Chemical Constituents of Bryophytes: Structures and Biological

Review pubs.acs.org/jnp

Chemical Constituents of Bryophytes: Structures and Biological Activity Yoshinori Asakawa*,† and Agnieszka Ludwiczuk‡ †

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 20-093 Lublin, Poland

‡

ABSTRACT: Comparatively little attention has been paid to the bryophytes for use in the human diet or medicine in spite of the presence of 23 000 species globally. Several hundred new compounds have been isolated from the liverworts (Marchantiophyta), and more than 40 new carbon skeletons of terpenoids and aromatic compounds were found. Most of the liverworts studied elaborate characteristic odiferous, pungent, and bitter-tasting compounds, of which many show antimicrobial, antifungal, antiviral, allergic contact dermatitis, cytotoxic, insecticidal, anti-HIV, plant growth regulatory, neurotrophic, NO production and superoxide anion radical release inhibitory, muscle relaxing, antiobesity, piscicidal, and nematocidal activities. The biological effects ascribed to the liverworts are mainly due to lipophilic sesqui- and diterpenoids, phenolic compounds, and polyketides, which are the principal constituents of their oil bodies. Some mosses and liverworts produce significant levels of vitamin B2 and tocopherols, as well as prostaglandin-like highly unsaturated fatty acids. The most characteristic chemical phenomenon of the liverworts is that most of the sesqui- and diterpenoids are enantiomers of those found in higher plants. In this review, the chemical constituents and potential medicinal uses of bryophytes are discussed.

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INTRODUCTION The bryophytes are found all over the world, except in the sea. They grow on wet soil or rocks, on the trunks of trees, in lakes and rivers, and even in Antarctica. The bryophytes are placed taxonomically between the algae and the pteridophytes. There are about 23 000 species worldwide, and they are further divided into three phyla, the Bryophyta (mosses; 14 000 species), the Marchantiophyta (liverworts; 6000 species), and the Anthoceratophyta (hornworts; 300 species). Scientists take the view that liverworts are the earliest plants, evolving 472 million years ago, so these are the ancestors of all land plants that we now know.1 Among the bryophytes, almost all liverworts possess cellular oil bodies, which are membrane-bound cell organelles that consist of ethereal terpenoids and aromatic oils suspended in a carbohydrate- or protein-rich matrix, while the other two phyla do not. These oil bodies are very important biological markers for the taxonomy of the Marchantiophyta.2 The phytochemistry of bryophytes has been neglected for a long time because they are morphologically very small and are difficult to collect in large quantities as pure samples. In addition, their identification is also very challenging, even under the microscope. They are considered to be nutritionally of limited value to humans, in spite of the presence of more than 23 000 species because there are so many other edible vegetables, mushrooms, algae, and even several ferns and lichens available. In fact, no references concerning their use as foods for humans have been seen. © XXXX American Chemical Society and American Society of Pharmacognosy

Many liverworts produce hot-tasting substances, like capsaicin or a piperine, attributed to some sesquiterpene and diterpene dialdehydes. Some mosses, such as Fissidens and Rhodobryum species, elaborate strong sweet tastes. These tasty liverworts and mosses may be useful as certain spices for foods or as food additives. Some of these plant species produce significant amounts of vitamins B2 and E and related compounds. Thus, it is considered that the bryophytes have potentially important food or spice properties that can be exploited. On the other hand, many moss species have been used as medicinal plants. Bryophytes are also dried and crushed, and the resulting powder is mixed with oil to make ointments that reputedly heal cuts, burns, and external wounds. North American Indians have used Bryum, Mnium, and Philonotis species and Polytrichum juniperinum as medicinal mosses to treat burns, bruises, and wounds.3 Marchantia polymorpha has been used as a diuretic in Europe. French liverworts were soaked with white liquor and patients drank the resulting mixture of liquor and extracts.4 In the literature on Chinese medicinal spore-forming plants, 24 lichens, 74 sea-algae, 22 mosses, five liverworts, 112 fungi, and 329 ferns have been listed with their Latin names, morphological characteristics, distribution locations, pharmacological Special Issue: Special Issue in Honor of Susan Horwitz Received: November 12, 2016

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DOI: 10.1021/acs.jnatprod.6b01046 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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activity and effects, and their prescription uses in detail.5 Several mosses have been widely used medicinally in China, to treat burns, bruises, external wounds, snake bite, pulmonary tuberculosis, neurasthenia, fractures, convulsions, scald, uropathy, pneumonia, and neurasthenia, among other uses.2f,g,5,6 Many species of liverworts possess characteristic fragrant odors and an intense pungent, sweet, or bitter taste. Generally, bryophytes are not damaged by bacteria and fungi, insect larvae and adults, snails, slugs, and small mammals. Furthermore, some liverworts cause intense allergic contact dermatitis and allelopathy.2c,d,l Although liverworts possess such potentially pharmacologically interesting substances, their isolation and structural characterization were neglected for almost a century. We have been interested in the application of bryophytes for human or domestic animal uses and in the isolation and the structural elucidation of biologically active substances from bryophytes and their biological assay. Since 1972, more than 1000 species of bryophytes collected around the world were chemically analyzed with respect to their chemistry, pharmacology, and application as sources of cosmetics and as medicinal or agricultural agents. The biological activities of liverworts are due to the terpenoids and aromatic compounds that are present in the oil bodies in each species.2c−g,j,l,7 Here, the biological and chemical diversity of liverworts and the chemical structures of the terpenoids, aromatic compounds, and polyketides found in several of these plants and their biological activity, including their characteristic odor and taste, as well as their possibility of use as foods or spices, are surveyed. A few biosynthetic pathways and the hemi- and total synthesis of selected biologically active compounds from liverwort constituents are also discussed.

exhibit a wide range of biological effects.2c,d,l The biological activity ascribed to the liverworts is mainly due to their lipophilic sesqui- and diterpenoids, phenolic compounds, and polyketides, which are the principal constituents of the oil bodies. The most characteristic chemical phenomenon of the liverworts is that most of the sesqui- and diterpenoids are enantiomers of those found in higher plants, although there are a few exceptions, such as the germacrane- and guaiane-type sesquiterpenoids. It is very noteworthy that different species of the same genus such as Frullania tamarisci and F. dilatata (Frullaniaceae), each produces sesquiterpene lactone enantiomers.2c,d,l Some liverworts, such as Lepidozia species (Lepidoziaceae), biosynthesize both enantiomers.2l However, the presence of nitrogen- or sulfur-containing compounds in the bryophytes is very rare.2d,g,l Recently, several nitrogen- and sulfur-containing compounds (1−4) were isolated from the Mediterranean liverwort, Corsinia coriandrina (Corsiniaceae, Marchantiales),11 and two prenylated indole derivatives (5, 6) from Riccardia species (Aneuraceae, Metzgeriales).2d

BIODIVERSITY OF BRYOPHYTES The Marchantiophyta (liverworts) include two subclasses, the Jungermanniidae and Marchantiidae, and six orders, 49 families, 130 genera, and 6000 species. There are 54 endemic genera in the Southern Hemisphere countries, such as in New Zealand and Argentina.8 In Asia, including Japan, a relatively large number of endemic genera (21) have been recorded, while South Africa, Madagascar, North America, and Europe are very poor regions of endemic genera.9 The richness of the endemic genera of bryophytes in the southern hemisphere suggests that these organisms might have originated in the Antarctic islands and migrated to the Northern Hemisphere during a long evolutionary process. In Southeast Asia and South America, there are rain forests where a large number of liverwort species have been found. However, many different species, such as those belonging to the Lejeuneaceae, are intermingled in colonies, and it is tedious and time-consuming work to purify each of these.2i

Figure 1. Nitrogen- and sulfur-containing compounds (1−10) from liverworts.

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Skatole (7) was isolated from or detected in the Malaysian Asterella or Mannia (Aytoniaceae)12 and the Tahitian Cyathodium foetidissimum (Cyathodiaceae) liverwort species.13 Benzyl- (8, 9) and β-phenethyl β-methylthioacrylates (10) were found in the New Zealand Balantiopsis rosea and the former two compounds isolated from the Japanese Isotachis japonica.2d Highly evolved liverworts belonging to the Marchantiaceae produce phytosterols, e.g., campesterol, stigmasterol, and sitosterols.2c,d,l Almost all liverworts elaborate α-tocopherol and squalene. The characteristic components of the Bryophyta are highly unsaturated fatty acids and alkanones, such as 5,8,11,14,17eicosapentaenoic acid, 7,10,13,16,19-docosapentaenoic acid, and 10,13,16-nonadecatrien-7-yn-2-one. The neolignans are one of the most important chemical markers of the Anthocerotophyta,2d while the presence of glycosylated terpenoids is very rare in the Marchantiophyta. A few bitter kaurene diterpene glycosides were found in Jungermannia species, and a number of flavonoid glycosides were detected in liverworts and mosses.2c,d,l Liverworts are also known to produce dictyopterenes, which are known as a sex pheromones of marine brown algae, such as Ectocarpus siliculosus, Dictyopteris membranaceae, and Cutleria multif idia.14 These pheromones are lipophilic, volatile acetogenins consisting of C8 or C11 linear or monocyclic hydrocarbons or their epoxides.15 The liverwort species Fossombronia angulosa, collected in Greece, produced dictyopterene (11) as the major volatile component, as well as dictyotene (12) and multifidene (14).2h,16 Dictyotene (12) and (E)-ectocarpene (13) were also

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CHEMICAL DIVERSITY OF BRYOPHTES Extraction of the oil bodies with n-hexane or ether, using an ultrasonic apparatus, is very convenient for stem-leafy liverworts in order to yield a large amount of crude extract. In the case of thalloid liverworts, the specimens are ground mechanically and then extracted with nonpolar solvents. Several hundred new compounds have been isolated from the liverworts (Marchantiophyta), and more than 40 new carbon skeleton terpenoids and aromatic compounds were found.10 Most of the liverworts studied elaborate characteristic odiferous, pungent, and bitter-tasting compounds, of which many B



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found in the Tahitian Chandonanthus hirtellus as the major volatiles.13 However, the same species collected in Japan contained both pheromones as very minor components.17 These chemical identifications suggest that some families of liverworts and algae may have an evolutionary relationship.

Figure 2. Brown algae sex pheromones (11−14) also isolated from liverworts.

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Figure 3. α-Tocopherol (15) and related compounds (16−19) from liverworts.

BRYOPHYTES AS POTENTIAL FOOD SUPPLEMENTS AND SPICES The mosses Barbella pendula, B. enervis, Floribundaria nipponica, Hypnum plumaeforme, and Neckeropsis nitidula and some of the liverworts contain quantitatively much vitamin B2. Chickens and puppies fed a diet including these powered bryophytes gained more weight than control animals. The supplement did not cause any sickness or adverse taste.18 Since there are more than 14 000 species of mosses, more species possessing vitamin B2 will undoubtedly be discovered. The liverworts Marchantia polymorpha and Pellia endiviifolia and the mosses Atrichum undulatum and Mnium hornum produce vitamin E (α-tocopherol) (15), vitamin K (16), plastoquinone (17), plastohydroquinone (18), and α-tocoquinone (19).7a,b The last compound was also found in the moss Racomitrium japonicum.19 Nishiki et al.20 analyzed 700 liverworts chemically and found that almost all of them contained α-tocopherol and squalene. Prostaglandin-like highly unsaturated fatty acids (20−24) have been found in many mosses, such as Dicranum scoparium, D. japonicum, and Leucobryum species.8,21 These and other unsaturated fatty acids are viscous liquids, and it is thought that they are instrumental in protecting herbivorous animals living in very cold places from the cold.22 Such unsaturated fatty acids, like those obtained from fish oils, play an important role as antioxidants in the human body. Acetylcholine (25) and a cytokinin-like compound, N6-(isopentenyl)adenine (26), have been found in the callus tissue from the hybrid of Funaria hygrometrica × Physcomitrium pyriforme.23 Many liverworts produce hot-tasting substances that may be able to be used as spices for foods and as food preservatives, because they also possess potent antimicrobial and antifungal activities.

Figure 4. Unsaturated fatty acids (20−24), acetylcholine (25), and an adenine derivative (26) from mosses.

its acetate (28). An unidentified Malaysian liverwort (Asterella or Mannia) emitted skatole (7), which is responsible for the unpleasant smell of this liverwort.12 The stink bug smell of Chiloscyphus pallidus is attributable to (E)-dec-2-enal (29) and its related unsaturated aldehydes (30−32).25 The characteristic mold-like smell of Leptolejeunea elliptica is due to 4-ethylanisol (33), 4-ethyl phenol (34), and 4-ethyl phenyl acetate (35).26 A mixture of (R)-dodec-2-en-1,5-olide (36) and (R)-tetradec-2-en-1,5-olide (37) proved to be responsible for the strong, milky smell of the liverwort Cheilolejeunea imbricata.26 Plagiochila sciophila elaborates bicyclohumulenone (38), which possesses an aroma reminiscent of a variety of scents based on a strong woody note, resembling the odors of patchouli, vetiver, cedar wood, irises, moss, and carnations. Tamariscol (39), isolated from Frullania tamarisci ssp. tamarisci, F. tamarisci ssp. obscura, F. nepalensis, and F. asagrayana, similarly possesses an aroma reminiscent of the woody and powdery green notes of mosses, hay, costus, violet leaves, and seaweeds. Both compounds are important in commerce, where they are used as perfumes as such and as fragrance components in the powdery floral, oriental bouquet, fantastic chypre, fancy violet, and white rose types in various cosmetics. It is

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BIOACTIVE COMPOUNDS FROM BRYOPHYTES The bryophytes elaborate a number of biologically active secondary metabolites. Furthermore, these metabolites have interesting biological properties that will be discussed in the following subsections. Characteristic Odor. All liverworts emit a very strong odor when crushed. Lipophilic terpenoids and aromatic compounds constituted in the oil bodies are responsible for the intense sweet-woody, turpentine, sweet-mossy, fungal-like, carrot-like, mushroom-like, or seaweed-like odors.7a,i,24 Almost all liverworts that smell of mushrooms contain 1-octen-3-ol (27) and C



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Figure 5. Characteristic odiferous components (27−37) from liverworts.

noteworthy that the Frullania species that produce tamariscol only grow in high mountain regions.27 There are three chemotypes of the liverwort Conocephalum conicum. Types 1, 2, and 3 emit (−)-sabinene, (+)-bornyl acetate, and methyl (E)-cinnamate (40) as the major components, respectively, and are responsible for the characteristic odors of each type.28 When C. conicum (type 2) was stored in a glass vessel for a long time (2−9 months), the liverwort changed morphologically to form fine and thin gametophytes appearing to be a totally different plant from the original one. The stressed C. conicum (type 2) also drastically changed its chemical composition. The chemical marker bornyl acetate of this type completely disappeared, and then methyl (E)-cinnamate (40) (16.9%) was newly produced after nine months.29 When Marchantia paleacea ssp. diptera was cultured on Nipp soil or Gamborg’s B5 medium including 2% sucrose, (S)-(−)-perillaldehyde (41), the most important aroma for Perilla f rutescens (Lamiaceae), was elaborated in 50% yield, along with 1perillyl alcohol, shisool, limonene-10-ol, and p-menthen-10-al.30 The intense, carrot-like odor of Jungermannia obovata is due to 4-hydroxy-4-methyl cyclohex-2-en-1-one (42).31 In turn, the strong and distinct mossy odor of Lophocolea heterophylla and L. bidentata is due to a mixture of (−)-2-methylisoborneol (43) and geosmin (44).32 The latter compound was also found in cultured Symphyogyna brongniartii.33 The strong, sweet mossy note of Mannia f ragrans is attributable to grimaldone (45),34 while the sweet turpentine-like odor of the French Targionia hypophylla is due to a mixture of cis- and trans-pinocarveyl acetates (46, 47).35 The strong sweet, mushroom-like odor of the ether extract of Wiesnerella denudata is due to (+)-bornyl acetate (48) and to a mixture of the monoterpene hydrocarbons α-terpinene, β-phellandrene, terpinolene, α-pinene, β-pinene, and camphene. Compound 48 was the major volatile component of the ether extract of W. denudata collected in Borneo.2i The steam distillate as well as the volatile extract from the Japanese specimen contains mainly neryl acetate (49) (27% and 42%, respectively).2i,7a Gackstroemia decipiens emits a characteristic odor that is due to the presence of a mixture of (−)-13-hydroxybergamota-2,11-diene (50) and four santalane derivatives (51−54). These compounds were characterized by their olfactory effects.36 Isoafricanol (55), isolated from Pellia epiphylla, is responsible for the typical odor of its sporophyte.37 A fresh and dried sample of Takakia lepidozioides, the most primitive liverwort, emits a strong coumarin-like odor when crushed. In fact, the fresh leaves contain coumarin and 1,4-dihydroquinone, as the major components, along with dihydrocoumarin, 1,4-benzoquinone, dihydrobenzofuran, α-asarone, and α-tocopherol (15), as the minor component.38



Pungency and Bitterness. Some genera of liverworts, such as the Hymenophyton, Pellia, Porella, Trichocoleopsis, and Wiesnerella species, elaborate potent pungent constituents,2d,7i,24c,39 which exhibit interesting biological activities described in subsequent sections. Most of the North American liverworts contain unpleasant substances, some of which taste like immature green pea seeds or pepper.40 Species in the genera Anastrepta, Lophozia, Scapania, and many other stem-leafy liverworts produce intensely bitter principles.2d,l Jamesoniella autumnalis contains an intense bitter principle for which the taste resembles that of the leaf of lilac or Swertia japonica or the root of Gentiana scabra var. orientalis, although the bitter principles have not yet been isolated. Porella vernicosa complex (P. arboris-vitae, P. fauriei, P. gracillima, P. obtusata ssp. macroloba, P. roellii, and P. vernicosa) produces intense pungent substances.2c,d,l The strong hot taste of Porella species is due to the drimane sesquiterpene dialdehyde (−)-polygodial (56).2b,c,7h,39,41 This compound is known as the constituent of the Japanese medicinal plant Polygonum hydropiper, the Malaysian P. minus, and the Argentinean P. punctatum var. punctatum (Polygonaceae).41 The sacculatane diterpene dialdehyde sacculatal (57), two eudesmanolides, diplophyllolide (59) and ent-7α-hydroxydiplophyllolide (60), and a germacranolide, tulipinolide (61), that possess pungent tastes were isolated from Pellia endiviifolia and Trichocoleopsis sacculata, Chiloscyphus polyanthos, and Wiesnerella denudata, respectively.2c,d,l An additional pungent constituent, 1β-hydroxysacculatal (58), was obtained from P. endiviifolia, together with several sacculatane-type diterpenoids.42 The hot taste of Pallavicinia levieri and Riccardia lobata var. yakushimensis, which belong to the Metzgeriales, is also due to sacculatal (57).43 Polygodial (56) and sacculatal (57) have been obtained from cell D



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Figure 8. Pungent sesqui- (56, 59−65) and diterpenoids (57, 58) from liverworts.

suspension cultures of Porella vernicosa and Pellia endiviifolia, respectively.44 When one chews a whole plant of the stem-leafy liverworts Plagiochila asplenioides, P. f ruticosa, P. ovalifolia, and P. yokogurensis, which contain plagiochiline A (62) and plagiochiline I (63), a potent pungent taste slowly develops. It is suggested that both compounds 62 and 63 might be converted into the pungent unsaturated dialdehyde by human saliva. In fact, enzymatic treatment of 62 with amylase in phosphate buffer or with human saliva produced two strong pungent components, plagiochilal B (64), of which the partial structure is similar to that of the pungent drimane-type sesquiterpene dialdehyde, polygodial (56), and furanoplagiochilal (65).45 The pungent taste of Porella acutifolia ssp. tosana is due to the presence of the hydroperoxysesquiterpene lactones 1α- (66) and 1β-hydroperoxy-4α,5β-epoxygermacra-10(14),11(13)-dien12,18α-olide (67).46 The New Zealand liverwort Hymenophyton f labellatum produces a different pungent-tasting substance, from the other aforementioned liverworts, namely, 1-(2,4,6trimethoxyphenyl)buta-(2E)-en-1-one (68).47

Figure 10. Bitter-tasting diterpenoids (69−77) from liverworts.

the sweet constituents have been characterized, except for the presence of sucrose (Asakawa, unpublished results). Allergenic Contact Dermatitis. Frullania species are notable as liverworts that can cause incidents of very intense allergenic contact dermatitis. The primary allergy-inducing substances are the sesquiterpene lactones (+)-frullanolide (78) and (−)-frullanolide (79), which were isolated from Frullania dilatata and F. tamarisci ssp. tamarisci, respectively.2c The dihydrofrullanolides (80 and 81), with α-methyl-γ-butyrolactone units isolated from the above-mentioned liverworts, do not cause an allergenic response. F. asagrayana, F. bolanderi, F. brasiliensis, F. eboracensis, F. f ranciscana, F. inf lata, F. kunzei, F. nisquallensis, F. riparia, and other Frullania species that contain sesquiterpenes (82−88) with α-methylene-γ-butyrolactone functionality also cause strong allergenic contact dermatitis.7n The allergens of Schistochila appendiculata are a mixture of long-chain alkylphenols, such as 3-undecyl- (89), 3-tridecyl(90), 3-pentadecyl- (91), and 3-heptadecyl phenol (99), longchain alkyl salicylic acids, inclusive of 6-undecyl- (92), 6-tridecyl- (93), and 6-pentadecyl salicylate (94), and their potassium salts, potassium 6-undecyl- (95), 6-tridecyl- (96), and 6-pentadecyl salicylate (97), as well as 6-undecyl catechol (98).7n The dermatitis symptoms produced are similar to that caused by the long-chain alkylphenols of the seeds of Ginkgo biloba and by plants in the Anacardiaceae, such as Toxicodendron vernicif luum and Rhus succedanea.51 Cytotoxicity against Cancer Cells. In Vitro Studies. Many of the compounds, especially sesqui- and diterpenoids, isolated from liverworts and mosses showed cytotoxic activity against cancer cells. Table 1 shows compounds with potent cytotoxic activity studied in vitro. Plagiochila is a rich source of the cytotoxic sesquiterpenoids belonging to the 2,3-seco-aromadendrane type.2l Aponte et al.52

Figure 9. Pungent sesquiterpene peroxides (66, 67) and a phenyl butanone derivative (68) from liverworts.

Most of the species belonging to the Lophoziaceae produce bitter substances. Gymnocolea inf lata is persistently bitter and induces vomiting when one chews a few leaves for several seconds. This surprisingly intense bitterness is due to the clerodane diterpene lactone gymnocolin A (69).7b Jungermannia inf usca has an intense bitter taste due to the presence of infuscasides A−E (70−74). These metabolites were the first glycosides to be isolated from liverworts.48 Anastrepta orcadensis, Barbilophozia lycopodioides, and Scapania undulata are also bitter liverworts from which the highly oxygenated, bitter diterpenoids anastreptin A (75), barbilycopodin (76),31a,49 and scapanin A (77)50 were isolated, respectively. Many Fissidens and Rhodobryum species belonging to the Bryophyta contain sweet-tasting substances. However, none of E



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(−)-Herbertene-1,2-diol (108) and its dimers (−)-mastigophorene C (109) and (−)-mastigophorene D (110), isolated from the ether and methanol extracts of the Tahitian Mastigophora diclados, were cytotoxic against HL-60 cells with IC50 values of 1.4, 1.4, and 2.4 μg/mL, respectively.57 Pinguisane sesquiterpenoids 7-keto-8-carbomethoxypinguisenol (111) from Porella perrottetiana and naviculyl caffeate (112) from Bazzania novae-zelandiae showed cytotoxicity against HL-60 cells (IC50 2.7 μM)58 and demonstrated growth inhibitory effects against P-388 murine leukemia cells with a GI50 value of 1.1 μg/mL,60 respectively. 13-Hydroxychiloscyphone (113), obtained from Chiloscyphus rivularis, showed cytotoxic activity against A-549 lung carcinoma cells (IC50 value 2.0 μg/mL).61 Longipinane-type sesquiterpenoids marsupellone (114) and acetoxymarsupellone (115) from Marsupella emarginata showed cytotoxicity (IC50 1 μg/mL) against P-388 cells.7e,62 Among diterpenoids isolated from liverwort species, it is worth mentioning sacculatal (57). This compound isolated from Pellia endiviifolia was cytotoxic for Lu1, KB, KB-V, LNCaP, and ZR-75-1 (IC50 2.7−5.7 μM) (Cordell, Pezzuto, Asakawa, unpublished results). The ethanol-soluble extract of Lepidolaena taylorii, which showed cytotoxicity against the P-388 cell line (IC50 1.3 μg/mL), was purified to give a series of ent-8,9-secokaurane diterpenoids and ent-kaur-16-en-15-one derivatives.63 The cytotoxicity of these 8,9-seco- and ent-kaurenes was tested against the murine P-388 leukemia and several human tumor cell lines, inclusive of six leukemia and a range of organ-specific cancer cell lines. Rabdoumbrosanin (116) and 8,14-epoxyrabdoumbrosanin (117) showed the most potent cytotoxic activity (mean IC50 values of 0.06 and 0.27 μg/mL, respectively). 16,17-Dihydrorabdoumbrosanin (118) also showed cytotoxicity against P-388 cells at 0.80 μg/mL.63a Compounds 116 (including 10% 118) and 117 showed differential cytotoxicity in vitro when tested against five leukemia cell lines, with 116 showing an average IC50 value of 0.4 μM, but the cells were not killed (LC50 > 50 μM).63a Compound 119 was the most active against the leukemia cell lines, with a mean GI50 value of 0.3 μM, and the least active against various central nervous system cancer cell lines. The mean GI50 values, from 60 different human tumor cell lines, for compounds 116, 117, and 119 were 1.2, 2.5, and 1.5 μM, respectively.63b The mode of action for the cytotoxicity of the ent-8,9-seco-kaur-16-en-15-one and ent-kaur-16-en-15-one series was supported by Michael addition of a thiol to the C-16−C-17 double bond of 116, but the C-8−C-14 double bond of 117 was relatively unreactive.63 The ent-16-kauren-15one derivatives (120−123) together with rearranged kaurenes jungermannenones A−D (124−127), isolated from the New Zealand Jungermannia species, inhibited HL-60 cells with IC50 values from 0.4 to 7.0 μM (Table 1).64,65 Since apoptosisinducing compounds are potential candidates as antitumor agents, the induction of apoptosis by these kaurane diterpenoids in HL-60 cells was tested. The results suggested that these compounds are potent inducers of apoptosis in these cells.64−66 Treatment with these compounds caused proteolysis of poly(ADP-ribose) polymerase, a sign of activation of the apoptotic machinery, whereas one feature of cell death induced by treatment with compounds 120 and 124−127 was apoptosis. Treatment with compounds 121 and 122 induced necrosis against HL-60 cells.64−67 Activation of caspase-8 and processing of Bid, a typical substrate of caspase-8, were also observed on treatment of compounds 120−122 and 124.67,68

Figure 11. Allergy-inducing sesquiterpene α-methylene-γ-lactones (78, 79, 82−88) and their inactive dihydro derivatives (80 and 81) from liverworts.

Figure 12. Allergy-inducing long-chain alkylphenols (89−99) from liverworts.

isolated plagiochilines A (62) and I (63) from the Peruvian Plagiochila disticha. Both compounds showed cytotoxic activity against a panel of human tumor cell lines (3T3, H460, DU145, MCF-7, M-14, HT-29, K562). Among them, compound 62 exhibited the most potent activity against all of these cell lines, showing GI50 values of 1.4−6.8 μM. These studies also showed the selectivity of plagiochiline A (62) against human prostate carcinoma and, interestingly, displayed marked preference for the hormone-dependent human prostate cancer.52 An ether extract of Plagiochila ovalifolia showed inhibitory activity against P-388 murine leukemia cells, and its constituents, plagiochiline A (62), plagiochiline A-15-yl octanoate (100), and 14-hydroxyplagiochiline A-15-yl (2E,4E)-dodecadienoate (101), exhibited IC50 values of 3.0, 0.05, and 0.05 μg/mL, respectively.53 Several sesquiterpene lactones isolated from liverworts, e.g., lepidozenolide (102) from Lepidozia fauriana54 as well as (−)-ent-arbusculin B (103) and (−)-ent-costunolide (104) from Hepatostolonophora paucistipula,55 and glaucescenolide (105) from Schistochila glaucescens,56 showed potent cytotoxicity when evaluated against the P-388 murine leukemia cell line (IC50 2.1, 1.1, 0.7, and 2.3 μg/mL, respectively). Two lactones obtained from Tahitian liverworts, (−)-diplophyllolide (59) from Mastigophora diclados and tulipinolide (61) from Frullania species, were shown to be cytotoxic against the HL-60 cell line.57,58 (−)-Diplophyllolide (59) also showed cytotoxicity against KB cells with an IC50 value of 3.3 μg/mL.57 The liverworts Porella perrottetiana and Chandonanthus hirtellus produced lactones 4α,5β-epoxy-8-epi-inunolide (106) and chandolide (107), respectively, which were evaluated for cytotoxic activity against the HL-60 leukemia cell line.58,59 F



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Table 1. Cytotoxic Compounds from Liverworts and Mosses compound Liverwort compounds plagiochiline A (62)

cell line

rabdoumbrosanin (116) 8,14-epoxyrabdoumbrosanin (117) 16,17-dihydrorabdoumbrosanin (118) ent-11α-hydroxy-16-kauren-15-one (120)

KB P-388 3T3 H490 DU145 MCF-7 M-14 HT-29 K562 U937 HCT116 A549 3T3 DU145 MCF-7 M-14 HT-29 K562 P-388 P-388 P-388 P-388 P-388 P-388 HL-60 KB HL-60 HL-60 HL-60 HL-60 HL-60 HL-60 HL-60 P-388 A-549 P-388 P-388 Lu1 KB KB-V LNCaP ZR-75-1 P-388 P-388 P-388 HL-60

ent-1β-hydroxy-9(11),16-kauradien-15-one (121) ent-9(11),16-kauradiene-12,15-dione (122) ent-6β-hydroxy-16-kauren-15-one (123) jungermannenone A (124)

P-388 HL-60 HL-60 HL-60 HL-60

jungermannenone B (125)

HL-60

jungermannenone C (126)

HL-60

jungermannenone D (127)

HL-60

marchantin A (132)

MCF-7

plagiochiline I (63)

plagiochiline A-15-yl octanoate (100) 14-hydroxyplagiochiline A-15-yl (2E,4E)-dodecadienoate (101) lepidozenolide (102) (−)-ent-arbusculin B (103) (−)-ent-costunolide (104) glaucescenolide (105) (−)-diplophyllolide (59) tulipinolide (61) 4α,5β-epoxy-8-epi-inunolide (106) chandolide (107) (−)-herbertene-1,2-diol (108) (−)-mastigophorene C (109) (−)-mastigophorene D (110) 7-keto-8-carbomethoxypinguisenol (111) naviculyl caffeate (112) 13-hydroxychiloscyphone (113) marsupellone (114) acetoxymarsupellone (115) sacculatal (57)

IC50 [μg/mL]

IC50 [μM]

GI50 [μg/mL]

GI50 [μM]

ED50 [μg/mL]

0.287a 3.053 1.752 4.852 1.452 6.852 3.952 1.952 4.852 2.652 4.752 9.452 3.852 4.852 9.052 6.452 6.152 8.752 0.0553 0.0553 2.154 1.155 0.755 2.356 1.457 3.357 4.658 8.558 5.359 1.457 2.457 2.557 2.758 1.160 61

2.0 162 162

5.7 3.2 2.7 5.7 5.7 0.0663a 0.2763a 0.863a

0.163b 0.2763b 1.963b

0.363b 0.863b 5.963b

0.4863b

1.663b

0.8266 0.4964

4.074 G

7.064 0.5964 0.4065 0.2864 1.372 1.2165 5.372 1.2865 7.872 0.7865 2.772 11.575 DOI: 10.1021/acs.jnatprod.6b01046 J. Nat. Prod. XXXX, XXX, XXX−XXX


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Table 1. continued compound

cell line

IC50 [μg/mL]

IC50 [μM]

GI50 [μg/mL]

GI50 [μM]

ED50 [μg/mL]

Liverwort compounds

marchantin B (133) marchantin E (136) 2α,5β-dihydroxybornane-2-cinnamate (137) α-zeorin (138) (+)-3α-(4′-methoxybenzyl)-5,7-dimethoxyphthalide (139) Moss compounds momilactone B (140) pallidisetin A (142) pallidisetin B (143) 1-O-methylohioensin B (144)

1-O-methyldihydroohioensin B (145) 1,14-di-O-methyldihydroohioensin B (146)

5.575 3.77b 3.27b 7.67b

A256 KB KB KB HepG2 P-388 HL-60 KB

4.579 1.180 0.9258 0.9658

HT-29 SW620 RPMI-7951 U-251 MG RPMI-7951 U-251 MG HT-29 RPMI-7951 U-251 MG U-251 MG A549 RPMI-7951

181 181 1.082 2.082 1.082 2.082 1.082 1.082 2.082 0.882 1.082 1.082

Figure 13. Cytotoxic sesquiterpenoids (100−115) from liverworts.

ent-11α-Hydroxy-16-kauren-15-one (120) isolated from the Japanese liverwort Jungermannia truncata showed cytotoxic activity against P-388 cells with a GI50 value of 0.48 μg/mL.63b The same compound together with other kauranes (128−131) were evaluated for cytotoxicity against HL-60 human leukemia cells. Of these, only compound 120 was cytotoxic with an IC50 value of 0.82 μM.66 The presence of an enone group at C-15/ C-16 in this class of metabolites appears to be essential for

cytotoxic activity as well as for the induction of apoptosis and the activation of caspases in human leukemia cell lines.66,69,70 The ent-kaurenes with an enone group, 120−122 and 124, selectively inhibited nuclear factor-κB (NF-κB)-dependent gene expression following treatment with TNF-α. Compound 120, in combination with TNF-α, caused an increase in apoptosis in human leukemia cells, accompanied by activation of caspases, and when combined with camptothecin, also resulted in an H



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increase in apoptosis.71 Jungermannenones A−D (124−127), obtained from Jungermannia species, induced cytotoxicity in HL-60 human leukemia cells accompanied by DNA fragmentation and nuclear condensation. Both are biochemical markers of apoptosis induction, which occurred through a caspaseindependent pathway. Compounds 124 and 127 showed inhibitory activity for NF-κB, which is a transcriptional factor of anti-apoptotic factors. Thus, ent-kaurene diterpenoids from liverworts may be promising candidates as lead components for future antitumor agents.72

48 h.76 Marchantin C (134) also decreased invasion ability in brain cancer cells at 6−10 μM and inhibited phosphorylation of the ERK.MAPK signaling pathway and 55.9% of angiogenesis at 10 μM in a CAM assay.77 Certain monoterpenoids found in liverworts, such as bornyl acetate (43), demonstrate potent apoptosis-inducing activities against cultured cells of Marchantia polymorpha. Apoptosis induced by monoterpenoids occurs through the production of active oxygen species, such as H2O2.78 2α,5β-Dihydroxybornane-2-cinnamate (137) from Conocephalum conicum exhibited weak cytotoxic activity against human HepG2 cells, with IC50 values of 4.5 μg/mL.79 Triterpene alcohol α-zeorin (138) has been isolated from several liverworts and displayed cytotoxic activity against P-388 cells with an IC50 of 1.1 μg/mL.80 The crude diethyl ether extracts of two unidentified Indonesian and Tahitian Frullania species were found to exhibit cytotoxic activity against both the HL-60 and KB cell lines, with EC50 values of 6.7 and 1.6 μg/mL (HL-60 cells) and 1.6 and 11.2 μg/mL (KB cells), respectively.58 Bioactivityguided fractionation of the Indonesian sample led to the isolation of (+)-3α-(4′-methoxybenzyl)-5,7-dimethoxyphthalide (139), which possessed cytotoxic activity against HL-60 and KB cells, showing IC50 values of 0.92 and 0.96 μM.58

Figure 14. Cytotoxic diterpenoids (116−131) from liverworts.

Among the aromatic compounds occurring in liverworts, the most characteristic for these spore-forming plants are macrocyclic bis-bibenzyls such as marchantins A−E (132−136).2c,d,l To date, more than 60 macrocyclic and acyclic bis-bibenzyls have been isolated from many liverworts and their structures established.2d,j,7a,d,o Bis-bibenzyls possess various biological activities, including cytotoxicity against KB cells and antitumor activity.2c,d,7b,73 Marchantins A (132), B (133), and E (136) showed cytotoxicity against KB cells (IC50 range 3.2−7.6 μM).7b Marchantin A (132), isolated from Marchantia emarginata ssp. tosana, induced cell growth inhibition in human MCF-7 breast cancer cells with an IC50 of 4.0 μg/mL. Fluorescence microscopy and a Western blot analysis indicated that compound 132 induces apoptosis of MCF-7 cells through a caspase-dependent pathway. The phenolic hydroxy groups at C-1′ and C-6′ are responsible for inducing both its cytotoxic and antioxidant activities.74 Marchantin A (132) from M. polymorpha induced a reduction in cell viability of the A256 breast cancer cell line (IC50 5.5 μM). The effect was increased in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously.75 Marchantin C (134) and its dimethyl ether, and 7,8-dehydromarchantin C and its dimethyl ether, were synthesized, and their possible modulatory effects on P-glycoprotein in VCRresistant KB/VCR cells were investigated.76 The results indicated that 134 was the most potent inhibitor of cell proliferation in both KB and KB/VCR cells among these four compounds, while the three synthetic derivatives of 134 did not show discernible antiproliferative activity. Potent apoptosis in KB/VCR cells was induced by treatment with 16 μM of the dimethyl ether of marchantin C (134) and 0.2 μM VCR for

Figure 15. Cytotoxic bis-bibenzyls (132−136), monoterpene derivative (137), triterpene (138), and bibenzyl phthalide (139) from liverworts.

Besides liverworts, mosses also biosynthesize components with cytotoxic activity. The two pimarane diterpenoids momilactones A (140) and B (141), which were identified as phytoalexins in rice, were isolated from the moss Hypnum plumaeforme (Hypnaceae).15 Momilactone B (141) was shown to be cytotoxic against human colon cancer HT-29 and SW620 cells at 1 μM.81 Pallidisetin A (142) and pallidisetin B (143), two bibenzyl derivatives isolated from the moss Polytrichum pallidiscetum, showed cytotoxicity against human melanoma (RPMI-7951) and human glioblastoma multiforme (U-251 MG) cells, with ED50 values of 1.0 μg/mL and 2.0 μg/mL, respectively.82 Three cytotoxic benzonaphthoxanthones, 1-O-methylohioensin B (144), 1-O-methyldihydroohioensin B (145), and 1,14-di-O-methyldihydroohioensin B (146), were also isolated from the moss P. pallidiscetum. Compound 146 proved to be cytotoxic for human colon adenocarcinoma (HT-29), human melanoma (RPMI-7951), and human glioblastoma multiforme (U-251 MG) cells, with ED50 values of 1.0, 1.0, and 2.0 μg/mL, respectively. Compound 145 showed inhibitory activity only against U-251 cells (ED50 0.8 μg/mL), while 146 inhibited the growth of the A549 lung carcinoma (A549) (ED50 1.0 μg/mL) and RPMI-7951 melanoma (ED50 1.0 μg/mL) cell lines.82 I



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Marchantin A (132) showed antibacterial activity against Acinetobacter calcoaceticus (MIC 6.25 μg/mL), Bacillus cereus (12.5 μg/mL), B. megaterium (25 μg/mL), B. subtilis (25 μg/mL), Cryptococcus neoformans (12.5 μg/mL), and Staphylococcus aureus (3.13−25 μg/mL).7a Marchantin A (132) isolated from the Hungarian M. polymorpha showed antimicrobial activity against the Gram-negative bacteria Pasteurella multocida, Pseudomonas aeruginosa, Haemophilus inf luenzae, and Neisseria meningitidis (MIC 4.5, 84.5, 72.7, and 72.7 nM, respectively) and against the Gram-positive Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus viridans (MIC 6.9, 9.1, and 18.1 nM, respectively).84 Marchantin C (134) and neomarchantins A (147) and B (148), isolated from Schistochila glaucescens, exhibited antimicrobial activity against the Grampositive bacterium B. subtilis with MIC values of 2, 1.5, and 2 μg/mL, respectively.85 In addition to the antimicrobial properties, the mentioned bis-bibenzyls also have an antifungal effect. Marchantin A (132) shows antifungal activity against Aspergillus niger (25−100 μg/mL), Pyricularia oryzae (12.5 μg/mL), Rhizoctonia solani (50 μg/mL), and the dermatophyte Trichophyton mentagrophytes (3.13 μg/mL).7a Marchantin C (134) and neomarchantins A (147) and B (148) were active against T. mentagrophytes, with MIC values, in turn, of 0.5, 1, and 0.5 μg/mL.85 Riccardin D (149), isolated from the Chinese Marchantia polymorpha, showed antifungal activity against the fluconazoleresistant C. albicans strains QL-14, QL-28, SDEY-24R, and SDEY-09R with MIC values of 16, 32, 16, and 16 μg/mL, respectively. When riccardin D (149) was mixed with fluconazole, the antifungal activities were substantially more potent (MIC values in the range 0.313−0.375 μg/mL).86 The antifungal activity of riccardin D (149) against C. albicans might be attributed to its inhibitory effect on cell wall chitin synthesis.87 Riccardin D (149) exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species (ROS) accumulation in C. albicans. Compound 149 also induced apoptosis in C. albicans through activating a metacaspase.2d,88 Reinvestigation of the antifungal activity of the bis-bibenzyls from Marchantia polymorpha using a bioautographic method showed that neomarchantin A (147), riccardin D (149), and 13,13′-O-isopropylidene-riccardin D (150) possessed antifungal activity against C. albicans with respective MID (minimum inhibitory dose) values of 0.25, 0.2, and 0.4 μg/mL, respectively, compared to the MID of 0.01 μg for the positive control miconazole. In turn, marchantin A (132), marchantin B (133), marchantin E (136), and riccardin H (151) showed moderate growth inhibitory activities against the same fungus, with MID values of 2.5, 4.0, 2.5, and 4.0 μg/mL, respectively.89 Direct TLC bioautographic detection of the antifungal activity of an ether extract of Asterella angusta showed activity against C. albicans. Riccardin D (149), riccardin B (152), 11-Odemethyl marchantin I (153), and dihydroptychantol (154) showed antifungal activity against C. albicans, exhibiting MIQ (minimum inhibitory quantity) values between 0.25 and 0.8 μg and MIC (minimum inhibitory concentration) values in the range 16−64 μg/mL.75,90 The free phenolic hydroxy group seems to play an important role in mediating antifungal activity since bis-bibenzyls possessing a methoxy group displayed decreased potencies in this regard.91 When riccardin C (155) isolated from Plagiochasma intermedium was combined with fluconazole, the synergistic or additive activity of 155 caused the MICs of fluconazole to be reduced from 256 μg/mL to 8 μg/mL against three resistant

Figure 16. Cytotoxic diterpenoids (140 and 141), bibenzyl derivatives (142 and 143), and benzonaphthoxanthones (144−146) from mosses.

Cytotoxicity against Cancer Cells. In Vivo Studies. 8,9-Secokauranes, rabdoumbrosanin (116), and 8,14-epoxyrabdoumbrosanin (117), isolated from a New Zealand Lepidolaena taylorii, were tested in an in vivo model system for antitumor activity. Unfortunately, both compounds were inactive at the doses tested (100 and 150 mg/kg for 116 and 18 and 12 mg/kg for 117).63b Marchantin C (134), isolated from Asterella angusta, strongly inhibited the growth of human cervical tumor xenografts in a nude mouse model and decreased the quantity of microtubules in a time- and dose-dependent manner at the G2/M phase in human glioma A172 cells and human cervical adenocarcinoma HeLa cells at 8−16 μM.83 The same compound (134) decreased the polymerization rate of tubulin, similarly to the potent known microtubule depolymerizer vincristine at 8−24 μM. These results indicated that 134 may play the same role in microtubule depolymerization in both its apoptotic effects in the cell and antitumor activity in vivo. Compound 134 is a novel microtubule inhibitor that induces mitotic arrest of tumor cells and suppresses tumor cell growth. The structure of marchantin C (134) is distinct from the classical microtubule inhibitors such as colchicine, podophyllotoxin, vinblastine, and vincristine, and it may be regarded as a potential antitumor agent for further in vivo study in more challenging antitumor models as a result of inhibiting microtubule polymerization.83 Antimicrobial, Antifungal, and Antiviral Activities. Sacculatal (57), isolated from Pellia endiviifolia, showed antibacterial activity against Streptococcus mutans (a causative organism of dental caries), exhibiting an LD50 value of 8 μg/mL.7n The herbertane sesquiterpenoids isolated from the Madagascan species Mastigophora diclados were tested against a Staphylococcus aureus strain, using an agar diffusion method. These sesquiterpenoids showed weaker activity than the standard antibiotics chloramphenicol (zone of inhibition, 22 mm) and kanamycin (23 mm). Of the compounds tested, mastigophorene C (109), a dimer of herbertene-1,2-diol (108), showed antibacterial activity (17 mm), while its monomer (108) also displayed some activity (13 mm).21 The crude ether and methanol extracts of a Tahitian specimen of M. diclados showed antimicrobial activity against B. subtilis and S. aureus (MIC 16 μg/mL).57 Bioactivity-guided fractionation of both extracts gave (−)-herbertene-1,2-diol (108), (−)-mastigophorene C (109), and (−)-mastigophorene D (110), which showed moderate antimicrobial activity against B. subtilis at MIC values of 2−8 μg/mL.57 J



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strains of C. albicans.92 Isoplagiochin D (156), 6′,8′-dichloroisoplagiochin C (157), and 6′-chloroisoplagiochin D (158) from Bazzania trilobata showed discernible antifungal activity in a microtiter plate test against P. oryzae with IC50 values of 4.0, 3.9, and 2.6 μg/mL and against Septoria tritici with IC50 values of 23.5, 15.9, and 4.5 μg/mL, respectively. Compounds 157 and 158 also demonstrated inhibitory activity against B. cinerea with IC50 values of 7.6 and 18.9 μg/mL, respectively. Free phenolic hydroxy groups on the aromatic rings of the bisbibenzyls play an important role in mediating inhibitory activity against fungi such as C. cucumerinum.91

ited growth inhibitory activity against P. infestans and S. tritici with IC50 values of

Chemical Constituents of Bryophytes: Structures and Biological

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