A reconsideration of the infrageneric classification of Pleurozia as based on morphology would be worthwhile but has not been attempted in the present study. As noted by Jack (I.c.), the most striking morphological variation in the genus is seen in the development of the lobule, which may be explanate or saccate. Saccate lobules are constantly present in P. conchifolia. P. gigantea and P. purpurea whereas explanate lobules are present in P. giganteoides. P. articulata and P. caledonica. P. acinosa and P. heterophylla are heterophyllous and produce leaves with watersacs as well as leaves with explanate lobules. In P. heterophylla the two kinds of leaves are sharply different (Fulford 197 I), but in P. acinosa a gradual transition from leaves with explanate lobules to leaves with highly modified saccate lobules is seen.

3. FLAVONOID CHEMISTRY The results on the flavonoid chemistry of Pleuroziaceae are summarized in Table 2. The flavonoid patterns of Pleurozia- and Eop/eurozia-species are simple compared to those of other liverwort species (e.g. Takakia, Markham & Porter 1979; Radula, Mues 1984), but nevertheless proved to be useful for chemotaxonomic considerations. Some compounds detected in traces on 2D-TLCs of the crude extracts could not be isolated and are therefore not taken into consideration. From the large variety of flavonoids R. MUES et a!.: Taxonomy of Pleuroziaceae by flavonoid chemistry 83

TABLE 2. Flavonoids from ten Pleuroziaceae-species.

Marker f1a vonoids* Species Differential f1avonoids* 2 3 4

Eopleurozia paradoxa ++ 9 ( + + ); 10 ( + + ) (Jack) Schust. Eopleurozia simplieissima I ( + ); 5 ( + );unknown ++ ++ ++ (Herz.) Sch ust. Luteolin-di-C-glycoside (+ + ) Pleurozia acinosa ++ ++ + 8 ( + + ) (Mitt.) Trev. Pleurozia ealedoniea ++ ++ + 8 ( + + ) (Gott. ex Jack) Steph. Pleurozia artieulala + + ++ 8 ( + + ) (Lindb.) Lindb. et Lackstr. Pleurozia eonehifolia (Hook. et Walker-Arnott) Aust. Hawai nr. 187 ++ ++ 6 ( + ); 7 ( + ) New Guinea Norris 65731 + ++ 6 (++ ); 7 ( + + ) New Guinea Koponen 29264 ++ ++ Pleurozia giganlea ++ ++ ++ (Web.) Lindb. Pleurozia giganleoides ++ + + ++ Horik. Pleurozia purpurea ++ + 1 + Lindb. Pleurozia helerophylla + ++ Steph ex. Fulf.

* for numbers see Fig. I. + + = main compound. + = minor compound. produced by Iiverworts (Markham 1988; Mues & Zinsmeister 1988) only flavones were detected in ag. alcoholic extracts of the investigated samples. Their structures, shown in Fig. 1, belong to four different types. Predominant are di-C-glycosides of luteolin (2), chrysoeriol (3) and tricetin (4) (Type I) (Fig. 1), which should be considered the "marker flavonoids" of the family. The other flavones represent the so-called "dif­ ferential flavonoids": further flavone di-C-glycosides (5- 7), an O-glycosylated di-C­ glycoside (8) (Type 11), a flavone O-glycoside (9) (Type Ill) and a free aglycone (10) (Type IV) (Fig. 1). Most of the compounds detected in the investigated samples are already known from liverworts and details on their chromatographic or spectral characteristics have been reported by Markham et al. (1978); Theodor et al. (1980; 1981a, b); Mues (1982a, b); Mues et al. (1983) and Markham et al. (1987). Some notes on the structure elucidation of (8) are given in Experimental. The genera Eopleurozia and Pleurozia are clearly distinguished by their flavonoid chemistry. Whereas Pleu­ rozia-species produce, besides the above mentioned "marker flavonoids", only flavone C-glycosides of a single aglycone type and the O-glycosyJated flavone C-gJycoside (8), 84 Journ. Hattori Bot. Lab. No. 70 1 99 1

HO

OH 0

1 Vicenin-2 A1 ,A2,R3 = H; } 2 lucenin-2 R1 = OH; R2,R3 = H; 3 Ste/larin-2 R1 =OCH3 ; R2,R3 = H; R4,RS = C-linked B-O-glucopyranose 4 Tricetin 6,8-di-C-glucoside R1 ,R3 = OH; R2 = H; 5 Tricin 6,8-di-C-glucoside R1 ,R3 = OCH3; R2 = H; 8 Carlinoside A1 = OH; R2 ,R3 = H; R4 = C-linked B-O-glucopyranose; As = C-linked a-l -arabinopyranose 7 lsocarlinoside R1 = OH ; R2,R3 = H; R4 = C-linked a-l-arabinopyranose; RS = C-linked B-O-glucopyranose

8 3'-O-glucosyl-lucenin-2 R1 = O-l inked glucose; ~,R3 = H; R4,RS = C-linked B-D-glucopyranose 9 luteolin 4' -O-glucuronide R1 = OH; ~ = glucuronic acid; R3,R4,Rs = H 10 luteolin R1 =OH ; R2,R3,R4,Rs =H FIG. I. Flavonoids from Pleuroziaceae.

the Eopleurozia-species exhibit a larger variety of additional structure types. Eopleu­ rozia simplicissima produces the three marker flavonoids (2- 4) and three further flavone di-C-glycosides: vicenin-2 (1), tricin 6,8-di-C-glucoside (5) and an undeter­ mined luteolin di-C-glycoside. Vicenin-2 as apigenin-type and tricin 6,8-di-C-glucoside as tricetin 3' ,5 '-dimethylether-type flavones characterize this species chemically, as they are only found in its extracts among all investigated Pleuroziaceae. This result should yet be regarded as preliminarily, however, because only one sample of E. simplicissima was available for analysis. This species is very rare and has never been recorded since 1932, when it was discovered in Borneo (Herzog 1950). E. paradoxa differs significantly in its flavonoid pattern from E. simplicissima. Among the "marker flavonoids" only (2) was detected, but as "differential flavonoids" luteolin 4'-O-glucuronide (9) and the free aglycone luteolin (10) were identified. The latter two compounds were only detected in E. paradoxa. As the six samples of E. paradoxa, from four countries in South America, proved to be identical in their flavonoid patterns, the compounds 9 and 10 are clear chemical species attributes, The only Pleurozia species producing at least one O-glycosylated flavone C­ glycoside are P. acinosa. P. caledonica and P. articulata . In all aq. alcoholic extracts of these species 3'-O-glucosyl-Iucenin-2 (8) has been detected besides the marker fla­ vonoids 2- 4 (P. acinosa and P. caledonica) and 2 and 3 (P. articulata). This is the first detection of 3' -O-glucosyl-lucenin-2 in any bryophyte species and to our best know­ ledge it seems to be a new natural product. The investigated samples of each species proved to be identical in their flavonoid patterns. In extracts of the other Pleurozia-species examined (P. conchifolia, P. giganteoides, P. purpurea, P. heterophylla) no flavone O-glycosylation was detectable. They are characterized only by synthesis of flavone di-C-glycosides belonging to the basic aglycone types luteolin, chrysoeriol and tricetin. Thus, these species show flavonoid R. MUES et al.: Taxonomy of Pleuroziaceae by flavonoid chemistry 85 patterns of less diverse structure types than the species discussed before. P. conchifolia is the only species which synthesizes, besides the "marker flavo­ noids", two other flavone di-C-glycosides: carlinoside (6) and isocarlinoside (7). These compounds are very similar to lucenin-2 and differ only in one C-linked sugar; instead of one glucose molecule an arabinose unit is attached to the C-6 or C-8-position. However, the two compounds have been detected as main flavonoids in only one of three investigated samples of this species. In a second sample they occur only in traces and in a third sample they were not detectable (Table 2). P. conchifolia appears to be the only species of Pleuroziaceae with a rather variable flavonoid pattern. Only tricetin 6,8-di-C-glucoside (4) was found in each sample. P. gigantea, P. purpurea, P. giganteoides and P. heterophylla show the most reduced flavonoid pattern of the family. Only the "marker flavonoids" were detected and "differential flavonoids" are completely absent. The most extensive flavonoid reduction is observed for P. heterophylla with only one main flavonoid, tricetin 6,8-di-C-glucoside (4) and a minor one, lucenin-2 (2). In one sample of P. purpurea from the Faeroe islands (Denmark) stellarin-2 (3) was undetectable and tricetin 6,8-di-C-glucoside (4) was a minor compound. All other samples of these species proved to have stable flavonoid patterns.

DISCUSSION The results from flavonoid chemistry research of ten Pleuroziaceae species gen­ erally agree with the taxonomic treatment of the family, based on morphology, discussed in the first part of this paper. The flavone di-C-glycosides lucenin-2, stellarin- 2 and tricetin 6,8-di-C-glucoside have been found to be the "marker flavonoids" for the family; no other liverwort family is as yet characterized by this compound pattern. These flavone di-C-glycosides were detected in most taxa and seem to represent the basic flavonoid pattern of the first liverworts with a character combination of extant "Pleuroziaceae". Parallel to species evolution and diversification the ability to produce a wider range of different flavonoid structure types was developed, followed by an evolutionary trend, possibly from specialization and isolation, to reduce this structure diversity to the basic "marker flavonoids" again. The largest variety of compounds of different structure types has been detected in Eopleurozia paradoxa and E. simplicissima, thus supporting the phylogenetically more primitive position of Eopleurozia compared to Pleurozia from comparative morpho­ logical studies. The differences in general habit and leaf characters between the two Eopleurozia species shown above, are also reflected in their different flavonoid chem­ istry. All Pleurozia species produce as detectable flavonoids neither apigenin- and tricin-type flavone C-glycosides nor any free aglycone or flavone mono-O-glycoside. Three species groups can be recognized based on flavonoid chemistry. The first group, composed of P. acinosa, P. caledonica and P. articulata, produces 3'-O-glucosylated lucenin-2 and the "marker flavonoids". P. articulata is distinguished from the other two species by the absence of stellarin-2 (Table 2). This classification according to the 86 Journ. Hattori Bot. Lab. No. 70 199 1

flavonoid pattern seems to support the morphological classification by Jack (1886), who placed P. acinosa and P. caledonica in section Florida and P. articulata in section Articulata. The second group, consisting only of P. conchifolia, produces besides the "marker flavonoids" two further flavone C-glycosides of the luteolin-type, viz. carlinoside and isocarlinoside (as main compounds at least in one sample). As the flavonoid pattern of this species is variable to a certain extent, it is not unequivocally useful as chemical species marker. Jack (1886) placed P. conchifolia together with P. gigantea and P. purpurea in section Sphagnoidea ( = sect. Pleurozia) . In this case the morphological classification is not fully supported by the results of flavonoid chemistry. The third and last species group of the present study, P. gigantea, P. purpurea, P. giganteoides and P. heterophylla, is characterized by the absence of any "differential flavonoids". Only the "marker flavonoids" were detectable. It thus appears that the placement, on morphological groups, of these four species in a common section Pleurozia ( = Sphagnoidea) and the return of Eopleurozia giganteoides to Pleurozia are fully supported by the chemical results. Because of their reduced flavonoid pattern the four species of the last group are regarded to be most advanced in the family. From the present results on flavonoid chemistry, conclusions as to the systematic position of the Pleuroziaceae within the liverwort system cannot be drawn as most detected compounds are widespresad in the J ungermanniales.

ACKNOWLEDGEMENTS We are grateful to colleagues providing us with plant material for this study; Or. B. H. Thiers, New York Botanical Garden (P. articu!ata), Or. P. Isoviita, University of Helsinki (P. conchiJolia. P. gigantea), Or. F. K. Meyer, Herbarium Haussknecht, Jena (E. simplicissima), Prof. J.-P. Frahm, University of Ouisburg, Prof. H. Geiger, University of Stuttgart-Hohenheim, Or. H. Hiirlimann, Basel, Mrs. A. Loken, Trondheim and Or. O. Long, Royal Botanic Garden, Edinburgh (P. purpurea). We thank Or. R. Graf, UniversitiH des Saarlandes, for recording the mass spectra.

EXPERIMENTAL Plant material: the investigated samples are listed in Table 3. The identity of the plants was checked by one of us (S.R.G). Voucher specimens are kept in the herbarium of the University of Utrecht (u). Chemical analysis of ftavonoids: the 20-TLC-screening analysis was performed according to Mues (1988). Only from one sample of P. purpurea a larger amount of plant material was available for analysis. 40 g air-died and carefully purified gametophytes were ground to a powder and

preextracted several times with CHCl3 to remove the chlorophyll and the Iipids. In the CHClrfraction no ftavonoids were detectable. Then the ground plant material was repeatedly extracted with 80% aq. EtOH and three times with 80% MeOH. The evaporated alcoholic extracts were subsequently shaken with EtOAc to remove further nonpolar compounds; all ftavonoids remained in the water-phase. The ftavonoids were separated and isolated by column

chromatography (CC), first with 3% HOAc, later with BA W ( = n-BuOH-HOAc-H20 = 4 : 1 : R. MUES et a!. : Taxonomy of Pleuroziaceae by fl avonoid chemistry 87

TABLE 3. Sources of investigated Pleuroziaccae for fla vonoid analysis.

Extracted Species Source amount in mg

Eopleurozia paradoxa Ecuador, Loja Zamora, I. Dec. 1874, E. Andre 4524 260 (Jack) Schust. Venezuela, Estado Bolivar, Kukenantepui, 2600 m, Jan. 80 1977, F. Delascie & C. Brewer 4932 Colombia, Dept. Risaralda, Cord. Occidental, Macizo 270 Tatama, 3700 m, 1983, J. Aguirre & A. M. Cleef 4342 Colombia, Pilfamo de Las Papas, 3530-3730 m, 180, 200 5.-19. Sept. 1958, H. Bischler 759, 858 Chile, Prov. Magallanes, Puerto Virtudes, 100 m, l OO 10. 2. 1976, S. W. Greene 1800a Eopleurozia simplicissima Borneo, Sarawak, Mt. Dulit, ca 1230m, 8. Oct. 1932, 360 (Herz.) Schust. P. W. R ichards 2193 Pleurozia acinosa Japan, Yakushima, Cryptomeria Forest Reserve, 1000 m, lOO (Mitt.) Trev. 27.0ct. 1979, S. R . Gradstein et al. 3212 Japan, Yakushima, Mt. Motchomu, 900 m, 26. Oct. 1979, 130 S. R . Gradstein et a f. 3185 Japan, Yakushima, A rakawa, lI00- 1200m, 17 . Jan. 1973, lOO W. B. Schofield 53533 Pleurozia articulata Australia, Queensland , Mt. Bellenden K er, 10. July 1984, 140, 180 (Lindb.) Lindb. & Lackstr. B. Thiers 2488 a, b Pleurozia caledonica New Caledonia, M t. des Sources N. of St. Louis, 800m, 70 (Gott. ex Jack) Steph. 10. March 1962, R . M. Schuster 23 12 New Caledoni a, Mt. Mome, Massif des Voghis, 750m, 180 M. G. Baumann-Bodenheimer 14850 Pleurozia conchifolia Hawai, Oahu, Underwood & Cook (ed.), Hepaticae (Hook. et Walker-Arnott) Americanae nr. 187 40 Aust. New Guinea, Morobe Prov., Mt. Buruman, lOOD- I550m, 50 28. May 1981, T Koponen 29264 New Guinea, Morobe Prov., Sarawaket Range, 1600- 50 1700 m, 24. August 1981, D. H. Norris 65731 Pleurozia gigantea R eunion, southwest of Piton, Mare-a-Boue, 1600 m, 90 (Web.) Lindb. 16. Dec. 1973, J. L. De Sloover 17302 R eunion, Plaine des Palmistes, 800 m, 28. Dec. 1963, 70 J. L. De Sloover 1 7803 Malaysia, Sabah, K inabalu National Park, 11000 ft, 190 5. April 1981 , T SalO 64 New Guinea, Boridi, 5000ft, 26. Nov. 1935, s.n. 80 New G uinea, Vogelkop, Mt. Nettoti, 1700 m, 29. Nov. 196 1, lOO, lOO P. van Royen & H. Sleumer 7487a, 7489 p.p. New Guinea, W. Highlands, Mt. Hagen, 2400m, 23. Nov. lOO 1965, S. Kurokawa 4424 New Guinea, C. Highlands, Mt. Wilhelm, 3750 m, 200 14.-17. Aug. 1981, S. R . Gradstein 4043 New Guinea, Morobe Prov., Mt. Kaindi, 5.- 11. Aug. 1981, lOO S. R. Gradstein 3863 88 Journ. Hattori Bot. Lab. No. 70 I 9 9 I

TABLE 3. (Continued)

Extracted Species Source amount in mg

Pleurozia gigantea New Guinea, Morabe Prov. Cromwell Mts., 2400m, 60 (Web.) Lindb. 21. June 1981 , T. Koponen 31458 New Guinea, Morobe Prov., Mt. Sarawaket, 2000 m; 90 11. July 1981 , D. H. Norris 63706 New Guinea, Morobe Prav., Cromwell Mts., 80 2100-2200 m, 11. June 1981, D. H. Norris 61388 New Guinea, Morobe Prov., Rawlinson Range, 70 1750rn, 21. May 1981, T. Koponen 29135 New Guinea, Morobe Prav., Mt. Kaindi, 2100m, 140 5. - 11. Aug. 1981,S. R. Gradstein 3770 Pleurozia giganteoides Japan, Yakushima, 1650rn, 27. June 1962, lOO, lOO Horik. Nakanishi 80-3 Japan, Yakushirna, Arakawa, l100-1200m, 17. Jan. lOO 1973, W B. Schofield 53559 Pleurozia heterophylla Guyana, Mt. Roraima, ca 2000m, 14.- 17. Feb. 1985, 180, 130 Steph. ex Fulf. S. R. Gradstein 5394 Pleurozia purpurea Lindb. Scotland, N-Harris, northwest part of Usgnaval, 540m, 200 1. June 1981 , D. Longs.n. Scotland, Highland-region, Sutherland, S-bank of 200 Loch Assynt, J.-P. Frahm s.n. Scotland, near Tarbert, South Harris, W-bank of Laxadale 40000 Loch, 2o-30m, 13. Sept. 1984, H. Geiger 414 Faeroe Islands, Streymoy, 2 km SE-Saksun, 50 m, 50 8. July 1973, H. Hurlimann 731281 Norway, Brernanger, Reisevatn, S0rvassbotn, 80 m, 900 Juli 1987, H. H. Blom s.n. orway, Rogaland, Forsand, near Manafossen, July 1989, 1050 A. Ldken s. n.

5, upper layer) on cellulose (Avicel, for CC, Merck). The last separation step was accomplished by ID-paper-chromatography (PC), again with BAW (upper layer) as solvent. For final purification each compound was run on a Sephadex LH-20 column (Pharmacia, Uppsala) with 80% aq. MeOH as solvent. The compounds tricetin 6,8-di-C-glucoside (1.9 mg) and lucenin-2 (9.1 mg) were crystallized from MeOHjH 10. All other compounds described in this paper were isolated by 2D-PC (Whatman 3MM) of the crude 80% aq. MeOH plant extracts. The papers were loaded with an extract amount equivalent to lOOmg (or less if no lOOmg available) extracted air-dried plant material. As solvents were used for the first dimension BA W, for the second one 15 % HOAc. After separation the papers were dried, the spots marked under UV-light (366nm) and cut. The cut paper was eluted with 80% MeOH and the compounds were finally purified on Sephadex LH-20 columns with 80% aq. or pure MeOH. All compounds isolated from 2D-PC were received in amounts less than I mg and could not be crystallized. Acid hydrolysis conditions: hydrolysis of O-linked sugars was performed with 1 N TFA R. MUES et a!.: Taxonomy of Pleuroziaceae by flavonoid chemistry 89

(trifuoroacetic acid) for 1 hour, Wessely-Moser rearrangement (Wessely & Moser 1930) was achieved by treatment with 2 N HCl for 2 hours, both under reflux. After cooling the mixture was evaporated several times with water for removing the TFA and finally dissolved in a MeOH/

H20 mixture for TLC. PM- and PDM-ethers were prepared according to Brimacombe et al. (1966), modified as described in Mues (1982a). Some notes on the structure determination of 3'-O-glucosylated lucenin-2(8): compound 8 appears under UV-light (366nm) on TLC and PC as a deep purple spot with a rather high Rf-value in 15% HOAc (0.77) suggesting a higher glycosylation degree than the remaining compounds. The reaction on TLC with NHJ (yellow fluorescence) and the increased intensity of the NaOMe-UV-spectrum compared to the MeOH-spectrum (Mabry et al. 1970) suggest a free 4' -OH-group in the molecule. An ortho-dihydroxy group can be excluded, because of the olive-green fluorescence on TLC after treatment with NA ( = Naturstoffreagenz A), the almost identical AlC1,/AlClJ-HCl UV-spectra and the lack of a bathochromic shift with HJBO) (Mabry et al. 1970). A free 7-0H-group is indicated by the maximum of 334nm in the NaOMe-spectrum and of 280nm in the NaOAc-spectrum compared to 273 nm in the MeOH-spectrum (Mabry et al. 1970; Markham 1982). After hydrolysis with 1 N TFA (conditions see above) lucenin-2 and glucose were detected on TLC in several systems. As no further hydrolysis products were observed, not even after milder hydrolysis conditions, the structure of the compound was determined as lucenin-2 with an additional glucose at the 3' -position.

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