INTRODUCTION

Lichens are complex symbiotic associations between fungi and algae that are important constituents of many ecosystems. The production of various unique extracellular secondary metabolites known as lichen substances is the result of this symbiosis. These compounds possess a wide range of biological activities, including antimicrobial, antifungal, antiviral, and anticancer activities.1 Historically, some lichen species have been used for the food, dye, cosmetics, and folk medicine industries. Among them, the genus (Parmeliaceae; lichenized Ascomycetes) is the largest genus of fruticose lichen, with more than 360 species, and is widely distributed from polar zones to tropical areas in the world.2 The lichen Vain. belonging to the genus grow hanging from tree branches in the damp forest at Bidoup Nui Ba National Park, Dam Rong district, Lam Dong province. According to Philippe Clerc3, this lichen was suggested to synonymy with Stirt. However, Kristiina Mark4 and his partner proposed synonymization of and under . In addition, this lichen has not yet been chemically and biologically studied. The primary goal of the present work was to study the chemical constituents of the lichen Vain.

MATERIALS AND METHODS

Lichen Material

Thalli of the studied lichen (Figure 1) were separated from tree branches and bark of various old trees in a damp forest at Bidoup Nui Ba National Park, Dam Rong district, Lam Dong province, Vietnam, in August 2018 and authenticated by Dr Vo Thi Phi Giao, Faculty of Biology, University of Science, Vietnam National University - Ho Chi Minh City. A voucher specimen, coded US-B040-DUNG, was deposited at the laboratory of the Faculty of Environmental Science, Saigon University.

RESULTS

The chemical investigation on the thalli of lichen Vain. collected in Lam Dong Province obtained six compounds using efficient separation techniques. These compounds were isolated from the EA1 fraction of EtOAc extract, and their structures are shown in Figure 2. The spectral properties of these compounds, including H and C NMR data, were identified and compared to those previously described in the literature.

Lecanorin (1): white amorphous powder. HRESIMS (positive mode) 297.0736 [M+Na] (calcd. for CHONa, 297.0739). Rf: 0.4 (silica gel plate; CHCl-MeOH, 95:5). The H and C NMR (recorded at 500 MHz for H and 125 MHz for C in DMSO–), see Table 1.

Isolecanoric acid(2): white amorphous powder. HRESIMS (positive mode) 341.0605 [M+Na] (calcd. for CHONa, 341.0637). Rf: 0.5 (silica gel plate; CHCl-MeOH, 90:10). The H and C NMR (recorded at 500 MHz for H and 125 MHz for C in DMSO–), see Table 1.

Norstictic acid (3): white amorphous powder. HRESIMS (positive mode) 395.0348 [M+Na] (calcd. for CHONa, 395.0379). Rf: 0.5 (silica gel plate; CHCl-MeOH, 95:5). The H and C NMR (recorded at 400 MHz for H and 100 MHz for C in DMSO–), see Table 2.

Methylstictic acid (4): white amorphous powder. HRESIMS (positive mode) 423.0699 [M+Na] (calcd. for CHONa, 423.0692). Rf: 0.5 (silica gel plate; CHCl, 100%). The H and C NMR (recorded at 500 MHz for H and 125 MHz for C in CDCl), see Table 2.

22,24-5,6-epoxyergosta-8,22-diene-3-diol (5): white amorphous powder. HRESIMS (positive mode) 451.3169 [M+Na] (calcd. for CHONa, 451.3188). Rf: 0.4 (silica gel plate; CHCl-MeOH, 95:5). The H and C NMR (recorded at 400 MHz for H and 100 MHz for C in CDCl), see Table 3.

Lupeol (6): white amorphous powder. HRESIMS (positive mode) 449.3772 [M+Na] (calcd. for CHONa, 449.3759). Rf: 0.4 (silica gel plate; CHCl, 100%). The H and C NMR (recorded at 500 MHz for H and 125 MHz for C in DMSO–), see Table 3.

DISCUSSION

Compound (1) was obtained as a white amorphous powder. Its molecular formula, CHO, was determined from its HRESIMS at 297.0736 [M+Na] (calcd. for CHONa, 297.0739). The H NMR data showed the presence of two singlets at 2.35 (3H) and 2.24 (3H) for two methyl groups, four singlets of five aromatic protons at 6.22 for protons H-3 and H-5 and at 6.51, 6.46, and 6.41 for protons H-1', H-5' and H-3', respectively. The C NMR spectrum showed signals of 15 carbons corresponding to two methyl groups at 21.0 and 21.5 (6'-CH and 6-CH), five aromatic methines at 100.5; 106.2; 109.9; 112.9, and 113.5 (C-3; C-3'; C-5; C-1', and C-5', respectively), seven quaternary carbons including four oxygenated carbons at 108.3; 139.7; 140.3; 151.1; 158.0; 160.3, and 161.1 (C-1; C-6'; C-6; C-2'; C-4'; C-2, and C-4, respectively), and one carboxyl carbon at 167.7 (C-7). The key HMBC correlations from 6-CH to C-1; C-5, and C-6 together with from 6'-CH to C-1'; C-5', and C-6' disclosed the position of these methyl groups at C-6 and C-6'. The exact location of the aromatic protons was also established based on 2D NMR. These spectroscopic data, along with its HRESIMS data at 297.0736 [M + Na] were consistent with the published data.5 Consequently, the structure of (1) was established as lecanorin (Figure 2).

Compound (2), giving the molecular formula CHO by HRESIMS at 341.0605 [M+Na] (calcd. for CHONa, 341.0637), a white amorphous powder was obtained. Its H NMR spectrum showed two singlets at 2.36 (3H) and 2.54 (3H) for two methyl groups, a pair of doublets for two -substituted aromatic protons at 6.32 (1H, , = 2.0 Hz, H-5ʹ) and 6.24 (1H, , = 2.0 Hz, H-3ʹ) and a singlet at 6.22 (2H, H-3 and H5). The C NMR spectrum was similar to that of (1) except for the presence of one more carboxyl carbon at 171.8 (C-7'). The position of the aromatic protons and the substituted functional groups were established based on HMBC correlations. These spectroscopic data were compatible with those published in the literature.6 Therefore, the structure of (2) was suggested to be isolecanoric acid. (Figure 2).

Compound 3 was isolated as a white amorphous powder and identified as norstictic acid, as shown in Figure 2, by comparison of its physical and spectral data with the literature7. The HRESIMS showed an [M+Na] peak at m/z 395.0348, which revealed the molecular formula of CHO. The H and C NMR spectra of (3) (Table 2) showed signals of two methyl groups [ 2.21 (3H, ); 2.45 (3H, ); 9.6; 21.4, respectively], an aldehyde group [ 10.46 (1H, ); 192.8], and a lactone group ( 163.6). Additionally, the presence of two methine groups [ 6.79 (1H, ); 95.0, and 6.85 (1H, ); 117.4] and an ester group supported the ether bridge between C-11a and C-12a, leading to a depsidone skeleton. The HMBC correlations (Figure 3) between these protons and carbons confirmed the structure of (3).

Compound 4 was isolated as a white amorphous powder. The H and C NMR spectra of (4) (Table 2) showed signals assignable to two methoxy groups [ 3.97 (3H, ); 3.70 (3H, ); 58.0; 56.8, respectively], a lactone group ( 169.7), two methyl groups [ 2.30 (3H, ); 2.56 (3H, ); 9.3; 22.4, respectively], an aldehyde group [ 10.50 (1H, ); 187.0], an aromatic proton [ 6.74 (1H, ); 112.1], and a methine proton [ 6.40 (1H, ); 103.1]. Furthermore, the HRESIMS of (4) showed a pseudomolecular ion peak at m/z 423.0699 [M+Na] which revealed the molecular formula of CHO. ItsH and C NMR resonances were similar to those of (3) except for two additional methoxy groups at C-1 [ 3.70 (3H, ); 56.8] and C-10 [ 3.97 (3H, ); 58.0] in (4). The HMBC correlations from 1-OCH ( 3.70) to C-1 and from H-10 ( 3.97) to C-10 confirmed the location of two additional methoxy groups. By comparing these data with those in the literature,8(4) it was suggested to be methylstictic acid.

Compound (5) was isolated as a white amorphous powder. The H NMR spectrum contained four doublet signals for secondary methyl groups at 1.01 (3H, , = 6.8 Hz, H-21), 0.90 (3H, , = 6.8 Hz, H-28), 0.82 (3H, , = 6.4 Hz, H-26), 0.81 (3H, , = 6.4 Hz, H-27) and two singlets of two tertiary methyls at 1.24 (3H, ) and 0.57 (3H, ). The signal at 3.93 (1H, ) was assigned to the proton on carbon bearing a hydroxyl group. Additionally, the signal of two olefinic protons at 5.18 (2H, , = 15.6; 7.6 Hz, H-22 and H-23) of a cholestane skeleton were also observed. TheC NMR spectrum showed the presence of 28 carbon signals, including four olefinic carbon signals at 135.7 (C-22), 134.6 (C-9), 132.2 (C-23), and 127.1 (C-8) and four oxygenated carbons at 68.7 (C-3), 67.2 (C-7), 65.8 (C-5), and 62.7 (C-6). Furthermore, the molecular formula of (5) was determined to be CHO through the sodium adduct ion at 451.3169 [M+Na] in the HRESIMS spectrum. On the basis of the above results, compound (5) was determined to be 22,24-5,6-epoxyergosta-8,22-diene-3-diol.9

Compound (6) was isolated as a white amorphous powder. The H NMR spectrum showed the presence of singlet signals for seven tertiary methyl groups at 0.65, 0.76, 0.77, 0.87, 0.91, 0.99, and 1.64. A doublet of triplets for one proton at 2.37 assigned to H-19 is characteristic of lupeol. The H-3 proton displayed a multiplet at 2.97, while a pair of doublet signals of two protons at 4.68 and 4.54 were assigned to olefinic protons at H-29. The structural assignment of (6) lupeol was further substantiated by the characteristic signals of lupeol at 76.8 (C-3), 109.6 (C-29) and 150.2 (C-20) in its C NMR experiments. The molecular formula of (6) was determined to be CHO through the sodium adduct ion at 449.3772 [M+Na] in the HRESIMS spectrum. The structure of (6) was confirmed through HSQC and HMBC experiments (Figure 3). On the basis of the above results, compound (6) was identified as lupeol. 10

CONCLUSION

In the investigation of the chemical constituents of lichen Vain. collected in Bidoup Nui Ba National Park, Dam Rong district, Lam Dong province, six known compounds were isolated, including lecanorin (1), isolecanoric acid (2), norstictic acid (3), methylstictic acid (4), 22,24-5,6-epoxyergosta-8,22-diene-3-diol (5) and lupeol (6). This is the first time these compounds have been reported in the lichen Vain.

ABBREVIATIONS

H- NMR: Proton nuclear magnetic resonance

C NMR: Carbon-13 nuclear magnetic resonance

CDCl: Deuterochloroform

CC: Column chromatography

: doublet

: doublet of double

: doublet of double of double

: multiplet

DMSO: dimethyl sulfoxide (CDSOCD)

EtOH: Ethanol (CHOH)

EtOAc: Ethyl acetate (CHCOOCHCH)

HMBC: Heteronuclear multiple bond correlation

HRESIMS: High-resolution electrospray ionization mass spectrometry

MeOH: Methanol (CHOH)

: Broad singlet

: singlet

TLC: Thin layer chromatography

COMPETING INTERESTS

The authors declare no competing financial interest.

AUTHORS' CONTRIBUTIONS

Nguyen Huu Tri and Nguyen Thi My Dung contributed to conducting the experiments, obtaining the data and writing the manuscript. Nguyen Thi My Dung (corresponding author) has contributed a significant explanation of the data and revised the manuscript.

ACKNOWLEDGMENTS

Sai Gon University funded this research under grant number CS2020-53.