HETEROCYCLES

Review | Special issue | Vol. 77, No. 1, 2009, pp. 99-150
Received, 17th July, 2008, Accepted, 1st September, 2008, Published online, 4th September, 2008.
DOI: 10.3987/REV-08-SR(F)3

■ Bryophytes: Bio- and Chemical Diversity, Bioactivity and Chemosystematics

Yoshiori Asakawa,* Agnieszka Ludwiczuk, Fumihiro Nagashima, Masao Toyota, Toshihiro Hashimoto, Motoo Tori, Yoshiyasu Fukuyama, and Liva Harinantenaina

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro-machi, Tokushima, 770-8514, Japan

Abstract

The bryophytes contain the Marchantiophyta (liverwort), Bryophyta (moss) and Anthocerotophyta (hornwort) among which the Marchantiophyta contain cellular oil body and they produce a number of mono-, sesqui- and diterpenoids, aromatic compounds like bibenzyl, bis(bibenzyls) and acetogenins. Several of these compounds show interesting biological activity such as insecticide, insect antifeedant, cytotoxic, piscicidal, muscle relaxing, allergenic contact dermatitis, anti-HIV, DNA polymerase β, 5-lipoxygenase, calmodium inhibitory, anti-obesity, neurotrophic, cyclooxygenase, hyaluronidase and NO production inhibitory, antimicrobial and antifungal activities. Each liverwort biosynthesizes peculiar components, which are valuable for classification of liverworts. The typical chemical structures and bioactivity of the selected liverwort constituents as well as chemosystematics of several species of the Marchantiophyta are surveyed.

INTRODUCTION
The bryophytes are found everywhere in the world except in the sea. They grow on the tree (Figures 1 and 2), on soil, lake, river even in Antarctic island. The bryophytes are placed taxonomically between algae and pteridophytes; there are about 24,000 species in the world. They are further divided into three
phyla, Bryophyta (mosses 14,000 species), Marchantiophyta (liverworts 6,000 species) and Anthocerotophyta (hornworts 300 species). They are considered to be the oldest terrestrial plants, although no strong scientific evidence for this has appeared in the literature. This hypothesis was mainly based on the resemblance of the present-day liverworts to the first land plant fossils, the spores of which date back almost 500 million years.

Among the bryophytes almost all liverworts possess beautiful cellular oil bodies (Figure 3) which are peculiar, membrane-bound cell organelles that consist of ethereal terpenoids and aromatic oils suspended in carbohydrates- or protein-rich matrix, while the other two phyla do not. These oil bodies are very important marker for the classification in the Marchantiophyta.

Phytochemistry of bryophytes has been neglected for a long time because they are morphologically very small and difficult to collect a large amount as pure samples, their identification is also difficult and they are considered to be nutritionally useless to humans. However, a number of bryophytes, in particular, mosses have been widely used as medicinal plants in China, to cure burns, bruises, external wounds, snake bite, pulmonary tuberculosis, neurasthenia, fractures, convulsions, scald, uropathy, pneumonia, neurasthenia etc.1-6
Many species of the Marchantiophyta show characteristic fragrant odors and an intense hot and bitter taste. Some bryophytes species produce sweet tasting secondary metabolites. Generally, bryophytes are not damaged by microorganisms, insects, snails, slugs, and other small mammals. Furthermore, some liverworts cause intense allergenic contact dermatitis and allelopathy. We have been interested in these biologically active substances found in bryophytes and have studied about 1000 species of bryophytes collected in North and South America, Australia, Europe, French Polynesia, India, Japan, Madagascar, Malaysia, Nepal, New Zealand, Pakistan, Taiwan and Turkey with respect to their chemistry, pharmacology, and application as sources of cosmetics, and medicinal or agricultural drugs. The biological activities of liverworts are due to terpenoids and aromatic compounds.7-14a-c
Bio- and chemical diversity of the Marchantiophyta and the chemical structures of heterocyclic terpenoids, aromatic and related compounds found in several liverworts and some biological activity including characteristic odor and taste are surveyed in this paper. Hemisynthesis of bioactive compounds from liverwort constituents and chemosystematics of several Marchantiophyta species are also discussed.

1. Biodiversity of Bryophytes
The Marchantiophyta (liverworts) includes two subclasses, the Jungermanniidae and Marchantiidae, and six orders, 49 families, 130 genera (Table 1) and 6,000 species. These small plant groups are distributed everywhere in the world. Still many new species have been recorded in the literatures. There are 54 endemic genera in southern hemispheric countries, such as New Zealand and Argentina (Figure 4).14d In Asia including Japan, relatively a large number of endemic genera (21) has been recorded, however, South Africa, Madagascar and both North America and Europe are very poor regions of endemic genera as shown in Figure 4. The richness of endemic genera of bryophytes in southern hemisphere suggests that the bryophytes might originate from the past Antarctic islands since 350,000,000 years ago and developed to the northern hemisphere with a long range evolutionary process. In Japan, Yaku Island is the most important place to watch many species of the Marchatiophyta. In the southern hemisphere, New Zealand is the most charming country to see many different species of the Marchantiophyta which are totally different from those found in Japan.

In the tropical regions, such as Borneo, Sumatra and Papua New Guinea, there are rain forests where so many liverworts species have been found, but many different species like the Lejeuneaceae species are intermingled each other and it is time consuming work to purify all of them. In Ecuador and Columbia, the Marchantiophyta species grow in the high mountains, over 2,000 m where people live, not in the lower level of their lands. In Table 1, each subclass, order, family and genus of the Marchantiophyta is shown. Each underlined genus is that chemically already studied in our laboratory.

2. Chemical Diversity of Bryophytes
The extraction of oil bodies with organic solvent using ultrasonic apparatus is very easy for stem-leafy liverworts to give a large amount of crude extract. In case of thalloid liverworts the specimens are ground mechanically and then extracted with non-polar solvents. At present, over several hundred new compounds have been isolated from bryophytes and their structures were elucidated.6-14a Most of compounds found in liverworts are composed of lipophilic mono-, sesqui- and diterpenoids and aromatic compounds, such as bibenzyls and bis(bibenzyls) which have been isolated from the Marchantiaceae and Aytoniaceae in the Marchantiales, Lejeuneaceae, Lepidoziaceae and Plagiochilaceae in the Jungermanniales and Blasiaceae, Pelliaceae and Riccardiaceae in the Metzgeriales.

However, the presence of nitrogen or sulfur-containing compounds in bryophytes was very rare; recently several nitrogen-containing compounds have been isolated from the Mediterranean liverwort, Corsinia coriandrina belonging to the Corsiniaceae (Marchantiales).14e

The most characteristic chemical phenomenon of liverworts is that most of sesqui- and diterpenoids are enantiomers of those found in higher plants although there are a few exceptions such as drimane, germacrane and guaianes. It is very noteworthy that the different species of the same genera, like Frullania tamarisci and F. dilatata (Frullaniaceae) each produces different sesquiterpene enatiomers.
Some liverworts, such as Lepidozia species (Lepidoziaceae), biosynthesize both enantiomers.　 Flavonoids are ubiquitous components in bryophytes and have been isolated from or detected both in the Marchantiophyta and the Bryophyta.

Almost all liverworts elaborate α-tocopherol, stigmasterol and squalene. The characteristic components of the Bryophyta are highly unsaturated fatty acids and alkanones, such as 5,8,11,14,17-eicosapentaenoic acid, 7,10,13,16,19-docosapentaenoic acid and 10,13,16-nonadecatrien-7-yn-2-one and triterpenoids. The neolignan is one of the most important chemical markers of the Anthocerotophyta.

The presence of hydrophobic terpenoids is very rare in the Marchantiophyta. A few bitter kaurene glycosides have been found in the Jungermannia species (see later). However a numbers of flavonoid glycosides have been detected both in Marchantiophyta and Bryophyta. In the Charts 1-5, the typical terpenoids and aromatic compounds found in liverworts are shown.

3. Bioactive Compounds from Bryophytes
The biological characteristics of the terpenoids and aromatic compounds isolated from the liverworts in our laboratory are: 1) characteristic scents, 2) pungency and bitterness, 3) allergenic contact dermatitis, 4) cytotoxic, anti-HIV and DNA polymerase β inhibitory, 5) antimicrobial and antifungal activity, 6) insect antifeedant activity, mortality, and nematocidal activity, 7) superoxide anion radical release inhibitory activity, 8) 5-lipoxygenase, calmodulin, hyaluronidase, cyclooxygenase inhibitory activity and NO production inhibitory activity, 9) piscicidal and plant growth inhibitory activity, 10) neurotrophic activity, 11) muscle relaxing activity, 12) cathepsins B and L inhibitory activity, 13) cardiotonic and vasopressin antagonist activity and 14) anti-obesity activity.

3.1. Characteristic Scents
A number of liverworts emit volatile terpenoids or simple aromatic compounds when crushed, which are responsible for intense sweet-woody, turpentine, sweet-mossy, fungal-like, carrot-like, mushroomy, or seaweed-like scents.11,15,16
Almost all liverworts which smell of mushrooms contain 1-octen-3-ol and its acetate, which is generally more abundant than the free alcohol. A small thalloid liverwort, Asterella species grown in Pulau Dayang Bunting island in Malaysia emits an intense unpleasant odor. Surprisingly, it produces two components: skatole which is responsible for this smell and is composes of 20% of the total extract, and 80% of 3,4-dimethoxystyrene.17 The stink bug smell of the New Zealand Cheilolejeunea pallidus is attributable to (E)-dec-2-enal, (Z)-dec-2-enal, (E)- and (Z)-pent-2-enals,18 although the major components of such insects are the cis and trans-2-hexenals. The characteristic mold like smell of Leptolejeunea elliptica is due to p-ethylanisol, p-ethylphenol, and p-ethylphenylacetate.19 The strong milk-like fragrance of Cheilolejeunea imbricata is due to a mixture of (R)-dodec-2-en-1,5-olide (42) and (R)-tetradec-2-en-1,5-olide (43).19
Bicyclohumulenone (44) isolated from Plagiochila sciophila (=P. acanthophylla subsp. japonica) as a crystal possesses an aroma reminiscent of a variety of scents based on a strong woody note, resembling the odor of patchouli, vetiver, cedar wood, iris, moss, and carnations. Tamariscol (23) from European Frullania tamarisci subsp. tamarisci, Japanese F. tamarisci subsp. obscura, Taiwanese F. nepalensis, and East American F. asagrayana similarly possesses a remarkable aroma reminiscent of the woody and powdery green notes of mosses, hay, costus, violet leaves and seaweeds. Both compounds are important in commerce. They are used as perfumes as such or as perfume components of the powdery floral-, oriental bouquet-, fantastic chypre-, fancy violet- and white rose-types in various cosmetics. It is noteworthy that Frullania species producing tamariscol grow in high mountains only.

Total synthesis of (±)-tamariscol (23) has been accomplished using commercially available p-methoxyacetophenone in 13 steps.20 After it had been shown that both the tertiary alcohol and the 2-methyl-1-propenyl group attached to the cyclohexane ring of tamariscol were necessary for the characteristic scent of (23), thirteen mini-tamariscols were synthesized by Grignard reactions of 2,7-dimethylcyclohexanone, 2-methylcyclohexanone, 4-methylcyclohexanone, cyclohexanone, and cyclopentanone with vinylmagnesium bromide, 2-methyl-1-propenylmagnesium bromide and 2-methyl-2-propenylmagnesium bromide, respectively. Among them, 1-hydroxy-1-(2-methyl- 1-propenyl)cyclohexane (45) had a sweet mossy aroma similar to that of tamariscol itself.21 There are three chemo-types of Conocephalum conicum. The types 1, 2 and 3 emit (-)-sabinene, (+)-bornyl acetate, and methyl cinnamate as the major components, respectively, which are responsible for the characteristic odor of each type.22 Jungermannia obovata contains a tris-normonoterpene ketone, 4-hydroxy-4-methyl cyclohex-2-en-1-one (46) which possesses an intense carrot-like odor.23,24

The strong and distinct mossy odor of Lophocolea heterophylla and L. bidentata is due to a mixture of (-)-2-methylisoborneol25 and geosmin (47). Geosmin, possessing a strong earthy-musty odor, has also been found in in vitro cultured Symphyogyna brongniartii.26 The strong sweet mossy note of Mannia fragrans is attributable to the cuparene-type sesquiterpene ketone, grimaldone (48).27
The sweet turpentine-like odor of the French Targionia hypophylla is due to a mixture of cis- and tans-pinocarveyl acetates.28 The strong sweet-mushroomy scent of the ether extract of Wiesnerella denudata is due to (+)-bornyl acetate and a mixture of the monoterpene hydrocarbons, α-terpinene, β-phellandrene, terpinolene, α-pinene, β-pinene, and camphene.11 The odor of the steam distillate of W. denudata is weaker than that of its ether extract. The steam distillate contains nerol (14%), neryl acetate (27%), and γ-terpinene (31%), but the content of 1-octen-3-ol (7%) and its acetate (2%) is lower than that of Conocephalum conicum belonging to the same genus of Wiesnerella.

3.2. Pungency and Bitterness
Some genera of the Marchantiophyta produce intense pungent and bitter substances which exhibit interesting biological activities described in subsequent sections. Most North American liverworts contain unpleasant substances, some of which taste like immature green pea seeds or pepper.29 Porella vernicosa complex (P. arboris-vitae, P. fauriei, P. gracillima, P. obtusata subsp. macroloba, P. roellii and P. vernicosa) contain very pungent substances, and Jamesoniella autumnalis contains an intense bitter principle whose taste resembles that of the leaf of lilac and Swertia japonica or the root of Gentiana scabra var. orientalis. The strong hot taste of Porella vernicosa complex is due to (-)-polygodial (49).7,16

Polygodial is the major component of the medicinal plant, Polygonum hydropiper, P. minus and P. punctataum var. punctatum (Polygonaceae). The Malagasy medicinal plant, Cinnamosma fragrans (Canellaceae) produces structurally similar pungent component, cinnamodial (=ugandensidial) (50).30,31 It is noteworthy that some ferns, Blechnum fluviatile collected in New Zealand and Argentinean Thelypteris hispidula elabolate the pungent component, polygodial (49), together with its related drimanes.32,33　
The sacculatane diterpenedialdehyde, sacculatal (26), two eudesmanolides, diplophyllolide (51) and ent-7α-hydroxydiplophyllolide (52), a germacranolide, tulipinolide (53) and two 2,3-secoaromadendrane-type sesquiterpene hemiacetals, plagiochiline A (21) and plagiochiline I (54) which possess potent pungency were isolated from Pellia and Trichocoleopsis, and Chiloscyphus, Wiesnerella and Plagiochila, species, respectively. An additional pungent 1β-hydroxysacculatal (55) was obtained from Pellia endiviifolia, together with several sacculatane-type diterpenoids.34 The hot taste of Pallavicinia levieri 14 and Riccardia robata var. yakushimensis 35 is also due to sacculatal (26).

Polygodial and sacculatal have been obtained from cell suspension cultures from each liverwort.36,37 Porella acutifolia subsp. tosana is a pungent stem-leafy liverwort. Its taste is due to the presence of hydroperoxysesquiterpene lactones, 1α- (56), and　1β-hydroperoxy-4α,5β-epoxygermacra- 10(14),11(13)-dien-12,18α-olides (57).38 When one chews a whole plant of the stem-leafy liverwort, Plagiochila asplenioides, P. fruticosa, P. ovalifolia, and P. yokogurensis which contain plagiochiline A (21) one feels a potent hot taste slowly. It is suggested that (21) might be converted into pungent unsaturated dialdehyde by human saliva. Enzymatic treatment of (21) with amylase in phosphate buffer or with human saliva produces two strong pungent 2,3-secoaromadendrane-type aldehydes, plagiochilal B (58) whose partial structure is similar to that of the pungent drimane-type sesquiterpene dialdehyde, polygodial (49), and furanoplagiochilal (59).39 The New Zealand liverwort, Hymenophyton flabellatum produces different pungent tasting substance from the other aforementioned liverworts. Fractionation of the ether extract resulted in the isolation of several phenyl butanones (62-67) and their related compounds (68-70). The hot taste of the species is due to compound (62).32

Most of the species belonging to the Lophoziaceae produce surprisingly intense bitter substances. Gymnocolea inflata is persistently bitter and induces vomiting when one chews a few leaves for several seconds. The earlier review already mentioned that this is due to gymnocolin A (71).11 It contains additional unknown minor bitter diterpenoids whose structures remains to be clarified. Jungermannia infusca has an intense bitter taste. This is due to the presence of the infuscasides A-E (72-76) which were the first reported isolation of glycosides from liverworts.40 The bitterness of Anastrepta orcadensis, Barbilophozia lycopodioides, and Scapania undulata are attributable to highly oxygenated diterpenoids, anastreptin A (77) and barbilycopodin (78),23,41 and scapanin A (79),42 respectively.

3.3. Allergenic Contact Dermatitis
Frullania species are notable as liverworts that cause very intense allergenic contact dermatitis.7,43-47 The allergy-inducing substances are sesquiterpene lactones, (+)-frullanolide (24) and (-)-frullanolide (80) which have been isolated from Frullania dilatata and F. tamarisci subsp. tamarisci, respectively.7 Both dihydrofrullanolides (89, 90) with a α-methylene-γ-butyrolactones isolated from the above mentioned liverworts do not cause allergy. F. asagrayana, F. bolanderi, F. brasiliensis, F. eboracensis, F. franciscana, F. inflata, F. kunzei, F. nisquallensis, F. riparia and the other Frullania species which contain sesquiterpenes (81-88) with α-methylene-γ-butyrolactones cause strong allergenic contact dermatitis as does Schistochila appendiculata.

The allergens of the latter are long chain alkylphenols, 3-undecyl- (91), 3-tridecyl (92), 3-pentadecyl (93), and 3-heptadecyl phenols (94), long chain alkyl salicylic acids, 6-undecyl- (95), 6-tridecyl- (96), 6-pentadecyl salicylates (97), and their potassium salts, potassium 6-undecyl- (98), 6-tridecyl- (99), and 6-pentadecyl salicylates (100) as well as 6-undecyl catechol (101).11

Such dermatitis is similar to that caused by the long chain alkylphenols of the fruit of Ginkgo biloba and Anacardiaceae plants. Marchantia polymorpha and Metzgeria furcata also cause allergenic contact dermatitis but their allergens have not been isolated yet.

3.4. Cytotoxic, Anti-HIV-1 and DNA Polymarase β Inhibitory
A few eudesmanolides and germacranolide possessing inhibitory activity against KB cells have been isolated from liverworts.11 Conocephalum conicum and Wiesnerella denudata contain guaianolides which exhibited cytotoxic activity against P-388 lymphocytic leukemia.11 The crude ether extract (4-20 µg/mL) of the following liverworts showed cytotoxicity against P-388 in vitro [Asakawa Y. unpublished results]: Bazzania pompeana, Kurzia makinoana, Lophocolea heterophylla, Makinoa crispata, Marsupella emarginata, Pellia endiviifolia, Plagiochila fruticosa, P. ovalifolia, Porella caespitans, P. japonica, P. perrottetiana, P. vernicosa, and Radula perrottetii. On the other hand, Frullania diversitexta, F. ericoides, F. muscicola, F. tamarisci subsp. obscura, Lepidozia vitrea, Pallavicinia subciliata, Plagiochila sciophila, Spruceanthus semirepandus, and Trocholejeunea sandvicensis were not active against P-388.
2,3-Secoaromadendrane-type sesquiterpenoids, plagiochiline A (21), plagiochiline A 13-octanoate (102), and 12-hydroxyplagiochiline A 13-2E,4E-dodecadienoate (103) isolated from P. ovalifolia showed cytotoxic activity (ID50 3, 0.05, 0.05 µg/mL, respectively) against P-388.48 Polygodial (49) isolated from P. vernicosa complex, sacculatal (26) from P. endiviifolia, and two 2,3-secoaromadendrane-type sesquiterpene hemiacetals (102), and plagiochiline A 13-decanoate (104) from P. ovalifolia showed cytotoxic activity (2-4 µg/mL) against melanoma.49 Sacculatal (26) also showed cytotoxic activity against Lu1 cell (IC50 5.7 µg/mL), KB cell (3.2), LN Cap cell (7.6) and ZR-75-1 cell (7.6), respectively [Cordel GA, Pezzuto JM, Asakawa Y. unpublished results].
Marsupellone (105) and acetoxymarsupellone (106) from Marsupella emarginata showed cytotoxicity (ID50 1 µg/mL) against P388.49,50 Riccardins A (107) and B (108) which was the first bis-bibenzyls from the Japanese liverwort, Riccardia multifida subsp. decrescens inhibited KB cells at a concentration of 10 and 12 µg/mL, respectively. Many Plagiochila species and Radula perrottetii contained cytotoxic plagiochiline A (21) (0.28 µg/mL) and perrottetin E (109) (12.5 µg/mL) against KB cells, respectively.
The thalloid liverwort, Marchantia polymorpha which can cause allergenic contact dermatitis, shows inhibitory activity against Gram-positive bacteria, and has diuretic activity. The methanol extract (105 g) of Japanese M. polymorpha was chromatographed on silica gel and Sephadex LH-20 to give cyclic bis-bibenzyls, marchantin A (MA) (36, 30 g) and its analogues (MB-G). The yield of MA (36) is dependent upon Marchantia species. 80 to 120 g of pure MA has been isolated from 6.67kg of dried M. paleacea var. diptera. This thalloid liverwort elaborates not only the marchantin series, marchantin A (36), B (114), D (115) and E (116), but also the acyclic bis-bibenzyls, perrottetin F (110) and paleatin B (117).

Marchantins A, B, D, paleatin B and perrottetin F show DNA polymelase β inhibitory (ID50 14.4-97.5 µM), cytotoxic (3.7-20 µM against. KB cells) and anti-HIV-1 (5.30-23.7 µg/mL) activity.

Marchantin A (36) also shows cytotoxicity (T/C 117) against P-388.11 Blasia pusilla produces the bis(bibenzyl) dimers, pusilatin A (118), B (39), C (119) and D (120). Pusilatins B (39) and C (119) possess DNA polymerase β inhibitory activity (IC50 13.0 and 5.16 µM), moderate cytotoxicity against KB cells (ED50 13.1 and 13.0 µg/mL) and weak HIV-RT inhibitory activity.51 Trichocolea species produce prenyl ethers, tomentellin (121), demethyltomentellin (122), and trichocolein (123).52 Compound 121 is the major cytotoxic component of T. mollissima, active against BSC cells at 15 µg/disk.52 Compound 122 isolated from T. tomentella also showed the same activity.52 The ent-kaurenes and modified ent-kaurenes isolated from the New Zealand liverwort, an unidentified Jungermannia species showed cytotoxic activity against HL-60 cells. Compound (124-129) induced DNA fragmentation in HL-60 cells.53-60

3.5. Antimicrobial and Antifungal Activity
Several liverworts, Bazzania species, Conocephalum conicum, Dumortiera hirsuta, Marchantia polymorpha, Metzgeria furcata, Pellia endiviifolia, Plagiochila species, Porella vernicosa complex, P. platyphylla, and Radula species show antimicrobial activity.11 Several such as Bazzania species, Conocephalum conicum, Diplophyllum albicans, Lunularia cruciata, Marchantia polymorpha, Plagiochila species, Porella vernicosa complex, and Radula species display antifungal activity.11
Marchantin A (36) from many Marchantia species, M. chenopoda, M. polymorpha, M. paleacea var. diptera, M. plicata, and M. tosana, shows antibacterial activity against Acinetobacter calcoaceticus (MIC 6.25 µg/ml), Alcaligenes faecalis (100), Bacillus cereus (12.5), B. megaterium (25), B. subtilis (25),
Cryptococcus neoformans (12.5), Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhimurium (100), and Staphylococcus aureus (3.13-25).11 They also have antifungal activity against Alternaria kikuchiana, Aspergillus fumigatus (MIC 100 µg/mL), A. niger (25-100), Candida albicans, Microsprorum gypseum, Penicillium chrysogenum (100), Piricularia oryzae (12.5), Rhizoctonia solani (50), Saccharomyces cerevisiae, Sporothrix schenckii (100), and the dermatophytes Trichophyton mentagrophytes (3.13) and T. rubrum (100).11
The prenyl phenyl ethers (121, 122) isolated from Trichocolea mollissima and T. tomentella, respectively were mildly antifungal against T. mentagrophytes.52 Compound 123 isolated from T. lanata showed similar mild antifungal activity.52 Sacculatal (26), isolated from Pellia endiviifolia showed strong antibacterial activity against Streptococcus mutans (dental caries) at LD50 8 µg/mL, however, polygodial (49) is less active (100 µg/mL) than sacculatal.

3.6. Insect Antifeedant, Mortality and Nematocidal Activity
As mentioned earlier, plagiochiline A (21), found in several Plagiochila species, is a strong antifeedant against the African army worm (Spodoptera exempta).7 Compound 21 shows nematocidal activity against Caenorphabdiitis elegans (111 µg/mL). The pungent sacculatal (26) kills tick species Panonychus citri. Compound 26, eudesmanolides (51, 52) from Chiloscyphus polyanthos, and gymnocolin (71) from Gymnocolea inflata also have antifeedant activity against larvae of Japanese Pieris species.11 A series of natural drimanes and related synthetic compounds was tested for antifeedant activity against aphids.61 Polygodial (49) from the Porella vernicosa complex and warburganal (61) from the African tree Warburgia ugandensis were the most active substances. Natural (-)-polygodial (49) and the synthetic (+)-enantiomer (60) showed similar levels of activity as aphid antifeedants. (-)-Polygodial killed mosquito larvae at a concentration of 40 ppm and had mosquito repellent activity which is stronger than the commercially available DEET. Plagiochilide (130) isolated from Plagiochila species killed Nilaparvata lugens (Delphacidae) at 100 µg/mL.

3.7. Superoxide Release Inhibitory Activity
Excess superoxide anion radical (O2-) in organisms causes various angiopathies, such as cardiac infarction, and arterial sclerosis. Infuscaic acid (clerod-3,13(16)-14-trien-17-oic acid) (131) from Jungermannia infusca and plagiochilal B (58) inhibit the release of superoxide from rabbit PMN at IC50 0.07 and 6.0 µg/mL, respectively and from guinea pig peritoneal macrophage induced by formyl methionyl leucyl phenylalanine (FMLP) at IC50 40 µg /mL, and 25.0 µg/mL respectively.11
Norpinguisone methyl ether (132) from Porella elegantula exhibits 50% inhibition of the release of
superoxide from the guinea pig peritoneal macrophage at 35 µg/mL. The same activity (IC50 7.5 µg/mL)
has been found in cyclomyltaylyl-3-caffeate (133) from Bazzania japonica. Other sesquiterpenoids, plagiochilide (96) isolated from Plagiochila fruticosa, P. ovalifolia and P. yokogurensis, norpinguisone (17) from Porella vernicosa, bicyclogermacrenal (134) from Conocephalum conicum, herbertenediol (135) and isocuparene-3,4-diol (136) from Mastigophola diclados, the diterpenoids, infuscaside A (72) and infuscaside B (73) from Jungermannia infusca, and perrottetianal A (137) from Porella perrottetiana also inhibit superoxide release from guinea pig peritoneal macrophage (IC50 12.5-50 µg/mL).11 Radulanin K (138) from Radula javanica inhibits the release of superoxide anion radical from guinea pig macrophage (IC50 6 µg/mL).48 Polygodial (49) and sacculatal (26) also show superoxide anion radical release inhibition at 4.0 µg/ml from guinea pig peritoneal macrophage.

3.8. 5-Lipoxygenase, Calmodulin, Hyaluronidase Cyclooxygenase Inhibitory Activity and NO Production Inhibitory Activity
A macrocyclic bis-bibenzyl, marchantin A (36) isolated from several Marchantia species showed 5-lipoxygenase inhibitory activity [(89% at 10-5 mol, 94% at 10-6 mol, 45% at 10-7 mol, 16% at 10-8 mol) against LTB3 (5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid)], (99% at 10-5 mol, 97% at 10-6 mol, 70% at 10-7mol, 40% at 10-8 mol) against 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid)] and calmodulin inhibitory activity at ID50 1.85 µg/mL.11 Perrottetins A (139) and D (140) from Radula perrottetii and prenyl bibenzyls (141-145), also from Radula species, riccardin A (107) from Riccardia multifida, and marchantins D (115) and E (116) from Marchantia species had calmodulin inhibitory activity (ID50 2.0-95.0 µg/mL).11 The simple bibenzyls (146-149) from Radula and Frullania species also showed weak calmodulin inhibitory activity (ID50 100 µg/mL) as did the labdane-type diterpene diol, labda-12,14-dien-7,8-diol (151) (ID50 82 µg/mL) isolated from Porella perrottetiana.11 Perrottetin A (139), prenylbibenzyls (140, 148, 40, 150), marchantins D (115) and E (116), and riccardin A (107) also inhibited 5-lipoxygenase (76-4% at 10-6 mol).7 The following phenolic compounds showed significant cyclooxygenase inhibitory activity: marchantin A (36) (IC50 46.4 µM), marchantin B (64) (55.9), marchantin E (116) (58.0), paleatin B (117) (45.2), perrottetin D (140) ((26.2), radulanin H (145) (39.7), isoriccardin C (152) (50.8), and riccardin C (153) (53.5).62

Lunularic acid (155) which is found in almost all liverworts as a minor component has anti-hyaluronidase activity (IC50 0.13 nM). This activity is stronger than that of tranilast (N-3',4'-dimethoxy- cinnamoylanthranilic acid) which is an anti-allergenic agent developed in Japan for oral administration. Lunularic acid (155) has been obtained from hydrangenol-β-glucoside via hydrangenol in good yield.63 Perrottetin E (109) exhibited inhibitory activity for thrombin (IC50 18 µM) which is associated with blood coagulation.64
Over production of nitric oxide (NO) is involved in inflammatory response-induced tissue injury and the formation of carcinogenic N-nitrosamines. Large amounts of NO were expressed and generated by induced iNOS on stimulation of endotoxins or cytokines involved in pathological responses. Thus inhibition of iNOS is very important to control inflammatory disease. The inhibition by macrocyclic bis-bibenzyls isolated from several liverworts of lipopolysaccharide-induced NO production in culture media on RWA 264.7 cells was tested and the IC50 values of each compound is reported in Table 2.65

The presence of 1-2’ and 14-11’ diaryl ether bonds is important for strong inhibition of NO production. The presence of phenolic hydroxyl groups also plays an important role in the inhibitory activity.
Compounds with 7,8-unsaturation dramatically decreased the inhibition of NO, while introduction of a hydroxyl group at C-7’ resulted in slightly decreased activity. The methyl ether (111, 112) of marchantin A (36) and marchantin B (114) showed weaker activity than the original compounds. Herbertane monomers (135, 156, 157) and dimers (161, 165) and cuparenes (159, 160) isolated from Mastigophora diclados also showed inhibition of LPS-induced production of nitrite and their IC50 values were reported in Table 3.66

3.9. Piscicidal and Plant Growth Inhibitory Activity
The strongest piscicides are the pungent (-)-polygodial (49) from Porella vernicosa complex and sacculatal (26) from Pellia endiviifolia, Pallavicinia levieri, Riccardia robata var. yakushimensis, and Trichocoleopsis sacculata. Killie-fish (Oryzia latipes) are killed within 2 hr by 0.4 ppm solution of (49) and (26).7,11 Sacculatal (26) and 1β-hydroxysacculatal (55) also kill killie-fish within 20 min at 1 ppm.34 Killie-fish are also killed within 2 hr by a 0.4 ppm solution of synthetic pungent (+)-polygodial (60). Hence, piscicidal activity is not affected by the chirality of polygodial. Polygodial is also very toxic to fresh water bitterlings, which are killed within 3 min by a 0.4 ppm solution.11 On the other hand, isopolygodial (162) from cultured cells of Porella vernicosa and the higher plant Polygonum hydropiper and isosacculatal (163) from Pellia, Riccardia and Trichocoleopsis species show neither piscicidal nor molluscicidal activity even at 10000 ppm.11 Almost all crude extracts from liverworts which contain bitter or pungent substances show phytotoxic activity. (-)-Polygodial (49) inhibits the germination and root elongation of rice in husk at 100 ppm. At a concentration less than 25 ppm, it dramatically promotes root elongation of rice.7,67

3.10. Neurotrophic Activity
Mastigophorenes A (5), B (164), and D (165) from Mastigophora diclados exhibit neurotrophic properties at 10-5-10-7 M, greatly accelerating neuritic sprouting and network formation in the primary neuritic cell culture derived from the fetal rat hemisphere.68 Plagiochilal B (58) and plagiochilide (130) from Plagiochila fruticosa show not only acceleration of neurite sprouting but also enhancement of choline acetyl transferase activity in a neuronal cell culture of the fetal rat cerebral hemisphere at 10-5 M.14 Plagiochin A (166) also shows the same activity at 10-6 M.69 Two bitter diterpene glucosides, infuscaside A (72) and B (73) show neurite bundle formation at 10-7 M [Asakawa Y. unpublished results].

3.11. Muscle Relaxing Activity
Marchantin A (36) and the related cyclic bis-bibenzyls are structurally similar to bis-bibenzyl-isoquinoline alkaloids such as d-tubocurarine (167) which are pharmacologically important muscle relaxant active drugs. Surprisingly, marchantin A (26) and its trimethyl ether (111) also show muscle relaxing activity.13,70 Nicotine in Ringer solution effects maximum contraction of rectus abdominus in frogs (RAF) at a concentration of 10-6 M. After pre-incubation of marchantin A trimethyl ether (111) (at a concentration of 2 x10-7-2 x 10-4 M) in Ringer solution, nicotine (10-5-10-4 M) was added. At a concentration of 10-6 M, the contraction of RAF decreased by about 30%. d-Tubocurarine chloride (167) exhibits similar effects as does (111) with acetyl choline.13,70

Although the mechanism of action of marchantin A (36) and its methyl ether (111) in effecting muscle relaxation is still unknown, it is interesting that these cyclic bis-bibenzyls possessing no nitrogen atoms, cause concentration dependent decrease of contraction of RAF. Marchantin A and its trimethyl ether also had muscle relaxing activity in vivo in mice. MM2 calculations indicate that the conformation of marchantin A and its trimethyl ether and the presence of an ortho hydroxyl group in (36) and an ortho methoxyl group in (111) contribute to the muscle relaxing activity.70 Marchantin triacetate (113) and 7’,8’-dehydromarchantin A (168) and acyclic bis(bibenzyls), such as perrottetin E (109) and F (110) did not show any muscle relaxing activity.

3.12. Cathepsin L and Cathepsin B Inhibitory Activity
Cathepsin L is correlated with osteoporosis71 and allergy.72 We are currently searching for enzyme
inhibitors from natural products to develop chemopreventive drugs for these diseases. The marchantin series showed both cathepsins L and B inhibitory activity. Isomarchantin C (169) was the strongest inhibitor against both enzymes (95% for cathepsin L and 93% for cathepsin B at 10-5 M). Infuscaic acid (131) exhibited the same activity as mentioned above (63% and 32% at 10-5 M).

The crude extract of Porella japonica showed potent inhibition of cathepsins B and L. Biological activity guided fractionation gave three guaianolides, 11-epiporelladiolide (170), 11,13-dehydroporelladiolide (171), and porellaolide (172), together with porelladiolide (173) and its epoxide (174). Only compound (171) possessed a weak inhibitory activity against cathepsin B (13.4% at 10-5 M) and cathepsin L (24.7% at 10-5 M.73

3.13. Cardiotonic and Vasopressin (VP) Antagonist Activity
MA (36) shows cardiotonic activity [increase coronary blood flow (2.5ml/min. at 0.1mg)].11 Prenyl bibenzyl (142) from Radula perrottetii indicates vasopressin antagonist activity (ID50 27 µg/mL). However, 2-geranylbibenzyl (40) from the same liverwort did not show VP antagonist activity.11

3.14. Liver X-receptor (LXR)α agonist and LXRβ antagonist activity
Plasma high density lipoprotein (HDL) level is inversely related to the risk of atherosclerotic cardiovascular disease. In the research for agents that increases HDL-production, we found that riccardin C (153) and riccardin F (154) isolated from Reboulia hemisphaerica and Blasia pusilla functions as a liver X-receptor (LXR)α agonist and an (LXR)β antagonist. Riccardin C increases plasma HDL level without elevating triglyceride level in mice. This compound also enhances cholesterol efflux from THP-1 cells.74 This compound may provide a novel tool for identifying subtype–function and drug development against obesity. From 1.25 kg of the dried liverwort Blasia pusila, 1 g of riccardin C (153) was obtained as a pure state.

3.15. Synthesis of Bioactive Compounds from Liverwort Constituents
Mastigophola diclados collected in Borneo produces herbertane dimers (20) and (164) possessing neurotrophic activity. Both compounds were obtained by the biotransformation of herbertanediol (135) using Penicillium sclerotiorum.75　

The large stem-leafy liverwort elaborates a large amount of labdanediol (151). We focused on this diterpene to transfer into ambrox (151e) which is an extremely expensive aroma originating from mammals. We succeeded in the hemisynthesis of this compound in seven steps.76

The Indian medicinal plant, Coleus forskolii which has been used to treat disorders of the digestive organs, biosynthesized a highly oxygenated labdane diterpenoid, forskolin (175a) showing blood pressure lowering and cardio protective properties and to have therapeutic potential in glaucoma, congestive heart failure and bronchial asthma. On the other hand, very similar (175), to forskolin highly oxygenated labdanes, for example, folskolin (175a) and its congener (175b) were found in the Japanese liverwort Ptychantus striatus belonging to the Lejeuneaceae as the major component.
We also succeeded in the synthetic transformation of ptychantin A (175) to forskolin (175a) in 12 steps (12% overall yield) and 1,9-dideoxyfolskolin (175b) in 8 steps (37% overall yield).77,78 The more expedient synthetic transformation to forskolin (175a) from ptychantins A (175) have been accomplished by our group.79
Gottsengen et al.80 synthesized riccardin A-C (108, 109, 153) by using a combination of Ulmann, Wittig and Wurz reaction and Ni(0)-assisted intermolecular coupling reaction.
The total synthesis of riccardin B (109) and marchantin A (36) in twelve steps using the intramolecular Wadsworth-Emmons olefination and Wittig reaction was accomplished by Kodama’s group.81-83 Recently, Harrowven et al.84 accomplished the shortest total synthesis of riccardin C (153).

3.16. Recent Reports of Antimicrobial, Cytotoxic and Insect Antifeedant Compounds from Liverworts
Bioactivity-guided fractionation of the crude ether extract of the Chinese M. polymorpha using TLC bioautography assay gave three new bis(bibenzyls), 13,13’-O-isopropylidenericcardin D (176), riccardin H (177) and plagiochin E (178), together with four known bis(benzyls), marchantin A (36), marchantin B (114), marchantin E (116) and neomarchantin A (179).85 Significant antifungal activity against C. albicans was found for 178, 179, 176, with relative MID values of 0.4, 0.2 and 0.25 μg/ml, respectively compared to that of 0.01 μg for positive control miconasole. Compounds (36, 114, 116, 177), showed the moderate activity against the same fungous at the concentration of 2.5, 4.0, 2.5 and 4.0 μg, respectively.85
The genus Asterella belongs to the Aytoniaceae and there are about 80 species in the world. Some species emits strong bad odor. The Chinese A. angusta produces two new bibenzofurane bis(bibenzyls), asterelin A (180) and asterelin B (181), 11-O-demethyl marchantin I (182) and dihydroptychantol A (185), together with known riccardin B (17), marchantin H (183), marchantin M (184), marchantin P (186), plagiochin E (178) and an acyclic bis(bibenzyls), perrottetin E (109).86 Compounds (109, 180-186) showed moderate antifungal activity against clinical pathogenic fungus Candida albicans with MIC values ranging from 16 μg/mL to 512 μg/mL.86

The New Zealand liverwort Lepidolaena taylorii elaborates 11-oxygenated 8,9-secokaurane diterpenoids (187-193) and their structurally related kaurene-15-ones (194-199) all of which were tested in vitro against mouse P388 leukemia and 60 human tumor cell lines including six leukemia cell lines and a range of organ-specific cancers. Compounds 187-189, 191-193 and 194-196, 198, 199 showed cytotoxic activity against P-388 at a concentration of 0.10-1.9 μg/mL.87,88 Ent-kaurene (124) and (200) from the New Zealand Jungermannia species53 showed weak cytotoxic activity against P-388 at 0.48 and 25 μg/mL, respectively. Among the isolated compounds, 8,9-secokaurenes (187, 189, 193) showed selective toxicity amongst human tumor cell lines at a concentration of 1.2, 2.5 and 1.5 μM, respectively. The mode action was explained by Michael acceptors which will act by alkylating cellular thiols for the α-methylene lactone, α,β-unsaturated ketone, α,β-epoxyketones or α,β-unsaturated dialdehyde like polygodial (49).87,88

Hodgsonox (201), a new class of sesquiterpene possessing cyclopenta[5,1]pyran ring system fused to an oxirane ring, was isolated with many other analogues.89,90 Compound (201) exhibits toxicity (LC50 0.27 mg/mL) against the larvae of the Australian green blowfly Lucilia cuprina. The activity of 201 was weaker than that of commercially available insecticide, diazinon (LD50 of 0.0016 mg/mL).89
The New Zealand Lepidolaena clavigera produces clavigerins A-C (202, 203, 206) and their derivatives (204, 205, 207) which are the artifacts formed by alcoholysis of the acetoxy acetal moiety of 203 and 206. Compounds 203 and 206 showed insect antifeedant activities against webbing clothes moth larvae, Tineola bisselliella and Australian carpet beetle larvae Anthrenocenrus australis. Compounds 207 displayed significantly less activity against webbing cloth moss larvae than compounds 203 and 206.91 Compounds 202, 203, 206, 207 showed a weak cytotoxicity (30 µg/disk) against BSC cells. The insect antifeedant activity of compounds 203 and 206 might be due to their facile hydrolysis to corresponding aldehydes which act with biological nuclerophiles such as thiol or amine group. This mode of action was similar to plagiochiline A (21) from the liverwort, Plagiochila species which was easily hydrolyzed to give dialdehyde possessing 1,4-dicarbonyl group as mentioned earlier.
Recently, a number of terpenoids, aromatic compounds and acetogenins which may have some more interesting biological activities were isolated by our group.92-112 The bioassays of several of new products are now in progress.

4. Chemosystematics
Although the morphological classification of liverworts is difficult, because of their small gametophytes, it has been demonstrated that the secondary metabolites, such as lipophilic terpenoids and aromatics in their cellular oil bodies can assist in their taxonomic differentiation. The pattern of terpenoids and aromatic compounds often depends not only on the developmental stage, season and altitudinal distribution, but also on sexual (male, female and sterile) forms of the same species and collection from different locations. Knowledge of chemical constituents of liverworts might serve to delineate not only chemical, but also evolutionary relationships within the Marchantiophyta at the genus or family level. At present, more than 1,000 species of liverworts were collected and investigated in the world.
The fresh liverworts were crushed and extracted with ether, followed by filtration through a Pasteur pipette packed with silica gel and evaporation of solvent to obtain the crude oil which was analyzed by TLC and GC/MS. The remaining crude extracts were chromatographed on silica gel or Sephadex LH-20 to isolate terpenoids and aromatic compounds and their structures elucidated by chemical and spectroscopic analyses. Many kinds of detected and isolated compounds could be used as taxonomic indicators of each species of the Marchantiophyta. Previously, the chemosystematics of 42 families, the Aneuraceae, Pelliaceae, Pallaviciniaceae, Blasilaceae, Fossombroniaceae, Hymenophytaceae and Metzgeriaceae belonging to the order, Metzgeriales, Takakiaceae to the Takakiaceae, Haplomitriaceae to Calybryales, Jungermanniaceae, Lophocoleaceae, Gymnomitraceae (=Marsupellaceae), Arnelliaceae, Plagiochilaceae, Geocalyaceae, Acrobolbaceae, Scapaniaceae, Balantiopsilaceae, Adelantaceae, Lepidolaenaceae, Schistochilaceae, Antheliaceae, Lepidoziaceae, Calypogeiaceae, Cepharoziaceae, Isotachidaceae, Trichocoleaceae, Ptilidiaceae, Lepicoleaceae, Herbertaceae, Radulaceae, Pleuroziaceae, Porellaceae, Frullaniaceae, Jubulaceae and Lejeuneaceae to the Jungermanniales, Monocleaceae to the Monocleales, and Targioniaceae, Aytoniaceae (=Grimaldiaceae), Conocephalaceae, Lunulariaceae, Marchantiaceae and Ricciaceae to the Marchantiales were discussed.9,10 Here we summarize the recent results of chemosystematics of several selected liverworts collected in Ecuador, Germany, Greece, Japan, and Mexico.111

4.1. Chemosystematics of the Jungermanniales
4.1.1. Frullaniaceae
4.1.1.1. Frullania tamarisci subsp. obscura
Frullania is a very large and complex genus with over 1000 taxa. Frullania species are divided chemically into five major groups: 1) sesquiterpene lactone-type, such as eudesmanolides and eremophilanolides, 2) bibenzyl-type, 3) sesquitepene lactone-bibenzyl-type, 4) aromadendorane-type and 5) monoterpene-type.10
Frullania tamarisci subsp. obscura is a rich source of sesquiterpenes, containing hinesene, β-caryophyllene, β-elemene, 4,5-di-epi-aristolochene, bicyclogermacrene (208), α-humulene and eremophilene, of which hinesene (35.5%) was the major component. This species is characterized by the presence of sesquiterpene lactones that cause allergic contact dermatitis. Significant amounts of 4-epi-arbusculin A (209) (11.8%), α-cyclocostunolide (210) (3.8%) and β-cyclocostunolide (211) (1.5%) have been detected. There are two district chemotypes of F. tamarisci subsp. obscura in Japan. The first, Type-T, contains tamariscol (23) which shows remarkable aroma reminiscent of woody note and powdery green note of moss and 5α,7β(H)-eudesma-4α,6α-diol as the major components, while the second, Type-O, produces eudesmane-type sesquiterpene lactones.10 Because of the lack of the former two compounds, the present liverwort belongs to the Type O of F. tamarisci subsp. obscura, belonging to sesquiteperpene lactone-type of the Frullania.

4.1.2. Plagiochilaceae
4.1.2.1. Plagiochila sciophila
There are more than 3,000 species of Plagiochila which are divided mainly into two chemo-types; pungent and non-pungent species. The former type produces 2,3-secoaromadendrane-type sesquiterpene
hemiacetals like, plagiochiline A (21) which was degradated enzymatically to give two pungent
components, plagiochilal (58) and furnoplagiochilal (59).39 The chemical constitution of P. sciophila collected in Japan is totally different from that of the pungent group since it does not elaborate any plagiochiline series. Bicyclohumulenone (44), possessing a strong sweet mossy fragrance as mentioned earlier, β-barbatene (212) and (+)-cyclocolorenone (213) are main components occurring in the Japanese P. sciophila. Occurrence of a high amount of aromadendrane and humulane sesquiterpenoids in this Japanese species was previously reported.10,14 In addition, the following constituents were also detected as minor components: 1-octen-3-yl acetate, bicyclogermacrene (208), cyclomyltaylane, α-barbatene, allo-aromadendrane, sesquisabinene A, and an unidentified sesquiterpene alcohol [M+ 220 (59 base)] possessing a 1,1-dimethyl carbinol (m/z 59). The geographical and seasonal variation of the chemical constituents of the present liverwort has not been observed.

4.1.3. Radulaceae
4.1.3.1. Radula perrottetii
In Asia, there are ca 60 species of Radula which is divided taxonomically into three groups, Radula, Cladoradula and Odotoradula. Their chemical constituents are totally different from the other genera of the Marchantiophyta. Generally they contain a large amount of bibenzyls and prenyl bibenzyls. R. perrottetii, belonging to the subgenus Cladoradula is rich in heterocyclic bibenzyl derivatives.10,14a 2,2-Dimethyl-7,8-dihydroxy-5-(2-phenylethyl) chromene (214), perrottetin A (139) and 3,5-dihydroxy-2-(3-methyl-2-butenyl) bibenzyl (142) have been detected. This liverwort also produced a large amount of bisabola-2,6,11-triene (15.2%). The presence of this compound in the Japanese collection was previously confirmed by Tesso, König and Asakawa.103 Other sesquiterpenoids, such as α- and β-chamigrene, eremophilene, cuparene, β-acoradiene, aristolene, thujopsene, isodauca-4,7(14)-diene and α-gurjunene have also been detected as minor components. Additionally, in the investigated Japanese material, as well as in the above-mentioned Plagiochila sciophila, bicyclohumulenone (44), cyclocolorenone (213) and β-barbatene (212) have been identified.

4.1.4. Lejeuneaceae
4.1.4.1. Trocholejeunea sandvicensis
There are more than 1,000 species of the Lejeuneaceae in the world. Almost all of the species belonging to the Lejeuneaceae is tiny except for Ptychantus species and their chemical constituents are very complex. One of the typical groups of this family is the genera which produce pinguisane-type sesquiterpenoids. T. sandvicensis is one of this groups to produce a large amount of pinguisane-type sesquiterpenoids, which are the most significant chemical markers of this species.10 There are two chemotypes of T. sandvicensis: type-1 contains pingusanin (16) and the other one produces dehydropinguisanin (216) as the major components. T. sandvicensis collected in Tokushima, Japan contains dehydropinguisenol (215) (39%) and an unidentified sesquiterpene lactone [M+ 234 (124 base)] (10%) as the main component, and also pinguisenene, dehydropinguisanin (216), deoxopinguisone (217), ptychanolide (218) and ent-dihydrodiplophyllin. The present species also produced sesquiterpene hydrocarbons, such as bicyclogermacrene (208), isogermacrene D, β-caryophyllene, β-barbatene (212) and valencene.

4.2. Chemosystematics of the Metzgeriales
4.2.1. Pelliaceae
4.2.1.1. Pellia endiviifolia (Figure 5)
The Japanese P. endiviifolia possesses a persistent pungent taste that is due to the sacculatane-type diterpene dialdehyde. There are sacculatal (26) – the major component 11 and 1β-hydroxysacculatal (55)34 – the minor one. In addition, the non-pungent isosacculatal, the C-9 isomer has been detected. In German P. endiviifolia we also identified sacculatal as the main compound and two other sacculatanes, sacculatanolide and 7,17-sacculatadien-11,12-olide. The latter two compounds were isolated previously from axenic cultures of the liverwort Fossombronia wondraczekii, which is classified into the Fossombroniaceae (Metzgeriales).113 Thus, P. endiviifolia is closely related chemically to Fossombronia. The Japanese and German collections also produced a large amount of bicyclogermacrene (208). The two collections differ from one another in the composition of the sesquiterpenes fraction. In Japanese material, β-acoradiene and (E)-β-farnesene have been identified, while in German samples, allo-aromadendrene, tritomarene, bourbon-11-ene, selina-4,7-diene and β-cubebene were detected.

4.2.1.2. Pellia epiphylla
This German Pellia epiphylla is chemically similar to P. endiviifolia since it contains four sacculatane diterpenoids, sacculatal (26) (32.6%), isosacculatal (33.0%), sacculatanolide (1.3%) and its isomer 7,17-sacculatadien-11,12-olide (0.2%); these last two differ in the orientation of the γ-lactone moiety. Minor components include 1-octen-3-yl acetate, β-acoradiene, (E)-β-farnezene, zizaene, dioctyl ether which have been found in the other liverworts.

4.2.1.3. Makinoa crispata
There are at least three chemotypes of M. crispata.10 The present species collected in Japan belongs to the second chemotype, because this type produces dactylol and bicyclogermacrene (208) as major compounds. It is noteworthy that this species elaborated non-pungent sacculatane type diterpene dialdehyde, perrottetianal A (137) (7.5%), which is a characteristic compound of the first chemotype of M. crispata. A significant amount of β-barbatene (212) has also been detected. Thus, chemically, M. crispata is closely related to Pellia endiviifolia. In the volatile fraction of this liverwort many other minor compounds have been identified, for example α-himachalene, α-cubebene, α-copaene, β-bazzanene, α-longipinene, isobazzanene, cis-calamenene, and cinnamolide which is also significant chemical marker of Makinoa. Previously a chlorine-containing drimane (12) as shown in Chart 2 was isolated from this species.10

4.2.1.4. Noteroclada confluens (Figure 6)
Noteroclada is classified in the order Metzgeriales and is a member of the family Pelliaceae within that order. Unlike Pellia and Makinoa, Noteroclada has a leafy appearance. The major component detected in this Ecuadorian species is an unknown sesquiterpene alcohol [M+ 222 (108 base)] (49%). It produced also a large amount of bicyclogermacrene (208) and an unidentified bibenzyl derivative [M+ 344 (249 base)]. Additionally, brasila-1,10-diene, brasila-5(10),6-diene, dactylol, sativene, α-isocomene, pacifigorgia- 6,10-diene, pacifigorgia-1(6),10-diene, and another two unidentified compounds [M+ 330 (139 base); M+ 314 (base)] were detected.
It is noteworthy that N. confluens produces brasilane- and gorgonane-type sesquiterpenoids which have been found in Conocephalum belonging to the Marchantiales and Frullania species to the Jungermanniales, respectively.10

4.2.2. Pallaviciniaceae
4.2.2.1. Symphyogyna brasiliensis
Previously, we placed this species in the Hymenophytaceae, however, it should be replaced in the Pallaviciniaceae.114 The Pallaviciniaceae has thee genera, Pallavicinia, Symphogyna and Allisonia in New Zealand.115 The Ecuadorian S. brasiliensis contains dihydroagarofurane (219) (36.3%) and δ-selinene (220) (20.6%) as the main compounds. Besides 220, this species also produced other eudesmanes: cascarilladiene, selina-4,7-diene and eudesma-5,7(11)-diene. Bicyclogermacrene (208), thujopsene, calarene, γ- and δ-cuprenene, β-cubebene, bourbon-7(11)-ene and trans-dauca-4(11),7-diene were also present. The Venezuelan S. brasiliensis contains a unique labdane, symphyogynolide (27),10 however it was not detected in the present Ecuadorian specimen.

4.2.3. Fossombroniaceae (Codoniaceae)
4.2.3.1. Fossombronia angulosa
The most interesting species belonging to the Metzgeriales order is F. angulosa. The species collected in Greece contained three compounds previously reported in brown algae.116 These are dictyotene (221), the main compound of the volatile fraction (14.1%), (Z)-multifidene (222) and dictyopterene (223). When the species belonging to the Metzgeriales are dried, they immediately emit a sea-shore algae odor. The genuine component of this odor is dimethyl sulfide, which is the most important odor of almost all algal species. This may suggest that some liverworts originate from algae.14 The marine algal components in Fossombronia species are extremely interesting since the species is morphologically quite similar to marine algae, for example Ulva species.
Besides dictyotene, F. angulosa also produced a large amount of an unknown compound [M+ 300 (91 base)] (13.4%), 2-tridecanone (13.0%) and β-sabinene (11.7%). This species also biosynthesized cyathane-type diterpenoids; 5,18-dihydroxy-epi-homo-verrucosane (224) and (-)-2β,9α-dihydro- xyverrucosane (225) which are distributed in some Jungermanniales liverworts (see Table 3). This is the second record of such compounds in Metzgeriales; previously cyathane diterpenoids were isolated from the Arctic liverwort F. alascana.117
Some species belonging to the Metzgeriales biosynthesize the same or the similar specific terpenoids, such as drimanes, pinguisanes, sacculatanes or cyathanes and bis(bibenzyls) to those found in the Jungermanniales as shown in Table 4. In the modern classification of the Marchantiophyta the Jungermanniales and the Metzgeriales are united within the subclass Jungermannidae.118-120 The chemical evidence that both F. angulosa and the Jungermannia genera produce typical cyatane diterpenoids supports the above classification.

4.3. Chemosystematics of Marchantiales
4.3.1. Aytoniaceae
4.3.1.1. Reboulia hemisphaerica
R. hemisphaerica has several chemotypes.109 The Japanese R. hemisphaerica, collected in two different places in Tokushima, is characterized by totally different chemical compositions. The collection from Tokushima Bunri University Campus produced cyclomyltaylane and chamigrane sesquiterpenoids. Cyclomyltaylan-5α-ol (18) (43.5%) was the major component, which was present with β-chamigrenes, β-chamigrene-1β-ol and β-chamigrene-10α-ol (226). This collection belongs to the cyclomyltaylane-type. It is noteworthy that the separately investigated sporophytes and thalli of this species are characterized by very similar chemical composition. The second collection, from Momijigawa (Aioicho, Tokushima), 50km apart from the University campus contained mainly gymnomitranes, of which (+)-gymnomitr-8(12)-en-9α-ol (47) (31.3%) was the main compound. The other characteristic components were β-barbatene (212) (14.7%), gymnomitr-8-en-12-ol (227), gymnomitr-8(12)-en-9-one, (8R)-(+)-gymnomitran-9-one and gymnomitrol. This collection also produced a significant amount of β-caryophyllene (16.8%).

4.3.1.2. Asterella echinella (Figure 7)
Reboulia and Asterella are both members of the Reboulioideae, subfamily Aytoniaceae. Asterella species are known to emit intense, characteristic scents, both pleasant and unpleasant.121 The Mexican A. echinella produces mainly sesquiterpenes. It elaborated the sesquiterpene hydrocarbons: β-barbatene (212) (43%), β-caryophyllene (7.6%), β-acoradiene (2.9%), α-barbatene, isobazzanene, and δ-cuprenene, and also significant amounts of the sesquiterpene alcohols 4β-hydroxygermacra-1(10),5-diene (228) (4.2%), β-sabinene (3.5%), 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol (5.3%) and its acetate (4.9%). The present species is chemically different from A. venosa since the major constituents of A. venosa are monoterpenoids.

4.3.1.3. Asterella venosa (Figure 8)
As mentioned above, monoterpenes were the most representative in A. venosa (77%). The main compound was geranyl acetate (229) (40%), together with β-myrcene (13.9%), α-pinene (9.7%) and myrtenyl acetate (9.2%). Besides monoterpenoids, β-barbatene (212), β-caryophyllene and squalene have been identified. This Mexican species is closely related to the Portuguese A. africana. Chemical analysis of the essential oils from this species indicated that they were dominated by the monoterpene fraction. Myrtenyl acetate and α-pinene were the major components of all the oil samples.122


4.3.2. Conocephalaceae
4.3.2.1. Conocephalum conicum
The Japanese C. conicum is chemically more advanced than C. japonicum, since the former elaborates not only monocyclic but also bicyclic monoterpenoids.10 There are three chemo-types of Conocephalum conicum. The types 1, 2 and 3 emit (-)-sabinene, (+)-bornyl acetate, and methyl cinnamate as the major
components, respectively, which are responsible for the characteristic odor of each type.22 In C. conicum collected in Momijigawa, Tokushima, myrcene, limonene, neryl acetate, camphene, β-sabinene and β-pinene were identified. This C. conicum, like the German collection, belongs to the β-sabinene chemotype, because it also produced a large amount of this compound (38.4%).10,22 β-Elemene, β-caryophyllene, germacrene D, cubebol and ent-1(10)E,5E-germacradien-11-ol (230) have also been identified.

4.3.2.2. Conocephalum japonicum
The Japanese C. japonicum produced only limonene, as a minor monoterpene metabolite. The major component is isolepidozene (231) (49%), the diastereomer of the widespread sesquiterpene hydrocarbon, bicyclogermacrene (208). In addition, β-sabinene (31.7%) and isolepidozene (231) (14.8%) are also the significant components of the German C. conicum. Bicyclogermacrene (208) and another two germacranes: bicyclogermacrene-14-al (134) and 4β-hydroxygermacra-1(10),5-diene (228) as well as costunolide (232) and dihydrocostunolide (233) and β-cyclocostunolide (211) have been detected as minor components. We reported that C. japonicum is chemically different from the Japanese C. conicum and rather similar to Wiesnerella denudata since C. conicum does not produce germacrane-type sesquiterpene lactones, while W. denudata elaborates the same lactones as those found in C. japonicum.10 It is noteworthy that the German C. conicum contains the same monoterpene hydrocarbon, β-sabinene and isolepidozene (231) like C. japonicum.

4.3.2.3. Wiesnerella denudata
There are three different chemical races of W. denudata, the costunolide-guaianolide-type, the costunolide-type and the guaianolide-type.123 Guaianolides, for example zaluzanin C (234), zaluzanin D (235) and 8α-acetoxyzaluzanin D (236), alongside a large amount of neryl acetate (40.7%), have been detected with tulipinolide (53), dihydrotulipinolie (237) and its cyclized β-cyclocostunolide (211) in the Japanese W. denudata. The present species belongs to the costunolide-guaianolide chemo-type. In addition, Δ3-carene, 1-octen-3-yl acetate, nerol, 4β-hydroxygermacra-1(10),5-diene (228), together with two unidentified sesquiterpenoids [M+ 230 (105 base); M+ 230 (107 base)] were also detected as minor components. The presence of tulipinolide and guaianolides in W. denudata indicates that this liverwort is more evolved chemically that C. japonicum since the latter species produces neither C-8 acetoxylated costunolide nor guaianolides.10

4.3.3. Marchantiaceae
4.3.3.1. Dumortiera hirsuta (Figure 9)
There are a few chemo-types of Dumortiera hirsuta. The Argentinean D. hirsuta elaborates three dumortane sesquiterpenoids (238-240), a rearranged dumortane (241) and a nordumortane (242), along with marchantin C (246).10 The Ecuadorian D. hirsuta elaborates the peculiar α-pyrone derivatives, dumortins A-C (243-245).124 The Japanese and Argentinean species do not produce such aromatic
products. The major component detected in the Japanese D. hirsuta is an unknown sesquiterpenoid [M+ 220 (43 base)] (33%). This species elaborates germacrane type sesquiterpenoids, especially ent-1(10)E,5E-germacradien-11-ol (230) (13.8%), germacrene D (8.7%) and 4β-hydroxygermacra- 1(10),5-diene (228) (3.6%). These compounds were also detected in Conocephalum conicum; the first and second in the Japanese collection and the third in the German liverwort. Minor components include 1-octen-3-yl acetate, β- and δ-elemene, β-barbatene (212), γ-muurolene, (Z,E)-α-farnesene and cubebol.

4.3.3.2. Marchantia polymorpha (Figure 10)
The GC-MS pattern of the ether extract of M. polymorpha which were collected in Hatacho, Tokushima and Toyama in Japan is almost the same as that of the Polish specimen collected in Wroclaw.

(–)-Cyclopropanecuparenol (247) is the main component identified in the Japanese M. polymorpha thalli and female sporophytes. This liverwort also produces other cuparane as well as thujopsane and chamigrane sesquiterpenoids. α-Cuprenene and also δ- and β-cuprenene, thujopsene, thujopsane-2β-ol, thujopsenone, α-chamigrene, ent-9-oxo-α-chami-grene have been identified. The sesquiterpenoid composition of M. polymorpha thalli was quite similar to that of female sporophytes. In contrast to thalli, female sporophytes elaborated hexadecanoic acid and its ethyl ester, (Z,Z) 9,12-octadecadienoic acid, oleic acid and also oxacycloheptadeca-2-one.

4.3.3.3. Marchantia paleacea var. diptera
The Japanese M. paleacea var. diptera showed at least 27 peaks on the gas chromatogram of the diethyl ether extract of among which β-caryophyllene (36.5%) and an unidentified diterpenoid [M+ 290 (81 base)] (15.3%) were the most abundant components. Additionally, two monoterpenes, β-pinene and limonene, 1-octen-3-yl acetate, β-elemene, calarene, β-selinene and β-caryophyllene oxide have been identified. Fatty acids and long chain butenolides (248, 249) which are characteristic secondary metabolite of this species 10 have also been detected. All terpenoids detected are ubiquitous component not only in liverworts but also in higher plants, however, the presence of a great amount of marchantin-type macrocyclic compounds, for example, marchantin A (36) is the chemical marker of this species and M. polymorpha.10

4.3.3.4. Marchantia tosana
There are two chemotypes of M. tosana. Type 1 is closely related chemically to M. polymorpha and M. paleaceae var. diptera since these three Marchantia species produce marchantin type macrocyclic compounds with 2,5-dimethoxy-3-hydroxyphenthrene and type 2 produces 2,7-dimethoxy- 3-hydroxyphenanthrene derivatives.10 The present specimen collected in Momijigawa, Tokushima produced large amounts of isolepidozene (231) (45.2%) and β-barbatene (212) (23.1%). It is noteworthy that isolepidozene has been found as a predominant component in Conocephalum japonicum and the German C. conicum belonging to the Conocephalaceae as mentioned earlier. M. tosana also contained β-elemene, α-barbatene, isobazzanene, β-acoradiene, germacrene D, α- and γ-cuprenene and (4S*,5S*,6R*,7R*)-1(10)E-lepidozen-5-ol (250) as minor constituents. The present species contains marchantin A, thus it is classified as type 1.

4.3.4. Monosoleniaceae
4.3.4.1. Monosolenium tenerum
M. tenerum was studied chemically first time. In the German M. tenerum only two components have been detected in GC-MS. Analysis of MS suggested that the first one was a new bibenzyl derivative, 3,5,4’-trimethoxybibenzyl (251) and the second one a phthalide, identified as 3-(4’-methoxybenzyl)- 5,6-dimethoxyphthalide (252). The latter compound was isolated previously from the Australian liverwort Frullania falciloba, belonging to the Jungermanniales.125 The Chinese specimen was also analyzed by GC-MS. The GC-MS pattern was completely identical to that of the German collection.
The chemical constitution of M. tenerum is absolutely different from that of all Marchantiales species so far investigated chemically. It is quite interesting to note that the present species is closely related chemically to the Frullania (Jungermannales) chemotype II, which produce bibenzyls as the major product.

The volatiles of the liverwort species are characterized by a large number of sesquiterpenoids, as shown
in Table 5. Several acyclic, mono-, bi-, and tricyclic sesquiterpene systems are present, including many unique combinations. The investigated liverworts produced mainly sesquiterpene hydrocarbons, with bicyclogermacrene (208) and β-barbatene (212) being the most widespread in the present and previously analyzed species. Because sesquiterpene hydrocarbons are prevalent throughout liverworts and most liverwort species elaborate the same compounds, it is difficult to use these compounds as chemosystematic markers. However, other sesquiterpenoids, such as eudesmane, eremophilane, germacrane and guaiane sesquiterpene lactones, pinguisane, cuparane and gymnomitrane sesqui- and sacculatane and cyatane diterpenoids can be used as chemosystematic markers.

Some specific monoterpenoids are also used as chemical markers. Monoterpenes amounted to 77% of all compounds present in the volatiles of Asterella venosa. The main component was geranyl acetate (229), a compound with a lemon-like odor. In contrast to the above mentioned species, which have a pleasant smell, Asterella species grown in Malaysia emit an intense, unpleasant odor, which is due to skatole, which composes about 20% of the total extract.14a
In several cases, aromatic compounds are valuable chemosystematic indicators, especially bibenzyls and bis(bibenzyl) derivatives.10,14a Among investigated liverworts, the presence of bibenzyls and prenyl bibenzyls (139, 142, 214) are characteristic of the Japanese Radula perrottetii (Jungermanniales). It is noteworthy that the German and Chinese Monoselenium tenerum, belonging to the Marchantiales, produced bibenzyls and phthalides, previously detected in the Jungermanniales.125

CONCLUSION
The bryophytes, in particular, the Marchantiophyta (liverworts) are charming plants as the sources of bioactive substances. Most of liverworts produce lipophilic terpenoids (mono-, sesqui-and diterpenoids) and aromatic compounds, of which only a few nitrogen or sulfur containing compounds have been found.7,14a,e It is noteworthy that ca, 80% of the sesqui- and diterpenoids found in liverworts are the enantiomers of those found in higher plants. Since the last century, 5% of the total bryophytes have been studied chemically. Although liverworts are tiny plant groups, they produce many peculiar terpenoids, such as homomonoterpenoids, 1,4-dimethylazulenenoids, sesquiterpenoids: africane, brasilane, drimane, eremophilane, eudesmane, germacrane, guaiane, gymnomitrane (=barbatane), herbertane, myltaylane, cyclomyltaylane, pacifigorgiane, pinguisane, 2,3-secoaromadendrane, striatane, trifarane and diterpenoids: cyatane, dolabellane, fusicoccane, sacculatane and aromatic compounds: bibenzyl, bi(bibenzyls), naphthalene, 4-mehylstyrenethiocyanate, β-methylthioacrylate etc, all of which are significant chemical markers of each liverwort genus and family.

ACKNOWLEDGMENTS
A part of this work was supported by a Grant-in-Aid for the Scientific Research (A) (No. 11309012) and the Open Research (for AL) from the Ministry of Education, Culture, Sports, Science and Technology.

This paper is dedicated to Professor Emeritus Keiichiro Fukumoto for his 75th birthday.

References

1. G. Garnier, L. Bezaniger-Beauquesne, and G. Debraux, ‘Ressources médicinales de la flore francaise’, Paris, Vigot Frères Éditeurs, 1969, Vol. 1, pp. 78-81.
2. C. Suire, Rev. Bryol. Lichenol., 1975, 41, 105.
3. H. Ding, ‘Zhong guo Yao yun Bao zi Zhi wu’, Kexue Jishu Chuban She, Shanghai, 1982, pp. 1-409.
4. P.C. Wu, The Bryological Times, 1982, 13, 5.
5. H. Ando and A. Matsuo, ‘Advances in Bryology’, ed. by W. Schultze-Motel, Vaduz, J. Cramer, 1984, Vol. 2, pp. 133-224..
6. Y. Asakawa, ‘Phytochemicals in Human Health Protection, Nutrition, and Plant Defense’, ed. by J. Romeo, New York, Kluwer Academic/Plenum Publishers, 1999, pp. 319-342.
7. Y. Asakawa, ‘Progress in the Chemistry of Organic Natural Products’, ed. by W. Herz, H. Grisebac, and G. W. Kirby, Vienna, Springer, 1982, Vol. 42, pp. 1-285.
8. L. Harinantenaina and Y. Asakawa, Nat. Prod. Commun., 2007, 2, 701.
9. Y. Asakawa, Phytochemistry, 2001, 56, 297. CrossRef
10. Y. Asakawa, Phytochemistry, 2004, 65, 623. CrossRef
11. Y. Asakawa, ‘Bryophytes: Their Chemistry and Chemical Taxonomy’, ed. by D. H. Zinsmeister and R. Mues, Oxford, Oxford University Press, 1990, pp. 369-410.
12. Y. Asakawa, ‘Bryophyte Development: Physiology and Biochemistry’, ed. by R. N. Chopra, S. C. Bhatla, Boca Raton, CRC Press, 1990, pp. 259-87.
13. Y. Asakawa, ‘Bioactive Natural Products: Detection, Isolation, and Structural Determination’, ed. by S. M. Colegate and R. J. Molyneux, Boca Raton, CRC Press, 1993, pp. 319-47.
14. (a) Y. Asakawa, ‘Progress in the Chemistry of Organic Natural Products’, ed. by W. Herz, G. W. Kirby, R. E. Moore, W. Steglich, and Ch. Tamm, Vienna, Springer, 1995, Vol. 65, pp. 1-562; (b) Y. Asakawa, Pure Appl. Chem., 2007, 79, 557; CrossRef (c) Y. Asakawa, Nat. Prod. Commun., 2008, 3, 77; (d) H. Inoue, Kagakuasahi, 1988, 116; (e) S. H. von Reuβ and W. A. Konig, Eur. J. Org. Chem., 2004, 1184. CrossRef
15. M. Mizutani, Misc. Bryol. Lichenol., 1975, 6, 64..
16. Y. Asakawa, Aroma Res., 2004, 15, 204.
17. Y. Asakawa, M. Toyota, H. Tanaka, T. Hashimoto, and D. Joulain, J. Hatt. Bot. Lab., 1995, 78, 183.
18. M. Toyota and Y. Asakawa, Flav. Fragr. J., 1994, 9, 237. CrossRef
19. M. Toyota, H. Koyama, and Y. Asakawa, Phytochemistry, 1997, 44, 1261. CrossRef
20. M. Tori, M. Sono, Y. Nishigaki, K. Nakashima, and Y. Asakawa, J. Chem. Soc., Perkin Trans. 1, 1991, 435. CrossRef
21. M. Sono, ‘M. Ph. D. Thesis’, Tokushima Bunri University, 1991, pp. 1-110.
22. M. Toyota, T. Saito, J. Matsunami, and Y. Asakawa, Phytochemistry, 1997, 44, 1265. CrossRef
23. J. D. Connolly, Rev. Latinoam. Quim., 1982, 12, 121.
24. J. D. Connolly, ‘Bryophytes: Their Chemistry and Chemical Taxonomy’, ed. by D. H. Zinsmeister and R. Mues, Oxford, Oxford University Press, 1990 pp. 41-58.
25. M. Toyota, Y. Asakawa, and J.-P. Frahm, Phytochemistry, 1990, 29, 2334. CrossRef
26. J. Sporle, ‘Ph. D. Thesis’ Universität des Saarlandes, 1990, pp. 1-179.
27. S. Huneck, J. D. Connolly, A. Freer, and D. S. Rycroft, Phytochemistry, 1988, 27, 1405.. CrossRef
28. Y. Asakawa, M. Toyota, and A. Cheminat, Phytochemistry, 1986, 25, 2555. CrossRef
29. H. Inoue, ‘Koke no Sekai (World of Bryophytes)’, Tokyo, Idemitsu Ltd., 1978, 178.
30. L. Harinantenaina and S. Takaoka, J. Nat. Prod., 2006, 69, 1193. CrossRef
31. D. Kioy, A. I. Gray, and P. G. Waterman, Phytochemistry, 1990, 29, 3535. CrossRef
32. Y. Asakawa, M. Toyota, Y. Oiso, and J. E. Braggins, Chem. Pharm. Bull., 2001, 49, 1380. CrossRef
33. C. Socolsky, N. Murunga, and A. Bardon, Chem. Biodiv., 2005, 2, 1105. CrossRef
34. T. Hashimoto, Y. Okumura, K. Suzuki, S. Takaoka, Y. Kan, M. Tori, and Y. Asakawa, Chem. Pharm. Bull., 1995, 43, 2030.
35. Y. Asakawa, L. J. Harrison, and M. Toyota, Phytochemistry, 1985, 24, 261. CrossRef
36. K. Ono, T. Sakamoto, and Y. Asakawa, Phytochemistry, 1992, 31, 1249. CrossRef
37. K. Ono, T. Sakamoto, H. Tanaka, and Y. Asakawa, Flav. Fragr. J., 1996, 11, 53. CrossRef
38. M. Toyota, A. Ueda, and Y. Asakawa, Phytochemistry, 1991, 30, 567.. CrossRef
39. T. Hashimoto, H. Tanaka, and Y. Asakawa, Chem. Pharm. Bull., 1994, 42, 1542.
40. F. Nagashima, M. Toyota, and Y. Asakawa, Phytochemistry, 1990, 29, 1619. CrossRef
41. D. S. Rycroft, ‘Bryophytes: Their Chemistry and Chemical Taxonomy’, ed. by D. H. Zinsmeister and R. Mues, Oxford, Oxford University Press, 1990, pp. 109-119.
42. S. Huneck, J. D. Connolly, L. J. Harrison, R. Joseph, W. Phillip, D. S. Rycroft, G. Ferguson, and M. Parvez, J. Chem. Res., 1986, 162.
43. F. Nagashima, H. Tanaka, S. Takaoka, and Y. Asakawa, Phytochemistry, 1997, 45, 555. CrossRef
44. M. Toyota, C. Nishimoto, and Y. Asakawa, Chem. Pharm. Bull., 1998, 46, 542.
45. A. Bardon, G. B. Mitre, N. Kamiya, M. Toyota, and Y. Asakawa, Phytochemistry, 2002, 59, 205. CrossRef
46. Y. Asakawa, M. Toyota, M. von Konrat, and J. E. Braggins, Phytochemistry, 2003, 62, 439. CrossRef
47. M. von Konrat, J. E. Braggins, Y. Asakawa, and M. Toyota, The Bryologist, 2006, 109, 141. CrossRef
48. M. Toyota, K. Tanimura, and Y. Asakawa, Planta Med., 1998, 64, 462. CrossRef
49. Y. Asakawa, Pure Appl. Chem., 1994, 66, 2193. CrossRef
50. F. Nagashima, Y. Ohi, T. Nagai, M. Tori, Y. Asakawa, and S. Huneck, Phytochemistry, 1993, 33, 1445. CrossRef
51. T. Yoshida, T. Hashimoto, T. Takaoka, Y. Kan, M. Tori, Y. Asakawa, J. M. Pezzuto, T. Pengsupaarp, and G. A. Cordell, Tetrahedron, 1996, 52, 14487. CrossRef
52. N. B. L. Perry, M. Foster, S. D. Lorimer, B. C. May, R. T. Weavers, M. Toyota, E. Nakaishi, and Y. Asakawa, J. Nat. Prod., 1996, 59, 729. CrossRef
53. F. Nagashima, M. Kondoh, T. Uematsu, A. Nishiyama, S. Saito, M. Sato, and Y. Asakawa, Chem. Pharm. Bull., 2002, 59, 808. CrossRef
54. F. Nagashima, M. Kondoh, M. Kawase, S. Simizu, H. Osada, M. Fujii, Y. Watanabe, M. Sato, and Y. Asakawa, Planta Med., 2003, 69, 377. CrossRef
55. F. Nagashima, W. Kasai, M. Kondo, M. Fujii, Y. Watanabe, J. E. Braggins, and Y. Asakawa, Chem. Pharm. Bull., 2003, 551, 1189. CrossRef
56. I. Suzuki, M. Kondoh, F. Nagashima, M. Fujii, Y. Asakawa, and Y. Watanabe, Planta Med., 2004, 70, 401. CrossRef
57. M. Kondo, I. Suzuki, M. Sato, F. Nagashima, S. Simizu, M. Handa, M. Fujii, H. Osada, Y. Asakawa, and Y. Watanabe, J. Pharm. Exp. Therap., 2004, 311, 115. CrossRef
58. I. Suzuki, M. Kondoh, M. Harada, N. Koizumi, M. Fujii, Y. Asakawa, and Y. Watanabe, Planta Med., 2004, 70, 723. CrossRef
59. M. Kondo, I. Suzuki, M. Harada, F. Nagashima, M. Fujii, Y. Asakawa, and Y. Watanabe, Planta Med., 2003, 71, 275. CrossRef
60. F. Nagashima, M. Kondoh, M. Fujii, S. Takaoka, Y. Watanabe, and Y. Asakawa, Tetrahedron, 2005, 61, 4531. CrossRef
61. Y. Asakawa, W. Dawson, D. C. Griffith, J.-Y. S. Lallemand, V. Ley, K. Mori, M. Mudd, M. Pezechk-Leclaire, A. J. Picektt, W. Watanabe, C. M. Woodcock, and Z. Zong-Ning, J. Chem. Ecol., 1988, 14, 1845. CrossRef
62. C. Schwartner, W. Bor, C. Michel, U. Franck, S. B. Muller-Jakic, A. Nenninger, Y. Asakawa, and H. Wagner, Phytomedicine, 1995, 2, 113. CrossRef
63. T. Hashimoto, M. Tori, and Y. Asakawa, Phytochemistry, 1988, 27, 109. CrossRef
64. F. Nagashima, S. Momosaki, Y. Watanabe, M. Toyota, S. Huneck, and Y. Asakawa, Phytochemistry, 1996, 41, 207. CrossRef
65. L. Harinantenaina, D. N. Quang, T. Nishizawa, T. Hashimoto, C. Kohchi, G.-I. Soma, and Y. Asakawa, J. Nat. Prod., 2005, 68, 1779. CrossRef
66. L. Harinantenaina, D. N. Quang, T. Nishizawa, T. Hashimoto, C. Kohchi, G.-I. Soma, and Y. Asakawa, Phytomedicine, 2007, 14, 486. CrossRef
67. Y. Asakawa, S. Huneck, M. Toyota, T. Takemoto, and C. Suire, J. Hatt. Bot. Lab., 1979, 46, 163.
68. Y. Fukuyama and Y. Asakawa, J. Chem. Soc. Perkin Trans. 1, 1991, 2737. CrossRef
69. Y. Fukuyama and Y. Kodama, Food Ingred. J., 1996, 45.
70. Z. Taira, M. Takei, K. Endo, T. Hashimoto, Y. Sakiya, and Y. Asakawa, Chem. Pharm. Bull., 1996, 42, 52.
71. N. Katsunuma, J. Bone Mineral Metab., 1997, 15, 1. CrossRef
72. Y. Matsunaga, T. Saibara, H. Kido, and N. Katsunuma, Fed. Europ. Biochem. Soc. Lett., 1993, 324, 325. CrossRef
73. T. Hashimoto, H. Irita, M. Yoshida, A. Kikkawa, M. Toyota, H. Koyama, Y. Motoike, and Y. Asakawa, J. Hatt. Bot. Lab., 1998, 84, 309.
74. N. Tamehiro, Y. Sato, T. Suzuki, T. Hashimoto, Y. Asakawa, S. Yokoyama, T. Kawanishi, Y. Ohno, K. Inoue, T. Nagao, and M. N. Nishimaki, Fed. Europ. Biochem. Soc. Lett., 2005, 579, 5299. CrossRef
75. L. Harinantenaina, Y. Noma, and Y. Asakawa, Chem. Pharm. Bull., 2005, 53, 256. CrossRef
76. T. Hashimoto, K. Shiki, M. Tanaka, S. Takaoka, and Y. Asakawa, Heterocycles, 1998, 49, 315. CrossRef
77. H. Hagihara, F. Takeuchi, T. Suzuki, T. Hashimoto, and Y. Asakawa, Tetrahedron Lett., 2003, 44, 2305. CrossRef
78. H. Hagihara, F. Takeuchi, M. Kudou, T. Hoshi, T. Suzuki, T. Hashimoto, and Y. Asakawa, J. Org. Chem., 2006, 71, 4619. CrossRef
79. H. Hagihara, M. Tsukagoshi, T. Hoshi, T. Suzuki, T. Hashimoto, and Y. Asakawa, Synlett., 2008, 929. CrossRef
80. A. Gottsegen, M. Norgadi, B. Vermes, M. Kajtar-Peredy, and E. Bihatsi-Karsai, J. Chem. Soc. Perkin Trans. 1, 1990, 315. CrossRef
81. M. Kodama, Y. Shiobara, K. Matsumura, and H. Sumitomo, Tetrahedron Lett., 1995, 26, 877. CrossRef
82. Y. Shiobara, H. Sumitomo, M. Tsukamoto, C. Harada, and M. Kodama, Chem. Lett., 1985, 1587. CrossRef
83. M. Kodama, Y. Shiobara, H. Sumitomo, K. Matsumura, M. Tsukamot, and C. Harada, J. Org. Chem., 1988, 53, 72. CrossRef
84. D. C. Harrowven, T. Woodcock, and P. D. Howes, Angew. Chem. Int. Ed., 2005, 44, 3899. CrossRef
85. C. Niu, J. B. Qu, and H. Lou, Chem. Biodiv., 2006, 3, 34. CrossRef
86. J. Qu, C. Xie, H. Guo, W. Yu, and H. Lou, Phytochemistry, 2007, 68, 1767. CrossRef
87. N. B. Perry, E. J. Burgess, and R. S. Tangney, Tetrahedron Lett., 1996, 37, 9387. CrossRef
88. N. B. Perry, E. J. Burgess, S. H. Baek, B. T. Weavers, W. Geis, and A. B. Mauger, Phytochemistry, 1999, 50, 423. CrossRef
89. G. D. Ainge, P. J. Gerard, S. F. R. Hinkley, S. D. Lorimer, and R. T. Weavers, J. Org. Chem., 2001, 66, 2818. CrossRef
90. A. J. Barlow, B. J. Compton, U. Hertewich, S. D. Lorimer, and R. T. Weavers, J. Nat. Prod., 2005, 68, 825. CrossRef
91. N. B. Perry, E. J. Burgess, L. M. Foster, P. J. Gerard, M. Toyota, and Y. Asakawa, J. Nat. Prod., 2008, 71, 258. CrossRef
92. M. Toyota, T. Shimomura, H. Ishii, and Y. Asakawa, Chem. Pharm. Bull., 2002, 50, 1390. CrossRef
93. T. Hashimoto, S. Takaoka, M. Tanaka, and Y. Asakawa, Heterocycles, 2003, 59, 645. CrossRef
94. R. Lu, C. Paul, S. Basar, W. A. Konig, T. Hashimoto, and Y. Asakawa, Phytochemistry, 2003, 63, 581. CrossRef
95. F. Nagashima, N. Matsumura, Y. Ashigaki, and Y. Asakawa, J. Hatt. Bot. Lab., 2003, 94, 197.
96. F. Nagashima, M. Murakami, S. Takaoka, and Y. Asakawa, Phytochemistry, 2003, 64, 1319. CrossRef
97. L. Harinantenaina and Y. Asakawa, Biochem. System. Ecol., 2004, 32, 1073. CrossRef
98. M. Toyota, I. Omatsu, J. E. Braggins, and Y. Asakawa, Chem. Pharm. Bull., 2004, 52, 481. CrossRef
99. F. Nagashima, T. Sekiguchi, and Y. Asakawa, Chem. Pharm. Bull., 2004, 52, 556.. CrossRef
100. F. Nagashima, M. Murakami, S. Takaoka, and Y. Asakawa, Chem. Pharm. Bull., 2004, 52, 949. CrossRef
101. L. Harinantenaina and Y. Asakawa, Chem. Pharm. Bull., 2004, 52, 1382. CrossRef
102. G. B. Mitre, N. Kamiya, A. Bardon, and Y. Asakawa, J. Nat. Prod., 2004, 67, 31.. CrossRef
103. H. Tesso, W. A. Konig, and Y. Asakawa, Phytochemistry, 2005, 66, 941. CrossRef
104. F. Nagashima, K. Mishi, Y. Hamada, S. Takaoka, and Y. Asakawa, Phytochemistry, 2005, 66, 1662.. CrossRef
105. F. Nagashima, T. Sekiguchi, and Y. Asakawa, Nat. Prod. Res., 2005, 19, 679. CrossRef
106. L. Harinantenaina, R. Kurata, and Y. Asakawa, Chem. Pharm. Bull., 2005, 53, 515. CrossRef
107. F. Nagashima, Y. Kuba, and Y. Asakawa, Chem. Pharm. Bull., 2006, 54, 902. CrossRef
108. L. Harinantenaina, Y. Takahara, T. Nishizawa, C. Kohchi, G.-I. Soma, and Y. Asakawa, Chem. Pharm. Bull., 2006, 54, 1046.. CrossRef
109. M. Furusawa, T. Hashimoto, Y. Noma, and Y. Asakawa, Chem. Pharm. Bull., 2006, 54, 996. CrossRef
110. M. Toyota, I. Omatsu, J. Braggins, and Y. Asakawa, J. Oleo Sci., 2006, 55, 579. CrossRef
111. A. Ludwiczuk, F. Nagashima, S. R. Gradstein, and Y. Asakawa, Nat. Prod. Commun., 2008, 3, 133.
112. Y. Asakawa, M. Toyota, F. Nagashima, and T. Hashimoto, Nat. Prod. Commun., 2008, 3, 289.
113. H. Feld, U. M. Hertewich, J. Zapp, and H. Becker, Phytochemistry, 2005, 66, 1094. CrossRef
114. S. R. Gradstein, S. P. Churchill, and N. Salazar-Allen, ‘Guide to the Bryophytes of Tropical America’, New York, The New York Botanical Garden Press, 2001.
115. K. W. Allison and J. Child, ‘The Liverworts of New Zealand’, Dunedin, University of Otago Press, 1975.
116. T. Kajiwara, A. Hatanaka, Y. Tanaka, T. Kawai, M. Ishihara, T. Tsuneya, and T. Fujimura, Phytochemistry, 1989, 28, 636. CrossRef
117. C. Grammes, G. Urkhardt, M. Veith, V. Huch, and H. Becker, Phytochemistry, 1997, 44, 1495. CrossRef
118. R. M. Schuster, ‘Bryophyte Systematics’, ed. By G. C. S. Clarke and J. G. Duckett, London, Academic Press, 1979, Vol. 14, pp. 41-82.
119. R. M. Schuster, ‘New Manual of Bryology’, ed. by R. M. Schuster, Nichinan, The Hattori Botanical Laboratory, 1984, Vol. 2, pp. 1971-1092.
120. B. Crandall-Stotler and R. E. ‘Bryophyte Biology’, ed. by A. J. Shaw and B. Goffinet, Cambridge, Cambridge University Press, 2000, pp. 21-70..
121. Y. Asakawa, J. Hatt. Bot. Lab., 1982, 53, 283.
122. A. C. Figueiredo, J. G. Barroso, L. G. Pedro, S. S. Fontinha, M. Sim-Sim, S. Sérgio, L. Luis, and J. J. C. Scheffer, Flav. Frag. J., 2006, 21, 534. CrossRef
123. M. Toyota, K. Kondo, M. Konoshima, and Y. Asakawa, J. Hatt. Bot. Lab., 2000, 89, 289.
124. L. Kraut, R. Mues, A. Speicher, M. Wagmann, and T. Eicher, Phytochemistry, 1997, 42, 1693. CrossRef
125. Y. Asakawa, K. Takikawa, and M. Tori, Phytochemistry, 1987, 26, 1023. CrossRef