INTRODUCTION

L. (Marchantiaceae) belongs to liverworts. It is known as a traditional medicine to cure poisonous snake bites, insect bites, inflammation, cuts, fractures, etc. 1. In biological studies, possessed inhibition of the enzymes -glucosidase and tyrosinase, antibacterial and antioxidant activities, and cytotoxicity against the MCF-7 cell line2. In phytochemical studies, was reported to contain many types of compounds comprising bis-bibenzyl, sesquiterpenoid3, flavonoid4, glycoside5, phenolic and coumarin6. Our previous study on the ethyl acetate extract of this species led to the isolation of one bibenzyl (lunularin), two bis-bibenzyl compounds (marchantin A and isoriccardin C), and two flavonoids (luteolin and apigenin). The antibacterial, antioxidant, and cytotoxic activities were evaluated. The compound lunularin showed an IC value of 4.59 ± 0.38 µg/mL and an SC of 3.99 ± 0.47 µg/mL in the experiments against MCF-7 cancer cells and DPPH radical scavenging activity, respectively. In comparison, isoriccardin C showed remarkable antibacterial activity against with an inhibition zone of 12.67 mm at a concentration of 50 µg/well7. This paper reports a continuous chemical study of the -hexane extract of this species.

MATERIALS AND METHODS

Extraction and isolation

The dried L. (2.0 kg) was ground into powder and macerated in methanol (3x7 L) at room temperature. The filtrated solution was then evaporated at reduced pressure to give a crude extract (110.0 g). This crude extract was separated into different polar fractions by a liquid–liquid partition method with -hexane and ethyl acetate.

The -hexane extract (31.3 g) was subjected to normal-phase silica gel column chromatography. The solvent systems used to elute this column were -hexane:ethyl acetate (stepwise, 99:1, 98:2, 95:5, 90:10, 50:50, 0:100) and ethyl acetate:methanol (1:1) to give eight subfractions (H1‒H8). A silica gel column chromatography was applied for fraction H3 (4.6 g), eluted with -hexane:ethyl acetate (stepwise, 99:1, 98:2, 95:5, 90:10, 50:50, 0:100) to give seven subfractions (H3.1‒H3.7). Subfraction H3.3 (650 mg) was then subjected to silica gel column chromatography and eluted with -hexane:ethyl acetate as the abovementioned eluent to afford six fractions (H3.3.1‒H3.3.6). Compound 4 (10.3 mg) was obtained from fraction H3.3.2 (110 mg) after applying this fraction to Sephadex-LH20 eluted with chloroform:methanol (1:4). Three compounds, 2 (5.6 mg), 3 (6.1 mg), and 5 (12.2 mg), were obtained from subfraction H3.4 (350 mg) by silica gel column chromatography eluted with -hexane:ethyl acetate (6:4) and further purified by Sephadex-LH20 chromatography eluted with a chloroform:methanol (1:4) solvent system. Fraction H4 (5.1 g) was separated into six subfractions (H4.1‒H4.6) by silica gel column chromatography and eluted with -hexane:ethyl acetate as described above. Compound 1 was then isolated from subfraction H.4.2 (1.2 g) by silica gel column chromatography eluted with -hexane:ethyl acetate (65:35), followed by Sephadex-LH20 chromatography eluted with chloroform:methanol (1:4). Fraction H8 (3.0 g) was subjected to silica gel column chromatography and then eluted with -hexane:ethyl acetate and then ethyl acetate:methanol as previously described. A precipitate (220 mg) appeared when the column chromatography of fraction H8 was eluted with ethyl acetate:methanol (50:50), which was then recrystallized in ethyl acetate to afford 6 (16.6 mg).

RESULTS

From the -hexane extract of L., collected in Da Lat city, Lam Dong Province, six compounds, 1 (7.8 mg), 2 (5.6 mg), 3 (6.1 mg), 4 (10.3 mg), 5 (12.2 mg) and 6 (16.6 mg), were isolated. Their physical properties and spectroscopic data were obtained as follows (Figure 1).

Marchantin G (1): A yellowish solid. HR-ESI-MS: 453.1352 [M‒H]. H-NMR (DMSO-) ( in Hertz): δ 6.60 (, 8.5, H-2/H-6), 6.98 (, 8.5, H-3/H-5), 3.01 (, H-7/H-8), 7.03 (, 5.0, H-12), 6.74 (, 5.0, 4.5, H-13), 7.03 (, 5.0, H-14), 6.47 (, 2.0, H-3'), 7.09 (, 2.0, H-5'), 3.76 (, H-8'), 6.08 (, H-10'), 6.82 (, 8.0, 2.0, H-12'), 7.11 (, 8.5, 8.0, H-13'), and 6.43 (, 8.0, H-14’). C-NMR (DMSO-): δ 153.3 (C-1), 119.6 (C-2/C-6), 129.6 (C-3/C-5), 137.1 (C-4), 33.4 (C-7), 28.5 (C-8), 134.0 (C-9), 137.4 (C-10), 149.5 (C-11), 125.0 (C-12), 114.3 (C-13), 119.6 (C-14), 140.7 (C-1'), 145.9 (C-2'), 111.5 (C-3'), 137.1 (C-4'), 108.4 (C-5'), 158.0 (C-6'), 195.6 (C-7'), 46.0 (C-8'), 136.5 (C-9'), 113.8 (C-10'), 157.7 (C-11'), 114.1 (C-12'), 129.1 (C-13'), and 119.4 (C-14'). Selected COSY and HMBC correlations: see Figure 2.

Quercetin (2): A yellowish solid. HR-ESI-MS: 301.0353 [M‒H]. H-NMR (DMSO-) ( in Hertz): δ 6.18 (, 1.5, H-6), 6.40 (, 1.5, H-8), 7.67 (, 2.0, H-2'), 6.88 (, 7.0, H-5'), 7.53 (, 7.0, 2.0, H-6'), and 12.49 (1H, , 5-OH). C-NMR (DMSO-): δ 146.8 (C-2), 135.7 (C-3), 175.8 (C-4), 160.7 (C-5), 98.1 (C-6), 163.9 (C-7), 93.3 (C-8), 156.1 (C-9), 103.0 (C-10), 121.9 (C-1'), 115.0 (C-2'), 145.0 (C-3'), 146.8 (C-4'), 115.6 (C-5'), and 119.9 (C-6'). Selected HMBC correlations: see Figure 2.

Kaempferol (3): A yellowish solid. H-NMR (DMSO-) ( in Hertz): δ6.19 (, H-6), 6.44 (, H-8), 8.04 (, 9.0, H-2'/H-6'), 6.93 (, 8.5, H-3'/H-5'), and 12.46 (, 5-OH). C-NMR (DMSO-): δ 146.7 (C-2), 135.5 (C-3), 175.8 (C-4), 156.1 (C-5), 98.1 (C-6), 163.8 (C-7), 93.4 (C-8), 160.6 (C-9), 102.9 (C-10), 121.6 (C-1'), 129.4 (C-2'/C-6'), 115.4 (C-3'/C-5'), and 159.1 (C-4').

Hexaconazole(4): White solid. HR-ESI-MS: 314.0819 [CHClNO+H]316.0792 [CHClClNO+H]and 318.0773 [CHClNO +H]. H-NMR (DMSO-) ( in Hertz): δ 4.83 (, 14.5, H-1a), 4.54 (, 14.5, H-1b), 2.47 (, 14.0, 5.5, H-3a), 1.61 (, 14.5, 12.5, 4.5, H-3b), 1.25 (, H-4a), 0.70 (, H-4b), 1.16 (, H-5), 0.77 (7.0, H-6), 7.73 (, H-3'), 8.26 (, H-5'), 7.52 (, 2.5, H-3''), 7.29 (, 8.5, 2.0, H-5''), 7.53 (, 8.0, H-6''), and 5.71 (, 2-OH C-NMR (DMSO-): δ 56.1 (C-1), 75.5 (C-2), 35.7 (C-3), 25.1 (C-4), 22.3 (C-5), 13.8 (C-6), 150.3 (C-3'), 144.7 (C-5'), 139.0 (C-1''), 132.3 (C-2''), 129.7 (C-3''), 130.9 (C-4''), 126.8 (C-5''), and 131.0 (C-6''). Selected COSY and HMBC correlations: see Figure 2.

Indole-3-carboxylic acid (5): White powder. HR-ESI-MS: 162.0557 [M+H]. H-NMR (DMSO-) ( in Hertz): δ7.98 (, 3.0, H-2), 8.00 (, 7.0, H-4), 7.14 (, 8.0, 7.0, 1.0, H-5), 7.18 (, 8.0, 7.0, 1.5, H-6), 7.45 (, 7.0, 1.0, H-7), and 11.77 (1H, , NH). C-NMR (DMSO-): δ 132.2 (C-2), 107.3 (C-3), 120.5 (C-4), 121.0 (C-5), 122.1 (C-6), 112.2 (C-7), 136.2 (C-8), 126.0 (C-9), and 165.9 (C-10). Selected COSY and HMBC correlations: see Figure 2.

Sitosterol 3--β-D-glucopyranoside (6): White powder. H-NMR (DMSO-) ( in Hertz): δ 3.45 (, H-3), 5.32 (, 4.5, H-6), 0.65 (, H-18), 0.95 (, H-19), 0.90 (6.5, H-21), 0.81 (7.0, H-26), 0.79 (7.0, H-27), 0.82 (7.0, H-29), 4.22 (7.5, H-1'), 2.91 (, H-2'), 3.12 (, 9.0, 9.0, H-3'), 3.01 (, 9.0, 9.0, H-4'), 3.07 (, H-5'), 3.64 (10.0, 5.5, H-6'a), and 3.41 (11.5, 6.0, H-6'b). The C-NMR (DMSO-): δ 36.8 (C-1), 31.3 (C-2), 76.9 (C-3), 38.3 (C-4), 140.5 (C-5), 121.2 (C-6), 31.4 (C-7), 31.3 (C-8), 49.6 (C-9), 35.4 (C-10), 20.6 (C-11), 39.2 (C-12), 41.8 (C-13), 56.2 (C-14), 23.8 (C-15), 27.7 (C-16), 55.4 (C-17), 11.6 (C-18), 18.9 (C-19), 36.2 (C-20), 18.6 (C-21), 33.3 (C-22), 25.5 (C-23), 45.1 (C-24), 28.7 (C-25), 19.1 (C-26), 19.7 (C-27), 22.6 (C-28), 11.8 (C-29), 100.8 (C-1'), 73.5 (C-2'), 76.8 (C-3'), 70.1 (C-4'), 76.9 (C-5'), and 61.1 (C-6').

DISCUSSION

Compound 1 was isolated as a yellowish powder. Its HR-ESI-MS showed a deprotonated ion peak at 453.1352 [M‒H] (calcd. for CHO‒H, 453.1338). The H-NMR spectrum of 1 showed two doublet proton signals with a large coupling constant of 8.5 Hz, each integrated two protons at δ 6.60 (H-2/H-6) and 6.98 (H-3/H-5) of a -disubstituted benzene ring (ring A), as shown in Figure 1. A set of three aromatic protons at δ 7.03 (, 5.0 Hz, H-12), 6.74 (, 5.0, 4.5 Hz, H-13), and 7.03 (, 5.0 Hz, H-14) were assigned for a 1,2,3-trisubstituted benzene ring (ring B). The connection of H-12/H-13/H-14 was confirmed by COSY correlations. Two -coupling doublet proton signals at δ 6.47 (, 2.0 Hz, H-3') and 7.09 (, 2.0 Hz, H-5') belonged to a 1,3,4,5-tetrasubstituted benzene ring (ring C). The four remaining aromatic proton signals at δ 6.08 (, H-10'), 6.82 (, 8.0, 2.0 Hz, H-12'), 7.11 (, 8.5, 8.0 Hz, H-13'), and 6.43 (, 8.0, H-14') along with the COSY connections H-12'/H-13'/H-14' confirmed the presence of -disubstituted benzene (ring D). In the higher magnetic resonance zone, there were signals of three methylene groups at δ 3.01 (, H-7/H-8) and 3.76 (, H-8'). From the above information, 1 was suggested to possess four benzene rings and three methylene groups. These corresponded to the presence of three carbon signals at the high magnetic field at δ 33.4 (C-7), 28.5 (C-8), and 46.0 (C-8') of three methylene groups in the C-NMR spectrum. In the higher frequency range, twenty-two carbon signals at 108‒158 ppm, including two signals at δ 119.6 (C-2/C-6) and 129.6 (C-3/C-5) appearing in the double intensity of a -disubstituted benzene ring, were assigned to four benzene rings. The last carbon signal at δ 195.6 was assigned to a conjugated carbonyl carbon. Therefore, 1 was suggested to be a bis-bibenzyl compound type that possessed two dihydrostilbenoid units, as normally found in species. HMBC correlations of protons H-3/H-5 with carbons at δ 153.3 (C-1) and 33.4 (C-7) demonstrated the position of ring A at C-7. The HMBC cross-peaks of proton H-8 with carbons at δ 134.0 (C-9), 137.4 (C-10), and 119.6 (C-14) suggested the position of ring B at C-8. Additionally, HMBC cross-peaks of both protons H-3' and H-5' with the carbonyl carbon at δ 195.6 (C-7'), along with correlations of proton H-8' with carbons at δ 195.6 (C-7'), 113.8 (C-10'), 119.4 (C-14'), confirmed the position of two remaining dibenzyl units. From all the above MS and NMR analyses as well as the good compatibility of the NMR data of 1 with published data8, 9, the chemical structure of 1 was suggested to be marchantin G.

Compound 2 was isolated as a yellowish powder. The molecular formula of 2 was identified to be CHObased on a quasi-molecular ion peak at 301.0353 [M‒H] (calcd. for CHO‒H, 301.0348) on the HR-ESI-MS spectrum. The proton spectrum of 2 showed the characteristic signals of a flavonoid, comprising a singlet proton signal at δ 12.49 (1H, ) of a chelated hydroxy group at C-5 as usual, two doublet proton signals with a small coupling constant value of 1.5 Hz at δ6.18 (H-6), 6.40 (H-8), and a set of three aromatic proton signals [at δ 7.67 (, 2.0 Hz, H-2'), 6.88 (, 7.0 Hz, H-5'), and 7.53 (, 7.0, 2.0 Hz, H-6')] of a 1,3,4-trisubstituted benzene ring. This corresponded to the presence of 15 carbon signals in the C-NMR spectrum and the molecular formula of CHO assigned by the HR-ESI-MS spectrum as above. The observation of the upfield shifted carbonyl carbon C-4 at δ 175.8 along with the carbon C-3 at δ 135.7 suggested 2 to be a flavonol type. The HMBC spectrum of 2 revealed the correlations of protons H-6, H-8, H-2', H-5', and H-6' with carbons via two or three bonds, as shown in Figure 2. Compound 2 was elucidated as quercetin by the good compatibility of its NMR data of 2 with those of quercetin10.

Compound 3 was obtained as a yellowish powder. The comparison of the NMR spectra of 2 and 3 revealed that 3 was also a flavonol. The only difference between 2 and 3 was the presence of a pair signals at δ 8.04 (, 9.0 Hz, H-2'/H-6'), 6.93 (, 8.5 Hz, H-3'/H-5') of a -disubstituted benzene ring in 3, instead of signals of the 1,3,4-trisubstituted ring in 2. The C-NMR spectrum of 3 displayed 15 carbon signals, including two carbon signals at δ129.4 (C-2'/C-6') and 115.4 (C-3'/C-5') appearing in double intensity in a symmetrical benzene ring possessing two substitutions at the position. Therefore, 3 was determined to be kaempferol due to the good compatibility of its NMR data with those published in the literature11.

Compound 4 was a white powder. HR-ESI-MS showed three protonated ion peaks at 314.0819, 316.0792, and 318.0773 with a relative intensity ratio of 9:6:1, which were calculated for (CHClNO+H, 314.0827), (CHClClNO+H, 316.0797), and (CHClNO+H, 318.0768), respectively. These results suggested that 4 possessed two chlorine atoms in the molecular formula. In the high magnetic zone, the proton spectrum of 4 showed signals of a terminal methyl (δ 0.77, 7.0 Hz, H-6), three methylene groups (δ 2.47, , 14.0, 5.5 Hz, H-3a; 1.61, , 14.5, 12.5, 4.5 Hz, H-3b; 1.25, , H-4a; 0.70, , H-4b; 1.16, , H-5). The COSY spectrum showed a connection of H6/H5/H4/H3 (Figure 2). These NMR data suggested that they belonged to an -butyl group linked to a certain chiral quaternary carbon. In the mediated magnetic zone, the proton spectrum also showed two doublet signals at δ 4.83 (, 14.5 Hz, H-1a) and 4.54 (, 14.5 Hz, H-1b) whose carbon signal was at δ 56.1 (C-1) via an HSQC experiment. These two nonequivalent hydrogens should be between a nitrogen atom and a certain quaternary carbon. The HMBC experiment showed cross-peaks of protons H-1 to C-3 and a quaternary carbon, of H-3 to C-1, and to the same quaternary carbon; therefore, this quaternary carbon should be C-2 (δ 75.5). Up to this point, compound 4 contained a six-carbon chain and a 2-hydroxyhexyl moiety. At a low magnetic field, the proton spectrum displayed a set of three aromatic signals at δ 7.52 (, 2.5 Hz, H-3''), 7.29 (, 8.5, 2.0 Hz, H-5''), and 7.53 (, 8.0 Hz, H-6'') of a 1,2,4-trisubstituted benzene ring. The HMBC experiment revealed cross-peaks of proton H-6'' to C-2 and of the hydroxy proton (δ 5.71,2-OH) to C-1''; therefore, this benzene ring joined to the abovementioned six-carbon chain at its C-2. In the proton spectrum, two left signals at δ 7.73 (1H, , H-3') and 8.26 (1H, , H-5'), which showed HSQC correlations to carbons C-3' (δ 150.3) and C-5' (δ 144.7), respectively, were hydrogens of a 1,2,4-triazole ring. The HMBC cross-peak of H-1 of the six-carbon chain to C-5' proved that this five-membered heterocyclic moiety was linked to C-1 of the chain. All mentioned data were in agreement, as shown in Figure 2. The chemical structure of 4 was determined to be hexaconazole by comparing its NMR data with those reported in the literature12. Although local residents used fungicides to kill parasitic fungi, the material was washed carefully before extraction. Therefore, hexaconazole, a triazole fungicide, isolated from this species could be an artifact that was absorbed by this liverwort. This noted that the bioactivities of extracts of , such as antibacterial activity in a previous preliminary study2, could be a result of either the second metabolites of this species or the artifact. Therefore, the chemical investigation was meaningful to understanding the bioactivities of the surveyed species.

Compound 5 was isolated as a white powder. Its HR-ESI-MS spectrum showed a pseudomolecular ion peak at 162.0557 [M+H] (calcd. for CHON+H, 162.0555). The proton spectrum of 5 showed a set of four signals at 8.00 (, 7.0 Hz, H-4), 7.14 (, 8.0, 7.0, 1.0 Hz, H-5), 7.18 (, 8.0, 7.0, 1.5 Hz, H-6), and 7.45 (, 7.0, 1.0 Hz, H-7) of a 1,2disubstituted benzene ring, which was further confirmed by the COSY correlations of H-4/H-5/H-6/H-7. In addition, the H-NMR spectrum of 5 displayed an olefin methine proton at δ7.98 (1H, , 3.0 Hz, H-2) and an amino proton at δ11.77 (1H, , NH). The C-NMR spectrum revealed nine carbon signals, including six signals belonging to the benzene ring, two olefinic carbons at δ 132.2 (=CH-, C-2) and 107.3 (=C<, C-3) of a double bond -CH=C<, and one carboxyl carbon at δ 165.9. The HMBC cross-peaks of the olefinic proton (H-2) with both quaternary carbons C-8 and C-9 of the benzene ring suggested the presence of an indole skeleton. The good compatibility of its NMR data with those published in the literature13 confirmed the chemical structure of 5 to be indole-3-carboxylic acid.

Compound 6 was a white powder. NMR spectra of 6 showed signals characterized for a sitosterol 3--β-D-glucopyranoside. The C-NMR spectrum displayed two olefinic carbons at δ 121.2 (=C<) and 140.5 (=CH-) of a double bond at C-5, an oxygenated carbon at δ 76.9 of C-3 as usual, one anomeric carbon at δ 100.8 (C-1') and five oxygenated carbons from 61.0 to 77.0 ppm of a sugar unit. Moreover, the proton spectrum showed signals supported for sitosterol 3--β-D-glucopyranoside, comprising one olefinic methine proton at δ 5.32 (, 4.5 Hz, H-6), one oxygenated methine proton at δ 3.45 (, H-3), two singlet, three doublet and a triplet proton signals from 0.65 to 0.95 ppm of six methyl groups in the sitosterol skeleton. An anomeric proton signal at δ 4.22 (1H, 7.5 Hz) and oxygenated proton signals from 2.9 to 3.7 ppm belonged to a β-D-glucose unit. 6 was thus determined to be sitosterol 3--β-D-glucopyranoside by comparing its NMR data with those reported in the literature14.

CONCLUSION

From the -hexane extract of L., six compounds were isolated, consisting of one bis-dibenzyl (marchantin G), two flavonols (quercetin and kaempferol), two alkaloids (hexaconazole and indole-3-carboxylic acid), and a phytosterol (sitosterol 3--β-D-glucopyranoside). Their chemical structures were elucidated by NMR and HR-MS data analysis in comparison with published data. This was the first time that the full NMR data of compound 1 were published.

ABBREVIATIONS

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

H-NMR: Proton nuclear magnetic resonance

C-NMR: Carbon-13 nuclear magnetic resonance

COSY: Homonuclear correlation spectroscopy

HSQC: Heteronuclear single quantum coherence

HMBC: Heteronuclear multiple bond correlation

: singlet

: broad singlet

: doublet

:doublet of doublets

: doublet of doublet of doublets

: broad doublet

: triplet

: multiplet

calcd.: calculated

DMSO: dimethyl sulfoxide

COMPETING INTEREST

The authors declare no competing financial interest.

AUTHORS’ CONTRIBUTION

Nguyen T.H.L., Tran Q.T. contributed to conducting the experiments and acquiring the data. Nguyen C.T.S., Nguyen K.P.P. interpreted NMR and MS data. Quach N.D.P., Nguyen T.H.T. provided final approval of the manuscript to be submitted.