INTRODUCTION

genus distributes in the subtropical regions, Mediterranean and mainly in the Northern hemisphere1. species is commonly found along the Coromandel coast and in the mangrove forest in Indian and in the South of Vietnam2. The previously chemical investigations on this species showed the presence of depside, depsidone, quinone, sterol, usnic acid derivatives, and phenolic compounds3, 4, 5. The bioactive assessments on extracts and isolated compounds from this species revealed anti-inflammatory, antimicrobial, anti-arthritic, antioxidant, and anticancer activities2, 4, 6, 7. The phytochemical investigation on this species was further studied on -hexane extract led to the isolation and structural elucidation of 5 compounds.

MATERIALS AND METHODS

Extraction and isolation

The fresh lichen (7.5 kg) was cleaned, air-dried, and ground into powder. Then, 876.8 g of crude extract was prepared using the above lichen powder in ethanol (3x10L) at room temperature. Next, this crude extract was applied to liquid-liquid partition step with -hexane (100%), -hexane:ethyl acetate (1:1, v/v), and ethyl acetate (100%) in turn to afford extracts, including -hexane (H, 200.9 g), -hexane:ethyl acetate (1:1) (H:EA, 225.5 g), and ethyl acetate (EA, 313.6 g).

The -hexane extract was subjected to silica gel column chromatography which was eluted with -hexane: ethyl acetate (13:1) to give seven sub-fractions (coded, PH1-PH7). PH2 (43.7 g) was applied to Sephadex-LH20, eluted with methanol to obtain five sub-fractions (coded, 2.1-2.5). Then, two compounds, 1 (10 mg) and 5 (8.7 mg) were isolated from sub-fraction 2.2 (4.5 g) by the silica gel column chromatography method, eluted with the solvent system of -hexane:chloroform: ethyl acetate (15:1:1). Next, PH4 (6.7 g) was separated into four sub-fractions 4.1 to 4.1 by the Sephadex-LH20 column chromatography, eluted with methanol. Afterward, sub-fractions 4.3 (2.4 g) was subjected to the C-18 reversed-phase silica gel column chromatography, eluted with the solvent system of Me: HO (15:1) to obtain 3 (11 mg) and 4 (7 mg). The same procedure was applied to sub-fraction PH7 (50.8 g) to afford 2 (9.5 mg).

RESULTS

From the -hexane extract of the lichen , collected at Binh Thuan Province, five compounds, 1 (10 mg), 2 (9.5 mg), 3 (11 mg), 4 (7 mg), and 5 (8.7 mg), were isolated. Their physical properties and spectroscopic data were performed as follows.

3-Hydroxy-7-methoxystigmast-5-ene (1): Colorless powder. The H-NMR (CDCl): δ 3.62 (, 5.0, 11.0 Hz, H-3), 2.36 (, 1.8, 5.0, 13.0, H-4a), 2.30 (, 1.5, 11.0, 13.0, H-4b), 5.74 (, 2.0, 5.0, H-6), 3.29 (, 4.0, H-7), 1.96 (, 3.5, 12.5, H-12), 0.66 (, H-18), 0.98 (, H-19), 0.92 (, 6,5 Hz, H-21), 0.81 (, 7.0 Hz, H-26), 0.83 (. 6.5 Hz, H-27), 0.86 (, 7.5 Hz, H-29), 3.35 (, -OCH). The C-NMR (CDCl): δ 36.9 (C-1), 31.6 (C-2), 71.6 (C-3), 42.5 (C-4), 146.3 (C-5), 120.9 (C-6), 74.1 (C-7), 37.4 (C-8), 42.9 (C-9), 37.6 (C-10), 21.0 (C-11), 39.2 (C-12), 42.3 (C-13), 49.2 (C-14), 24.4 (C-15), 28.4 (C-16), 55.9 (C-17), 11.6 (C-18), 18.4 (C-19), 36.3 (C-20), 19.0 (C-21), 34.1 (C-22), 26.2 (C-23), 46.0 (C-24), 29.4 (C-25), 19.2 (C-26), 20.0 (C-27), 23.3 (C-28), 12.2 (C-29), 56.9 (-OCH).

Sekikaic acid (2): Colorless solid. The H-NMR (CDCl): δ 6.38 (, H-3), 6.38 (, H-5), 2.99 (, H-8), 1.74 (, H-9), 0.94 (, 7.2 Hz, H-10), 3.83 (, 4-OCH), 6.43 (, H-5′), 2.97 (, H-8′), 1.64 (, H-9′), 1.00 (, 6.8 Hz, H-10′), 3.89 (, 4′-OCH). The C-NMR (Acetone-): δ 105.5 (C-1), 165.8 (C-2), 99.7 (C-3), 165.4 (C-4), 111.4 (C-5), 149.0 (C-6), 169.3 (C-7), 39.2 (C-8), 26.1 (C-9), 14.6 (C-10), 55.9 (4-OCH), 107.0 (C-1′), 157.3 (C-2′), 125.6 (C-3′), 156.2 (C-4′), 106.7 (C-5′), 146.8 (C-6′), 174.2 (C-7′), 39.2 (C-8′), 25.7 (C-9′), 14.6 (C-10′), 56.5 (4′-OCH). Selected HMBC correlations: see Figure 2.

Lichenxanthone (3): White crystal. The H-NMR (CDCl): δ 6.30 (, 2.0 Hz, H-2), 6.34 (, 2.4 Hz, H-4), 6.69 (, 2.4 Hz, H-5), 6.67 (, 2.0 Hz, H-7), 2.85 (, H-9), 3.87 (, 3-OCH), 3.90 (, 6-OCH), 13.39 (, 1-OH).

(+)-6,8-Dihydroxy-3-propyl-3,4-dihydroisocoumarin (4): Colorless needle crystal, = + 170 ( 0.1, MeOH). HR-ESI-MS: 245.0794 [M+Na]. The H-NMR (CDCl): δ 4.53 (, H-3), 2.82 (, 4.4, 16.4 Hz, H-4a), 2.88 (, 10.4, 16.0 Hz, H-4b), 6.31 (, H-5), 6.21 (, H-7), 1.68 (, H-1′a), 1.88 (, 5.2, 7.6, 10.0, 13.6 Hz, H-1′b), 1.49 – 1.58 (, H-2′), 0.97 (, 7.2 Hz, H-3′), 6.00 (, 6-OH), 11.20 (, 8-OH).

Ar-turmerone (5): Yellow solid, = 0 ( 0.2, MeOH). The H-NMR (CDCl): δ 2.31 (, H-1), 7.10 (, H-3/H-7; H-4/H-6), 3.29 (, H-8), 2.72 (, 8.0, 16.4 Hz, H-9a), 2.60 (, 8.0, 16.4 Hz, H-9b), 6.02 (, H-11), 2.10 (, 1.6 Hz, H-13), 1.85 (, 1.6 Hz, H-14), 1.24 (, 6.8 Hz, H-15). The C-NMR (CDCl): δ 21.1 (C-1), 135.9 (C-2), 129.2 (C-3/C-7), 126.8 (C-4/C-6), 143.5 (C-5), 35.4 (C-8), 52.8 (C-9), 200.1 (C-10), 124.2 (C-11), 155.3 (C-12), 20.9 (C-13), 27.9 (C-14), 22.1 (C-15). Selected HMBC correlations: see Figure 2.

DISCUSSION

Compound 1 was isolated as a colorless powder. The H-NMR spectrum showed an olefinic methine proton signal at δ 5.74 (, 2.0, 5.0, H-6), two oxygenated methine protons at δ 3.62 (, 5.0, 11.0 Hz, H-3) and 3.29 (, 4.0, H-7), a methoxy proton signal at δ 3.35 (, -OCH) and the rest proton signals resonating at high magnetic field including two singlets (δ 0.66 and 0.98), three doublets (δ 0.92, 0.81, and 0.83) and a triplet (δ 0.86) methyl signals which characterized for the stigmastane skeleton. It corresponded to the presence of a methoxy carbon and 29 carbons of the stigmastane. Two olefinic carbon signals resonated at δ 146.3 and 120.9 were a quaternary olefinic carbon (=C<, C-5) and a methine carbon (=CH-, C-6), respectively, in stigmast-5-ene as usual. The proton H-3 showed the large coupling constant value of 11.0 Hz with both axial protons H-2 and H-4, which approved the -configuration of the hydroxy group at C-3. Pettit and co-worker reported that the 7-methoxystigmast-5-ene compounds displayed a large coupling constant of 8.2 Hz of proton H-7 and a small coupling constant of 4.8 Hz of proton H-7. The compound 1 revealed the signal at δ 3.29 (, 4.0) of H-7, demonstrating the -configuration of the 7-methoxy group. Based on the above analysis and the good compatibly NMR data with those published in the literature8, 1 was suggested to be 3-hydroxy-7-methoxystigmast-5-ene.

Compound 2 was isolated as a colorless solid. The H-NMR spectrum displayed aromatic proton signals at δ 6.38 (2H, , H-3/H-5) of a tetra-substituted benzene ring and 6.43 (, H-5′) of a penta-substituted one. Two methoxy proton signals were observed at δ 3.83 (, 4-OCH), 3.89 (, 4′-OCH). At the higher magnetic field, it showed signals of two -propyl moieties at δ [2.99 (, H-8), 1.74 (, H-9), 0.94 (, 7.2 Hz, H-10)] and [2.97 (, H-8′), 1.64 (, H-9′), 1.00 (, 6.8 Hz, H-10′)] which were further identified by the HMBC cross-peaks as shown in Figure 2. The C-NMR spectrum showed 22 signals consisting of twelve signals of two benzene rings from 99.7 to 165.8 ppm, two carboxyl carbons at δ 169.3 (C-7) and 174.2 (C-7′), two methoxy carbons at δ 55.9 (4-OCH) and 56.5 (4′-OCH), and six final carbon signals from 14.6 to 39.2 ppm belonging to two -propyl chains. The position of two methoxys were C-4 and C-4’, which were confirmed by HMBC correlations of the methoxy protons to carbons at δ 165.4 (C-4) and 156.2 (C-4′). The HMBC correlations of H-8 to carbon C-1, C-5, and C-6, of H-8’ to carbons C-1’, C-5’, and C-6’ determined the positions of two n-propyl groups at C-6 and C-6’. All other HMBC correlations confirmed the chemical structure of 2. Additionally, the comparison of NMR data of 2 with the published data9 showed good compatibility; therefore, 2 was assigned as sekikaic acid.

Compound 3 was isolated as a white crystal. The H-NMR spectrum showed a signal at δ 13.39 (, 1-OH) of the proton of a phenolic hydroxy group which was chelated to an -carbonyl group, two -doublet signals which had the multiplet skewing effect together at δ 6.30 (, 2.0 Hz, H-2), 6.34 (, 2.4 Hz, H-4) of a tetra-substituted benzene ring, two other -doublet signals δ 6.69 (, 2.4 Hz, H-5), 6.67 (, 2.0 Hz, H-7) of the second tetra-substituted benzene ring. The moderate magnetic zone revealed signals of two methoxy groups at δ 3.87 (, 3-OCH) and 3.90 (, 6-OCH). The final proton signal at δ 2.85 (, H-9) was belonged to a methyl group connected to a benzene ring. Besides, the NMR data of 3 showed highly relevant to the published data10, 3 was thus determined as lichenxanthone.

Compound 4 was isolated as a colorless needle crystal. The HR-ESI-MS of 4 showed the ion peak at 245.0794 [M+Na] (calcd. for CHONa, 245.0790) which deduced its molecular formula to be CHO. The H-NMR spectrum displayed a singlet proton signal at δ 11.20 (, 8-OH) of a phenolic hydroxy group which was chelated to an -carbonyl group. Two broad-singlet proton signals at 6.31 (, H-5) and 6.21 (, H-7) suggested the presence of a 1,2,3,5-tetra-substituted benzene ring. A very broad-singlet signal integrated one proton at δ 6.00 (, 6-OH) was deduced to a hydroxy group which did not exist the intramolecular hydrogen bond. There was a multiplet signal at δ 4.53 (H-3) of an oxygenated methine group in the magnetic zone of protons on the carbon attached to the single-bonded oxygen. Signals of two non-equivalent protons of a methylene group deshielded δ 2.82 (, 4.4, 16.4 Hz, H-4a) and 2.88 (, 10.4, 16.0 Hz, H-4b) due to the attachment to sp quaternary carbon and a chiral methine group. At highest magnetic field, the observation of a triplet signal, integrated three protons at 0.97 (, 7.2 Hz, H-3′) belonged a methyl group which was adjacent one methylene group. Four remaining protons at δ 1.68 (1H, , H-1′a), 1.88 (1H, , 5.2, 7.6, 10.0, 13.6 Hz, H-1′b), 1.49 (1H, , H-2’a), and 1.58 (1H, , H-2′b) were deduced the presence of two methylene groups. Based on the above information and the good compatibility of its NMR data with those published in the literature11, along with its dextrorotatory activity = + 170 ( 0.1, MeOH), 4 was determined as (+)-6,8-dihydroxy-3-propyl-3,4-dihydroisocoumarin.

Compound 5 was isolated as a yellow solid. The H-NMR spectrum of 5 displays a singlet proton signal integrated four-protons at δ 7.10 (, H-3/H-7; H-4/H-6) para-disubstituted benzene ring, a singlet olefinic proton at δ 6.02 (1H, , H-11) of a methine group =CH- attached to two quaternary carbons. At the high magnetic field, the proton spectrum displayed signals at δ 2.10 (, 1.6 Hz, H-13) and 1.85 (, 1.6 Hz, H-14), which possessed HMBC correlations to the same olefinic carbons at δ 124.2 (C-11), 155.3 (C-12). These signals suggested the presence of -CH=C(CH) moiety. A methyl proton signal resonated at 2.31 (, H-1) was suggested to attach to the benzene ring (as -CHCH-), which was further confirmed by HMBC cross-peaks of this proton signal to aromatic carbons at δ 135.9 (C-2) and 129.2 (C-3/C-7). A proton signal appearing as a doublet, integrated three-protons at 1.24 (, 6.8 Hz, H-15) was assigned to the methyl group attached to the other methine group. This methyl proton signal showed HMBC correlations to a quaternary aromatic carbon C-5 at 143.5 (C-5), a methine carbon C-8 (35.4), and a methylene carbon C-9 (52.8), which demonstrated the presence of CH-CH(CHCH)-CH- fragment. The C-NMR spectrum showed a ketone carbon signal shielded at 200.1 (C-10), deduced to be the ketone group conjugated to the double bond. It corresponded to the sole quaternary olefinic carbon C-12 shifted to the higher frequency at 155.3 ppm. All the rest of HMBC correlations supported the chemical structure as shown. Additionally, the comparison of its NMR data to those reported in the literature12 gave further evidence to confirm the chemical structure of 5 as ar-turmerone. The optical rotation value of = 0 suggested 5 to be a racemic mixture of ar-turmerone. Ar-turmerone was known as the major bioactive compound of species and possessing anti-inflammatory and neuroprotective property13.

CONCLUSION

From the -hexane extract of the lichen , five compounds were isolated consisting of a sterol (3-hydroxy-7-methoxy-stigmast-5-ene), a depside (sekikaic acid), a xanthone (lichenxanthone), a coumarin derivative ((+)-6,8-dihydroxy-3-propyl-3,4-dihydroisocoumarin), and an ar-turmerone (5). Their chemical structures were elucidated by NMR, MS spectroscopic data analysis, optical rotations, and the comparison to the published data. Four isolated compounds 1, 2, 4, and 5 were reported to presence in the genus for the first time while 3 had been already isolated from other species of this genus. The ongoing studies on this species are in progress.

ABBREVIATIONS

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

H-NMR: Proton nuclear magnetic resonance

C-NMR: Carbon-13 nuclear magnetic resonance

HSQC: Heteronuclear single quantum coherence

HMBC: Heteronuclear multiple bond correlation

: singlet

: broad singlet

: doublet

: triplet

: multiplet

COMPETING INTEREST

The authors declare no competing financial interest.

AUTHORS’ CONTRIBUTION

Duong T.H contributed to conducting experiments, acquisition of data, and interpretation of data. Nguyen T. H. T interpreted NMR and MS data as well as gave final approval of the manuscript to be submitted.

Corresponding author: Dr. Nguyen Thi Hoai Thu, University of Medicine and Pharmacy at Ho Chi Minh City, 217 Hong Bang Str., District 5, Ho Chi Minh City. Email: nguyenthihoaithu@ump.edu.vn or hoaithudhyd@gmail.com

ACKNOWLEDGMENT

This research was supported by NAFOSTED under grant number 104.01-2019.45.