Bioactive Terphenyls Isolated from the Antarctic Lichen Stereocaulon alpinum

Three p-terphenyls (2–4)—2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)—were isolated for the first time as natural products along with seven known compounds (1, 5–10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 μM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 μM.


Introduction
Lichens, which represent complex symbiotic associations between fungi (mycobiont) and algae (photobiont), produce various unique secondary metabolites arising from the symbiosis [1]. The two association partners play crucial roles in providing each other the conditions necessary for their continual existence [1]. Owing to this unique relationship, both mycobionts and photobionts are able to grow in environments where it is considered impossible for them to survive by themselves [1]. There are records for the use of lichens as traditional medicines by cultures in Africa, Europe, Asia, America, and Oceania [2]. People commonly use lichens to treat skin disorders, wounds, digestive and respiratory issues, obstetric, and gynecological concerns [2]. The Stereocaulon genus is one of the fruticose lichen groups found in a broad spectrum of regions, including tropical areas and polar zones [3]. The genus is known as the source of common depsides encountered in many lichens and it also contains unique secondary metabolites [3]. Some species of this genus are used in treating wounds, ulcers [3], urinary infection [4], and symptoms of type 2 diabetes [5]. Previous studies also reported bioactive secondary metabolites from Stereocaulon alpinum, including lobaric acid as an antimitotic inhibitor [6]; a cyclic depsipeptide exhibiting cytotoxicity against human tumor cell lines [7]; a depsidone with anti-proliferative activity [8]; and pseudodepsidones with antibacterial, antioxidant, and tyrosine phosphatase inhibitory activities [9,10]. However, to the best of our knowledge, there have been no reports on Stereocaulon genus about structural analysis and biological activities of p-terphenyls, which are known to restrictedly appear in fungi and lichens, and are reported to exhibit cytotoxic, antibacterial, anti-inflammatory, and antioxidant properties [11][12][13][14].
As part of our work to discover potential bioactive compounds from polar natural products, chemical analysis of the Antarctic lichen S. alpinum was performed and led to the isolation of four p-terphenyls (1-4) along with four phenolic compounds (5)(6)(7)9), a dibenzofuran derivative (8), and a steroid (10). Three (2)(3)(4) of the four p-terphenyls are reported for the first time as natural products in the current study. Herein, we describe the isolation and structural elucidation of the new compounds as well as the biological activity of all isolated compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).
As part of our work to discover potential bioactive compounds from polar natural products, chemical analysis of the Antarctic lichen S. alpinum was performed and led to the isolation of four p-terphenyls (1-4) along with four phenolic compounds (5)(6)(7)9), a dibenzofuran derivative (8), and a steroid (10). Three (2)(3)(4) of the four p-terphenyls are reported for the first time as natural products in the current study. Herein, we describe the isolation and structural elucidation of the new compounds as well as the biological activity of all isolated compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).

Structure Elucidation of the Compounds
Compound 2 was obtained as a yellow crystal. The molecular formula was determined as C20H18O3 from the [M] + peak at m/z 306.1249 (calcd. for C20H18O3, 306.1256) in the HREIMS analysis. The 1 H NMR data (Table 1) showed signals attributable to a hydroxy group (δH 5.74), 10 aromatic protons corresponding to single-substituted benzene rings (δH 7.37-7.67), one singlet aromatic proton at δH 6.75 (1H, s) and two methoxy groups at δH 3.37 (3H, s) and 3.72 (3H, s). The 13 C NMR spectrum (Table 1)

Biological Evaluation
All isolated compounds (1-10) were evaluated for cytotoxicity and anti-inflammatory activity. Compound 3 exhibited significant cytotoxicity against HCT116 cells, human colorectal carcinoma cell line, with the IC 50 value of 3.76 ± 0.03 µM (Table S1), and it also displayed inhibitory activity on NO production in LPS-induced RAW264.7 macrophages, in which the IC 50 value was 22.82 ± 0.015 µM (Table S2). Four compounds (1-4) possess similar structures, but only compound 3-which has a hydroxy group attached to C-5 and a methoxy group attached to C-6 of the central benzene ring-showed its activities through the assays. Consequently, although the structure-activity relationships of the p-terphenyls have not been investigated thoroughly, our results suggested the possibility of these functional groups and their positions to be responsible for the biological activities of p-terphenyls.
Reversed-phase TLC was carried out on glass plates pre-coated with RP-18 F 254 (0.25 mm, Merck, Darmstadt, Germany), mobile phase MeOH:H 2 O (50:50 to 100:0), and MeCN:H 2 O (50:50 to 100:0). Spots on TLC plates were detected using UV lamp (254 nm and 365 nm), and heating after dipping in 20% sulfuric acid in H 2 O. The sample was injected into the semi-prep. HPLC was carried out by multiple injections with the concentration of each single injection being around 10 mg/1 mL and the injected volume being approximately 200 µL.

Lichen Material
The lichen was collected from King George Island, Antarctica, and identified by Dr. Ji Hee Kim. A voucher specimen was deposited at the Polar Natural Product Chemistry Laboratory of the Korea Polar Research Institute.

Extraction and Isolation
The air-dried and chopped lichen (300 g) was macerated with methanol (3 L × 3 times) at room temperature for three days. The crude extract was concentrated under a vacuum to yield a brown slurry (22 g). The methanol extract was then suspended in H 2 O and sequentially partitioned with n-hexane, ethyl acetate (EtOAc), and n-buthanol (BuOH) to afford the hexane (1.05 g), EtOAc (3.44 g), and BuOH (1.91 g) soluble fractions. The hexane fraction (1.05 g) was adsorbed onto a silica gel column chromatography (CC), and gravity elution was performed with the gradient solvent system of hexane with increasing amounts of EtOAc (5-100%), followed by mixture of chloroform (CHCl 3 ) and MeOH (from 0% to 50% MeOH in CHCl 3  The EtOAc fraction (3.44 g) was subjected to silica gel CC, and gravity elution was performed with a gradient solvent system of hexane with increasing amounts of EtOAc (5-100%), followed by CHCl 3 and MeOH (from 0% to 50% MeOH) to afford eight fractions (E1-E8). Purification of E2 (46.0 mg) was conducted by HPLC (MeOH:H  2,6-Dimethoxy-5-hydroxy-p-terphenyl (2)

Cell Culture
RAW264.7 macrophages and HCT116 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin at 37 • C in a humidified CO 2 incubator. In this study, macrophages were subjected in the absence or presence of the isolated compounds with different concentrations. Isolated compounds were added 1 h prior to LPS (0.5 µg/mL) stimulation. HCT116 cells were seeded 5 × 10 3 cells/well on a 96-well plate in triplicate, and then incubated in 5% CO 2 supplement at 37 • C.

MTS Assay
MTS assay was conducted to determine the cytotoxic effects of the isolated compounds against HCT116 cells. The cells were seeded at a density of 2 × 10 5 cells/mL on a 96-well plate. After incubating in 24 h, 10% MTS solution was added to the cell culture medium, and then it was further incubated at 37 • C for 1 h. The concentration of the treated compounds ranged from 50 µM to 6.25 µM, using serial dilution. The absorbance was measured after 24 h using a microplate reader (Promega, Madison, WI, USA) at 490 nm.

Measurement of Nitric Oxide (NO) Production
NO concentration in the RAW264.7 cell culture supernatant was measured using Griess reagent. Briefly, 100 µL of the collected supernatant was mixed with equal amounts of Griess reagent (1% sulfanilamide in 5% phosphoric acid, 0.1% N-(1-naphthyl) ethylenediamine). The mixtures were incubated for 10 min at room temperature, and then the absorbance value of each well was determined at a wavelength of 540 nm using a microplate reader. Nitrite concentration was determined using a sodium nitrite calibration curve (0-100 µM).