Structurally Diverse Sesquiterpenoids with Anti-neuroinflammatory Activity from the Endolichenic Fungus Cryptomarasmius aucubae

Graphic Abstract Two new sterpurane sesquiterpenoids named sterpurol D (1) and sterpurol E (2), and one skeletally new sesquiterpene, cryptomaraone (3), bearing a 5,6-fused bicyclic ring system, along with five known ones, sterpurol A (4), sterpurol B (5), paneolilludinic Acid (6), murolane-2α, 9β-diol-3-ene (7) and (–)-10,11-dihydroxyfarnesol (8) were isolated from an endolichenic fungus Cryptomarasmius aucubae. The structures of the new compounds were elucidated by analysis of NMR spectroscopic spectra and HRESIMS data. The absolute configurations of 1 and 2 were established by spectroscopic data analysis and comparison of specific optical rotation, as well as the biosynthetic consideration. Additionally, compounds 1, 2, 4–6, and 8 showed significant nitric oxide (NO) production inhibition in Lipopolysaccharide (LPS)-induced BV-2 microglial cells with the IC50 values ranging from 9.06 to 14.81 μM. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00299-9.

In continuation of our research on new and/or bioactive secondary metabolites from the endophytic fungi [26][27][28], a lichen-forming fungus Cryptomarasmius aucubae was isolated from the lichen collected from Hua Mountain in Shaanxi Province. After the cultivation of this fungus in cooked rice medium, eight sesquiterpenes (1-8) (Fig. 1), including three unreported and five known compounds, were obtained. Among them, compounds 2 and 5 were demonstrated to be potent anti-neuroinflammatory agents in lipopolysaccharide (LPS)-induced BV-2 microglial cells with the IC 50 values of 9.93 and 9.06 μM, respectively, which were comparable to that of quercetin (IC 50 = 9.75 μM) used as a positive control. Herein, the details of isolation, structure elucidation, and anti-neuroinflammatory activities of these compounds are presented.

Results, Discussion and Conclusion
The molecular formula of 1 was established to be C 19  The 1 H spectrum of 1 was very similar to that of the coexisting known sterpurol B (5), the only difference between them was that the hydrogen atom on the 12-OH in 5 was replaced by an acetyl group in 1 at δ H 1.95 and δ C 20.8/171.2 (Tables 1, 2). This indicated that 1 was an acetylated derivative of 5. In the HMBC spectrum of 1 (Fig. 2), the correlations from H-7 to C-5, C-6, C-8 and C-15, from H-9 to C-3, C-5, C-7, C-8 and C-10, from H-12 to C-1, C-2, C-11, C-13 and C-18, from CH 3 -13 to C-1, C-2, C-11 and C-12, from CH 3 -14 to C-3, C-4, C-5, C-8 and C-10, and from CH 3 -15 to C-5, C-7, C-8 and C-9, established the planar structure of 1 as a sterpurane-type sesquiterpene with two acetyl groups attached at C-5 and C-12. The absolute configuration of 1 (1R, 5R, 8S, 10R) was evidenced to be identical with that of 5, due to the same optical rotation for 1 ( (Tables 1 and 2) were similar to those of 5 except for a hydroxyl group (δ H 3.91; δ C 63.1). The location of the hydroxyl group at C-7 in 2 was determined by the observation of HMBC correlations from H-7 to C-6, C-8, C-9 and C-15. Detailed analysis of HSQC and HMBC spectra confirmed the structure of 2 as shown in Fig. 1. Furthermore, the relative configuration of 2 was determined by analysis of NOESY data. The obvious NOESY correlations (Fig. 2) of H-7 with H-15, H-15 with H a -9 (δ H 1.40) and H-17, indicated that they were all positioned on the same face of the tricyclic structure. In addition, the correlations of H-10 with H b -9 (δ H 1.71) indicated they were opposite orientation. The absolute configuration of 2 was determined using the modified Mosher's method [29] but failed, due to the instability of the sample. Nevertheless, based on the consideration of the biogenesis, the absolute configuration of 2 was deduced to be identical to that of 5, and was thus determined as 1R, 5R, 7S, 8S, 10R.
All of the compounds were tested for their anti-inflammatory activities by restraining the production of NO in lipopolysaccharide (LPS)-induced BV-2 microglial cells ( Table 3). As a result, compounds 1, 2, 4, 5, 6, 7 and 8 exhibited 75.9, 85.4, 73.1, 99.3, 79.1, 51.7 and 76.8% inhibition at 20 μM, respectively, whereas the positive control quercetin showed 95.6% inhibition at 20 μM. As shown in Table 3, the isolated compounds (except for 7) exhibited inhibitory effect with IC 50 values ranging from 9.06 to 14.81 μM, of which 5 was the most active compound with the IC 50 value of 9.06 μM. In addition, in vitro these sesquiterpenes were also assayed for other bioactivities, such as α-glucosidase inhibition, and antibacterial, however, they were inactive. In summary, eight secondary metabolites, including three new sesquiterpenoids, sterpurols D (1) and E (2), and cryptomaraone (3), and five known sesquiterpenes (4-8) were isolated and identified from the endolichenic fungus C. aucubae in rice solid-substrate fermentation. Compound 5 showed significant anti-inflammatory activity by reducing the release of NO in LPS-induced BV-2 Microglial cells without cytotoxicity at 50 μM. Besides, compounds 1, 2, 4, 6 and 8 displayed moderate anti-inflammatory activity. These findings are of value in searching for new anti-neuroinflammatory agents.

Fungal Material
The fungus, isolated from the crustose lichen collected in Hua Mountain, Huayin county, Shaanxi Province, China, in May 2017, was identified as Cryptomarasmius aucubae based on the DNA sequencing of the ITS of rDNA (Gen-Bank: NO. MW174800). The strain was assigned the accession No. SF69 and deposited in the Shaanxi Key Laboratory of Natural Products and Chemical Biology, Northwest A&F University, Yangling, China.

Fermentation and Extraction
The strain was activated by potato dextrose agar (PDA) medium in plates at 28 °C for 5 days. Then, the well-grown plate of the strain was cut into small pieces with a size of about 5 mm 2 , and the small pieces were inserted into 1000 mL Erlenmeyer flasks each containing 400 mL of potato dextrose (PD) liquid medium for culturing. The seed liquids were cultivated at 28 °C for 3 days on a shaking table at 120 rpm. Next, 20 mL seed liquid was poured into a rice medium (40 g rice, 60 mL distilled water) in 150 Erlenmeyer flasks (500 mL). After the fungi were fermented at 28 °C for 42 days, cultures were extracted two times with methanol. The methanol extract was vacuum filtered and dried under reduced pressure to yield a crude extract. The extract was dissolved and extracted with ethyl acetate and water in the volume ratio of 1:1 (4 L) for three times, and combined the organic layer, then it was concentrated under reduced pressure to give a crude extract (25.7 g).

Cell Viability Was Evaluated By MTT Assay
BV-2 murine microglial cells, acquired from Peking Union Medical College Cell Bank, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL) in carbon dioxide cell incubator. When cell growth density outnumbered 90%, BV-2 cells were seeded in 96-well plates at a density of 2 × 10 4 /well, 100 μL) and incubated for 24 h. Next, the cells were treated with the compounds (DMSO as solvent) at 20 μM for 24 h in DMEM with 1 μg/mL LPS. Cells treated with DMSO alone were used as the negative control. After adding 20 μL of 10 mg/mL MTT reagent to each well, the samples were shaken lightly and incubated at 37 °C for 4 h. The supernatant was removed, the blue-purple crystals were fully dissolved in DMSO (200 μL), and the absorbance of each well was read at 570 nm (Tecan Sunrise, Switzerland) [26,34]. Percentage of cell viability is calculated as: (absorbance of treated well/absorbance of control well) × 100%.

Nitric Oxide (NO) Production Inhibitory Assay
BV-2 cells were seeded into 96-well plates at 2 × 10 4 cells/100 μL of medium and incubated for 24 h. Then, cells were treated with 1 μg/mL of lipopolysaccharide (LPS) and various concentrations (0.1-20.0 μM) of test compounds (DMSO as solvent) for 24 h. An equal amount of DMSO and LPS were served as the controls; quercetin (J&K Scientific, Beijing, China) was taken as the positive control). The NO concentration in the medium was measured by using a Nitric Oxide Assay Kit, according to the accumulated levels of nitrite in the supernatants by a standard Griess reaction [26,34]. As follows, 50 μL of the culture supernatant of BV-2 cells was reacted with 50 μL of Griess reagent I and Griess regent II successively in a 96-well plate. The absorbance at 570 nm of the mixture was measured using a microplate reader. IC 50 values were calculated as the concentrations that reduced NO production by 50%. Quercetin was taken as the positive control.

α-Glucosidase Inhibitory Assay
α-Glucosidase Inhibitory assay was tested following the methods reported previously [35,36] with slight modification. The assay mixture (720 μL) contained 572.4 μL of 0.05 M phosphate buffer (pH 6.8), 3.6 μL of enzyme solution (10 U/mL), and 36 μL of 0.4 mM inhibitors (the tested compounds, genistein as positive control) were incubated at 37 °C for 10 min. Subsequently, 108 μL of 6 mM pNPG (4-nitrophenyl α-d-glucopyranoside) was added to the preincubated solutions, and the mixtures were incubated at 37 °C for 40 min. Then absorbance of the mixture at 405 nm was recorded. The negative control was prepared by adding PBS instead of α-glucosidase, the blank was prepared by adding solvent instead of tested compounds, and the inhibition rate was calculated as the following equation:

Antibacterial Assay
Antibacterial activities were evaluated according to the previously published report [37] with slight modification. Compounds 1 − 8 were tested in vitro for antibacterial activity against nine bacteria (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora pv.caratovora, Pseudomonas syringae, Erwinia carotovora subsp. Carotovora and Ralstonia solanacearum). The tested bacteria were incubated in the beef extract-peptone medium (BPA) at 30 °C at 120 rpm for 12 h and the spore concentration was diluted to approximately 2 × 10 6 CFU/mL with BPA medium. 50 µL of suspension was added to 96-well microplates, then 50 µL of compounds (Ampicillin and streptomycin as positive control) dissolved in DMSO-BPA medium was added to give a final concentration of 100 µM. After incubation at 30 °C for 24 h, the absorbance of the mixture at 600 nm was recorded.