Napthrene Compounds from Mycelial Fermentation Products of Marasmius berteroi

The metabolites of the genus Marasmius are diverse, showing good research prospects for finding new bioactive molecules. In order to explore the active metabolites of the fungi Marasmius berteroi, the deep chemical investigation on the bioactive compounds from its cultures was undertaken, which led to the isolation of three new naphthalene compounds dipolynaphthalenes A–B (1,2) and naphthone C (3), as well as 12 known compounds (4–15). Compounds 1, 2, and 4 are dimeric naphthalene compounds. Their structures were elucidated by MS, 1D and 2D NMR spectroscopic data, as well as ECD calculations. Compounds 2–4 and 7 exhibited acetylcholinesterase (AChE) inhibitory activities at the concentration of 50 μg/mL with inhibition ratios of 42.74%, 44.63%, 39.50% and 51.49%, respectively. Compounds 5 and 7,8 showed weak inhibitory activities towards two tumor cell lines, with IC50 of 0.10, 0.076 and 0.058 mM (K562) and 0.13, 0.18, and 0.15 mM (SGC-7901), respectively.


Introduction
Natural products are an important source of innovative chemical drugs [1]. Finding natural active ingredients for nerve protection and tumor suppression are also hot topics in the current research [2]. The genus Marasmius is a common basidiomycete in tropical and subtropical areas, which belongs to the family Marasmiaceae [3]. A few common species of Marasmius have been studied for searching of bioactive metabolites, and some active compounds such as terpenoids [4][5][6][7][8], steroids [9], cyclic peptides [10], isocoumarins [11] and piperidones [12] have been isolated. Many of these compounds showed antibacterial, cytotoxic and antihypertensive activities [9][10][11][12]. Marasmius berteroi is a small orange mushroom belonging to the genus Marasmius, which is widely distributed in the southeast of mainland China, mainly in Hainan, Guangdong, and Taiwan provinces. So far, few researches on chemical constituents of this fungus were reported [13]. To seek for new active small molecules from Marasmius berteroi, the deep chemical investigation on its cultures was thus undertaken, which led to the isolation of three new naphthalene ring compounds dipolynaphthalenes A-B (1,2) and naphthone C (3) along with twelve known analogues (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The isolation process and structural elucidation of three new compounds, as well as their AChE inhibitory and cytotoxic activity, are described in this paper.
The acetylcholinesterase inhibitory activity of compounds 1-15 were tested by previous method as described in the literature [28]. The results showed that compounds 2-4 and compound 7 exhibited anti-acetylcholinesterase activities at a concentration of 50 µg/mL with inhibition ratios of 42.74%, 44.63%, 9.50%, and 51.49%, respectively (Table 3). In the screening test of cytotoxicity [29], compounds 5, 7, 8 showed inhibitory activity against two tumor cell lines, with IC 50 of 0.10, 0.076 and 0.058 mM (K562) and 0.13, 0.18, and 0.15 mM (SGC-7901), respectively (Table 4). Binaphthalenes are dimeric compounds of naphthalene ring, mainly derivatives of DHN and juglone formed by different linking modes [30]. The acetylcholinesterase inhibitory activity evaluation results showed that compounds 2 and 4 exhibited anti-acetylcholinesterase activities, while compound 1 was inactive. Structurally, the active compounds 2 and 4 were naphthalene ring dimers with para-para ligation, while compound 1 was ortho-para ligation dimer, which indicated that the polymeric sites in binaphthalene compounds were very important for their biological activity. In the cytotoxic assay, compound 8 were active, while 9-15 were inactive, which may suggest that the 1,4-dione moiety is essential for their cytotoxicity.

Fungal Material
The fungus Marasmius berteroi was collected in the valley of Yinggeling, Hainan province of China, in November 2015, and identified by Associate Professor Sheng-zhuo Huang, the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. The mycelium was isolated from the Marasmius berteroi and its strain was maintained on potato dextrose agar (PDA) slant at 4 • C. A voucher specimen (YGL-18) was deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.

Fermentation, Extraction and Isolation
The fungus was cultured on slants of PDA medium at 28 • C for 5 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically to 200 × 1000 mL Erlenmeyer flasks each containing 300 mL medium. The flask was incubated at room temperature under static conditions for 30 days. The culture broth (60 L) was filtered to give the filtrate and mycelia. The filtrate was evaporated in vacuo to a small volume and then suspended in H 2 O and partitioned successively with EtOAc and n-BuOH. The EtOAc solution was evaporated under reduced pressure to give a crude extract (32.0 g), which was separated into fractions 1-6 on silica gel CC using a gradient eluent of petroleum ether-EtOAc (15:1-1:1, v/v, each 3 L). Fr. 2 (7.5 g) was subjected to repeated RP-18 CC (eluted with MeOH/H 2 O from 2:8 to 10:0, v/v, each 500 mL) and silica gel CC (eluted with petroleum ether-EtOAc from 5:1 to 1:1, v/v, each 600 mL) to afford compounds 1 (7.0 mg), 2 (5.0 mg) and 4 (3.0 mg).

Bioassay of AChE Inhibitory Activity and Cytotoxic Activity
AChE inhibitory activity of these compounds was assayed by the spectrophotometric method developed by Ellman [28]. Acetylthiocholine iodide (Sigma, St. Louis, MO, USA) was used as substrate in the assay. Na 2 HPO 4 (94.7 mL, 0.1 M) and NaH 2 PO 4 (5.3 mL, 0.1 M) were mixed to get phosphate buffer (PB, pH 8.0). Compounds were dissolved in DMSO (2% in PB). The reaction mixture contained PB (110 µL), test compound solution (10 µL, 2000 µM) and acetyl cholinesterase solution (40 µL, 0.1 U/mL), which were mixed and incubated for 20 min (30 • C). The reaction was initiated by the addition of DTNB (5,5-dithiobis-2-nitrobenzoic acid, 20 µL, 6.25 mM) and acetylthiocholine iodide (20 µL, 6.25 mM). The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s. Tacrine (Sigma-Aldrich 99%) was used as positive control (final concentration 0.33 µM), while 2% DMSO in PB was set as negative control (NC). All reactions were performed in triplicate. The percentage inhibition was calculated as follows: % age inhibition = (E − S)/E × 100 (E is activity of the enzyme without test compound and S is the activity of enzyme with test compound).

Conclusions
The secondary metabolites of fungi were various, which were good sources of active substances. The special compounds isolated from Marasmius berteroi also showed the diversity of the genus Marasmius. In the present study, three new naphthalene compounds dipolynaphthalenes A-B (1-2) and naphthone C (3), as well as 12 known compounds were isolated from the mycelial fermentation products of Marasmius berteroi. The bioactivity evaluation assays showed that dipolynaphthalene B (2), naphthone C (3), daldinone C (4) and 8-methoxynaphthalene-1,7-diol (7) showed anti-acetylcholinesterase activities.
Supplementary Materials: The following are available online. Figure S1: 1 H-NMR spectrum of dipolynaphthalene A (1); Figure S2