The bryophytes are widely distributed in the non-vascular terrestrial plants. Approximately, 25,000 species of bryophytes are found in the globe, and they are classified into three major groups such as mosses, liverworts, and hornworts (Lu et al., 2019). In general, bryophytes produce various specialized metabolites to defend themselves due to the absence of a vascular system and thick waxy cutin protection on the cell walls (Roberts et al., 2012). Apart from macromolecules, bryophytes also contain a number of specific compounds, including fatty acids, terpenoids, flavonoids, and polyphenols (Klavina et al., 2015; Lu et al., 2019; Tosun et al., 2015). In particular, mosses also produce a pleasant odor from volatile components in their fresh state and have been traditionally used in China and India for the treatment of various diseases, including burns, external wounds, snake bite, pulmonary tuberculosis, cardiovascular diseases, bone fractures, hepatic disorders, skin diseases, etc (Chandra et al., 2017). Moreover, Vicherova et al. (2020) reported that bryophytes can communicate with their neighbors via volatile organic compounds. However, studies on the phytochemistry and pharmacological potentials of bryophytes have been meager (Asakawa and Ludwiczuk, 2018).
Mosses are the largest group of bryophytes, play a considerable role in the biodiversity of terrestrial ecosystems. Mosses have been traditionally used to treat wounds and burns and other ailments. The extracts from mosses also exhibited strong antimicrobial activity against various pathogens (Singh et al., 2006; Kang et al., 2007). Previous studies reported that numerous moss species contain an appreciable amount of essential oils (Valarezo et al., 2018). Among mosses, Racomitrium (Grimmiaceae) species are important components of various terrestrial ecosystems (Stech et al., 2013). Racomitrium canescens is widely distributed from the northern temperate to arctic zones. R. canescens can be easily differentiated from the other Racomitrium species based on morphological characters, including papillose laminal cells, peristome teeth, hyaline alar cells, etc (Frisvoll, 1983).
Liverworts are another important group of bryophytes with about 6,000 species distributed worldwide (Asakawa and Ludwiczuk, 2018). Liverworts contain a variety of bioactive secondary metabolites, chiefly volatile components. In past decades, about 3,000 compounds have been identified in different liverworts. Certain terpenoid components such as pinguisane group of sesquiterpenoids and sacculatane group of diterpenoids have not been found in other plant species (Ludwiczuk and Asakawa, 2015). It is well known that the fragrance of liverwort is mainly connected with the presence of oil bodies. Among various liverwort species, the common liverwort Marchantia polymorpha L. is widely distributed in temperate regions (Romani et al., 2020). This plant is used for the treatment of boils, fractures, poisonous snakebites, abscesses, wounds, and hepatic disorders. In Europe, M. polymorpha has been used as a diuretic (Asakawa, 2007; Chandra et al., 2017; Mewari and Kumar, 2008).
To our knowledge, there has been no study on the essential oil composition of M. polymorpha and R. canescens. Hence, the present study aimed to determine the chemical composition of essential oils of these two species obtained from steam distillation by gas chromatography-mass spectrometry (GC-MS) analysis.
Materials and Methods
Whole plant samples of M. polymorpha and R. canescens (fresh weight 5 kg each-1) were collected from Songjung-ri (N37o37'14.66" E128o34'12.80"), Jinbu-Myeon, Pyeongchang in Korea during October 2020 (Fig. 1). These samples were collected under trees in a pine forest. They were already in a colony that had formed independent gametophyte. Samples were selectively collected by brushing off the soil without damaging the surrounding ecosystem. Collected samples were kept in a 4℃ freezer box and transported to the laboratory. The soil and plant debris in the samples were removed by washing in running tap water. The samples were authenticated and deposited in the Herbarium, National Institute of Biological Resources (NIBR), Korea with voucher numbers NIBRMS 0000107499 (M. polymorpha) and NIBRMS 0000107500 (R. canescens), respectively.
Essential oil isolation
The essential oils from entire parts of M. polymorpha and R. canescens were isolated by steam distillation extraction using a Clevenger-type apparatus. The steam distillation was performed at 100℃ for 90 min. The essential oil isolation was observed every 15 min for calculating the yield of the oil. The essential oil isolation was carried out in triplicates and the yield (%) was calculated as volume (mL) of the isolated oil per 100 g of the fresh plant material. The isolated essential oil was dried using anhydrous sodium sulfate and stored at 4℃ for further analysis.
Gas chromatography-mass spectrometry (GC-MS) analysis
The identification of the essential oil components from M. polymorpha and R. canescens was performed using a Varian CP3800 gas chromatograph coupled with a Varian 1200L mass detector (Varian, CA, USA). The GC-MS was equipped with a VF-5MS polydimethylsiloxane capillary column (30 m×0.25 mm×0.25 μm). The oven temperature programmed from 50℃ to 250℃ at a rate of 5℃ min-1. The injector temperature was 250℃ and the ionization detector temperature was 200℃. Helium was the carrier gas (1 mL min-1) and the injection volume was 1.0 mL of 1% solution diluted in n-hexane with a split ratio of 10:1. For mass spectra, an electron ionization system with ionization energy of 70 eV was used. The mass range was 50-500 m/z. The identification of the essential oil components in M. polymorpha and R. canescens was based on the comparison of their retention indices (RIs) relative to a homologous series of n-alkanes (C8-C22) and mass spectra from National Institute of Standards and Technology (NIST, 3.0) library and published literature data (Adams, 2007). Calculation of peak area of each compound relative to the total peak area of the whole chromatograph presented the percentage of each essential oil (EO) constituent.
The collection of essential oil was started after 30 minutes using the steam distillation extraction system and extracted for 90 minutes. The effect of extraction time on the yield of essential oils is presented in Fig. 2. The yield of essential oils from M. polymorpha and R. canescens was 0.40±0.003% and 0.03±0.001%, respectively. The GC-MS results revealed that the essential oils of M. polymorpha and R. canescens registered different chemical compositions. The essential oil components were identified based on their retention indices and mass spectra. The area percentage, retention index, and CAS number of the identified compounds from M. polymorpha and R. canescens essential oils are presented in Tables 1 and 2.
In total, 18 different volatile components were identified in M. polymorpha essential oil, which accounted for 87.30% of the total oil (Table 1). This essential oil consists of 11 sesquiterpene hydrocarbons, 4 alcohols, 2 oxygenated sesquiterpenes, and 1 aldehyde. It was observed that M. polymorpha essential oil contains a higher amount of sesquiterpene compounds followed by alcohols. The most abundant component in the essential oil of M. polymorpha is widdrol (35.45%). Thujopsene (11.20%), cuparene (7.67%), β-chamigrene (7.16%), α-bisabolol (6.69%), sativene (5.86%), α-chamigrene (5.59%), and α-acoradiene (4.63%) were also recorded as major components in M. polymorpha.
On the other hand, the essential oil of R. canescens contains 29 different volatile components, which accounted for 81.81% of the total oil (Table 2). In these, 10 aldehydes, 5 sesquiterpenes, 3 alcohols, 2 furans, 2 ketones, 2 monoterpenoid, 1 monoterepene, 1 phenylpropanoid, 1 ester, 1 sesquiterpenoid, and 1 phenylpropene were included. The essential oil of R. canescens predominantly contains a phenylpropene compound, estragole (58.86%). A considerable amount of D-limonene (7.20%), α-muurolol (3.28%), and pentadecanal (2.01%), were detected in this oil. A minimum concentration of sesquiterpene hydrocarbons such as phytol (1.63%) hexahydrofarnesyl acetone (1.56%), nonanal (1.48%), and β-caryophyllene (1.19%) were also found in the oil.
In general, bryophytes are distributed throughout the world with the exception of sea. The studies on the phytochemistry of bryophytes have been avoided for a long period due to difficulties in collecting a large number of samples. In recent times, numerous studies revealed the presence of a variety of bioactive metabolites from bryophytes. In particular, many species of liverworts have typical fragrant odors because they secrete different volatile components. Further, the biological activities attributed to many bryophyte species are owing to volatile components, especially terpenoids (Asakawa and Ludwiczuk, 2018). In this context, the present study was carried out to investigate the chemical composition of essential oils of M. polymorpha and R. canescens. The essentials oils were isolated by steam distillation technique and components were identified using GC-MS. Fig. 3 illustrates the structure of major components in the essential oils of M. polymorpha and R. canescens.
The results revealed that widdrol (35.45%) was the most abundant component in the essential oil M. polymorpha. In addition, α-longipinene, α-santalene, (+)-cuparene, β-chamigrene, and β-himachalene were each found over 5% in this oil. Widdrol, (+)-thujopsene and (-)-thujopsenone were also isolated from Japanese M. polymorpha (Asakawa et al., 1984; Matsuo et al., 1985). Thujopsene was also reported as the major component in Riccardia prehensilis (Hook. f. & Taylor) C. Massal (Cuvertino-Santoni et al., 2017). Further, this component was reported in the essential oils of different bryophytes such as Breutelia tomentosa (Sw. ex Brid.) A. Jaeger (Valarezo et al., 2018) and Reboulia hemisphaerica (L.) Raddi (Asakawa et al., 2013). In the present study, M. polymorpha essential oil is mainly characterized by sesquiterpene group of components. Sesquiterpenoids are the most diverse group of terpenoids occurring in liverworts. Chen et al. (2019) studied the essential oil composition of M. polymorpha from five regions in Chengdu and reported that sesquiterpenes were the main components. Caryophyllene oxide was the most abundant component in these essential oils followed by β-chamigrene and α-bulnesene. A study reported that thalli M. polymorpha emitted high amounts of 1-octen-3-ol and octan-3-one, after mechanical wounding (Kihara et al., 2014). About 60 structural types of components were detected from different liverworts species. So far, above 900 sesquiterpenoid compounds have been detected in the Marchantiophyta (Asakawa et al., 2013; Chen et al., 2018). Among them, eudesmane and aromadendrane skeletons are the most predominant (Ludwiczuk and Asakawa, 2019). A characteristic structural phenomenon of liverwort constituents is that most sesqui- and diterpenoids are enantiomers of those found in higher plants. Asakawa and Ludwiczuk (2018) suggested that components unique to liverworts are associated with several biotic interactions and act as defense to herbivores.
The present study revealed that a phenylpropene, estragole (methyl chavicol) was the most abundant component in the essential oil of R. canescens. Estragole was also detected in Rhodobryum giganteum (7.37%) (Li and Zhao, 2009). It is one of the important components in essential oils of several plants, including Ravensara anisata, Ocimum ciliatum, Foeniculum vulgare, Artemisia dracunculus, etc (Leal-Cardoso et al., 2004). However, Saritas et al. (2001) reported that β-cyclocitral is the most common monoterpenoid identified in different mosses followed by α-pinene, β-pinene, limonene, and camphor. Estragole was also detected in Rhodobryum giganteum (Schwaegr.) Par. (7.37%) (Li and Zhao, 2009). The chemical structure of estragole consists of a benzene ring with a methoxy and a propenyl groups. Estragole-rich essential oils exhibited a wide range of biological activities including antioxidant, anti-inflammatory, antimicrobial and cytotoxic against cancer cell lines (Santos et al., 2018).
The essential oil of M. polymorpha was mainly dominated by a sesquiterpene alcohol, widdrol and R. canescens was mainly characterized by a phenylpropene, estragole. This is the first report on the chemical composition of essential oils of M. polymorpha and R. canescens in Korea. The GC-MS profiling of the essential oils extracted from these two species has been used in differentiating chemotypes within species. This study may shed new light on the utilization of secondary metabolites from various bryophytes.
This study was supported by the Korean Ministry of Environment (grant no. 2018002270002). The authors would like to express thanks to Mr. Yong Su Kim for collecting samples and Dr. Min Ha Kim at the National Institute of Biological Resources, Korea for authenticating the samples.