INTRODUCTION

Liverworts, the species of the division Marchantiophita, belong to Bryophytes, which are known as nonvascular plants. They are mostly spread in high moisture areas, humid tropical forests, and the banks of rivers and streams. 1 The genus, liverwort, comprises approximately 250 species.2, 3 In the world, only a few of them, 8 species, have been chemically and biologically studied, and none have been studied in Vietnam. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Previous chemical studies on the eight mentioned species showed that they contained sesquiterpenes, diterpenes, and lignans. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Some possess cytotoxic14 and nitric oxide inhibitory activity. 17 Herein, we report the investigation of the n-hexane and chloroform extracts of . collected in Lac Duong district, Lam Dong province, Vietnam. We isolated six compounds, including a triterpenoid compound, cycloartenol (1), and five phenolic derivatives, p-coumaric acid (2), p-hydroxybenzoic acid (3), methyl 2,4-dihydroxy-3,6-dimethylbenzoate (4), barbatic acid (5), and methyl 2-hydroxy-4-methoxy-6-tetradecylbenzoate (6). Six compounds were isolated via silica gel chromatographic processes, and their chemical structures were elucidated by HR-ESI-MS, 1D, and 2D NMR spectroscopic methods.

MATERIALS AND METHODS

RESULTS

Using silica gel column chromatography, the chemical investigation of the -hexane and chloroform extracts of the liverwort sp. led to the isolation of six compounds. Their structures are shown in Figure 1 by using 1D and 2D NMR and HR-ESI-MS.

DISCUSSION

The chemical structures of six isolated compounds were elucidated based on modern spectroscopic methods such as HR-ESI-MS and 1D and 2D NMR, and their data were compared with those in the literature. Compounds possessing a similar framework were structurally discussed.

Compound 1 (5 mg) was obtained as a white solid that was highly soluble in chloroform. The HR-ESI-MS spectrum (positive mode) showed a pseudomolecular ion peak at 427.4030 [M+H] (calcd. for CHO+H, 427.3934). The H-NMR spectrum showed signals of seven methyl groups [ 0.81 (3H, , H-29), 0.96 (6H, , H-18, H-28), 0.91 (3H, 6.5 Hz, H-21), 0.93 (3H, , H-30), 0.96 (3H, , H-18), 1.60 (3H, , H-27), 1.66 (3H, , H-26)], two nonequivalent methylene protons [ 0.37 (1H, , 3.5 Hz, H-19a) and 0.58 (1H, , 3.5 Hz, H-19b)], one oxymethine [ 3.22 (1H, , 10.0, 5.5 Hz, H-3)] and one olefinic proton [ 5.12 (1H, , H-24)]. The C-NMR spectrum showed thirty signals with two olefinic carbons [ 125.98 (C-24) and 131.25 (C-25)], one oxygenated methine carbon [ 78.45 (C-3)], and many signals in the high-field zone (14‒53 ppm). The good compatibility of its NMR and MS data with those in the literature19, 20proposed that 1 was cycloartenol.

Compound 2 (3 mg) was obtained as colorless needles and was highly soluble in methanol. The NMR data (Table 2, Table 3) showed two aromatic signals [6.84 (2H, , 8.6 Hz; 130.3, C-3, C-5); 7.43 (2H, , 8.6 Hz; 116.1, C-2, C-6)] of a 1,4-disubstituted benzene ring and two olefinic protons [6.25 (1H, 15.6 Hz, H-8) and 7.63 (1H, , 15.6 Hz; H-7)]. The large coupling constants suggested the -configuration for this double bond. The other chemical shifts for the remaining carbons are presented in Table 3. The molecular formula of 2 was confirmed by the pseudomolecular ion peak (negative mode) at 163.0241 [M–H] (calcd. for CHO‒H, 163.0401), as well as the good compatibility in the comparison of its NMR data with those in the literature21, suggested that 2 was -coumaric acid.

Compound 3 (4 mg) was isolated as a white solid that was highly soluble in MeOH. Its HR-MS spectrum showed a deprotonated molecular ion peak at 137.0103 [M–H] (calcd. for CHO‒H, 137.0244), as well as good compatibility in the comparison of its NMR data with those in the literature22,suggested that 3 was -hydroxybenzoic acid.

Compound 4 (7 mg) was isolated as a white amorphous powder. Its molecular formula was determined to be CHO through its pseudomolecular ion peak at 195.0673 [M–H] (calcd. for CHO‒H, 195.0662) in the HR-ESI-MS spectrum. The H-NMR spectrum showed one aromatic methine proton at 6.21 (3H, ), two methyl groups at 2.10 (3H, ) and 2.46 (3H, ), and one methoxy group at 3.92 (3H, ). The C-NMR spectrum showed 10 resonances of one benzene ring, two methyl groups, and one methoxycarbonyl group (Table 2, Table 3). HMBC cross-peaks of the methyl protons at 2.10 (3H, , H-8) with C carbon signals at 108.7 (C-3), 158.2 (C-4), 163.3 (C-2), and 2.46 (3H, , H-9) with 110.7 (C-5), 140.3 (C-6) and 172.7 (C-7) (Figure 2) evidenced their positions. The presence of the methoxycarbonyl group in 3 was proven by cross-peaks of 3.92 (H-10) with 172.7 (C-7). The spectrum also confirmed the position of the chelated hydroxyl group through cross-peaks of 12.00 (1H, , 2-OH) with 105.5 (C-1), 108.7 (C-3), and 163.3 (C-2). Therefore, 4 was identified as methyl 2,4-dihydroxy-3,6-dimethylbenzoate, as shown in the literature.23

Compound 5 (4 mg) was isolated as a white solid that was highly soluble in acetone. The H-NMR spectrum showed four methyl signals [1.99 (3H, , H-8′), 2.03 (3H, , H-8), 2.61 (3H, , H-9′), 2.69 (3H, , H-9)], one methoxy group [ 3.93 (1H, , 4-OCH)] and two aromatic protons of two benzene rings at 6.43 (1H, , H-5′) and 6.59 (1H, , H-5). The spectrum also showed one chelated hydroxyl group at 11.59 (1H, , 2-OH). The C-NMR spectrum (Table 3) showed 18 signals of 19 carbons of four methyl groups ( 7.8, 9.4, 23.5, 24.8), one methoxy group( 55.1), nine signals of 12 aromatic carbons, two carboxyl carbons including one ester( 171.1), and one carboxylic acid( 177.2). The HMBC experiment (Figure 2) revealed that 5 was composed of two benzene rings. This was well supported by the HR-ESI-MS spectrum (negative mode) with a pseudomolecular ion peak at 359.1129 [M–H] (calcd. for CHO‒H, 359.1125). The good compatibility of its NMR data with those in the literature24 suggested that 5 was a barbatic acid.

Compound 6 (6 mg) was isolated as a white amorphous solid that was highly soluble in chloroform. Its NMR data (Table 1, Table 3) showed a benzene ring with four substitutions via two -coupled aromatic proton signals (6.33, 1H, , 2.7 Hz;H-3) and 6.28 (1H, , 2.7 Hz;H-5), a chelated hydroxyl at 11.69 (1H, , 2-OH), and two methoxy groups (3.92, 3H, , 7-OCH) and ( 3.80, 3H, ; 4-OCH). The data also showed that 1 possessed an alkyl side chain with some methylene groups ( 1.25‒2.84, 22.8‒37.1) and one terminal methyl group (0.89, 3H, , 6.6 Hz; 14.2). These results revealed that 6 contained one benzene ring, one methoxy group, one methoxycarbonyl group, and one aliphatic side chain. The HMBC spectrum (Figure 2) suggested that the chemical structure of 6 was similar to that of 4, except that it further possessed a side chain. The HR-ESI-MS spectrum of 6 showed a pseudomolecular ion peak at 377.2642 [M–H] (calcd. for CHO–H, 377.2682), which suggests that the alkyl chain moiety is a tetradecyl group. Therefore, 6 was elucidated as methyl 2-hydroxy-4-methoxy-6-tetradecylbenzoate. It is a new compound from the plant.

CONCLUSION

Six compounds were isolated from the -hexane and chloroform extracts of the liverwort , collected at Lac Duong district, Lam Dong province, Vietnam, using chromatographic methods. Those compounds are cycloartenol (1) and five phenolic derivatives, -coumaric acid (2), -hydroxybenzoic acid (3), methyl 2,4-dihydroxy-3,6-dimethyl benzoate (4), barbatic acid (5), and methyl 2-hydroxy-4-methoxy-6-tetradecyl benzoate (6). Although 1-5 were known in other species, this is the first time they have been reported in . After being checked in Scifinder (on September 30, 2022), compound 6 was determined to be new. Further research on the remaining extracts is ongoing.

ABBREVIATIONS

HR-ESI-MS: High resolution- Electrospray ionization-Mass spectrometry

H-NMR: Proton nuclear magnetic resonance

C-NMR: Carbon-13 Nuclear Magnetic Resonance

HMBC: Heteronuclear Multiple Bond Correlation

: singlet

: doublet

: doublet of doublets

: triplet

: multiplet

COMPETING INTEREST

The authors declare no competing financial interest.

AUTHORS’ CONTRIBUTION

Tran Q. T. collected samples and prepared extracts. Nguyen N. N., Nguyen T. B. T., and Nguyen H. C. performed column chromatography on the -hexane extract and isolated compounds. Nguyen X. K., Phan T. T., and Le H. K. performed column chromatography on the chloroform extract and isolated compounds. Duong T. H and Huynh N. V. interpreted the NMR and MS data. Nguyen X. K. prepared the manuscript and searched the bibliography. Nguyen K. P. P. gave the final correction for the manuscript.