A new terrein dimer and a new meroterpene from the mangrove endophytic fungus Lichtheimia sp.

Abstract A new terrein dimer named lichtheicol A (1) and a new meroterpene named lichtheiterpene A (2), were isolated from the mangrove endophytic fungus Lichtheimia sp. J2B1, together with 10 known compounds (3–12). The planar structures and absolute configurations of 1 and 2 were established by a combination of extensive spectroscopic data analyses and electronic circular dichroism (ECD) calculations. Compounds 4 and 5 exhibited marked inhibitory effects against butyrylcholinesterase (BuChE) with IC50 values of 0.71 and 0.53 μM, respectively.

In summary, two previously undescribed compounds, along with ten known compounds, were obtained from the mangrove endophytic fungus Lichtheimia sp.J2B1.All compounds were isolated from this fungal genus for the first time.Lichtheicol A was a rare terrein dimer.To our knowledge, only four terrein dimers have been reported from the culture of Eupenicillium javanicum up to the present [20].From the standpoint of biosynthesis, lichtheicol A could be generated from terrein via the Diels-Alder [4 þ 2] cycloaddition (Figure S12).The discovery of lichtheicol A and lichtheiterpene A in this study enriched the structural diversity of natural products obtained from fungi of the genus Lichtheimia.

Fungal material
The fungal strain J2B1 was isolated by one of the authors (J.W.) from mangroves collected in Penang, Malaysia, in February 2012.The strain was deposited in the Marine Drugs Research Center, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China.The fungus was identified as a species of the genus Lichtheimia according to its morphological characteristics and by comparison of the ITS sequence with those in the National Center for Biotechnology Information (NCBI).
The ITS sequence was submitted to GenBank with the entry number HM104211.1.

Fermentation
Agar plugs (5 mm i.d.) of the fungal strain were inoculated into flasks (500 ml) containing 200 ml of potato dextrose broth (PDB; 20 g glucose and 3 g sea salt in 1 L of potato infusion).Flasks with the inoculated fungal strain were placed in a rotary shaker (150 rpm) at 28 C for 4 days.Then the fungal mycelia were aseptically transferred into the solid rice media, which were prepared by autoclaving treatment of 75 g rice, 0.24 g sea salt, and 80 ml distilled water in an Erlenmeyer flask (1 L).Fermentation was performed under static conditions in 200 Erlenmeyer flasks (1 L per flask) at 25 C for 30 days.

Extraction and isolation
The culture media of Lichtheimia sp.J2B1 were dried and soaked in ethyl acetate at room temperature (3 Â 15 L, 24 h for each time) to afford the EtOAc extract, which was then dissolved with MeOH/H 2 O (90:10) (500 ml) and extracted with petroleum ether (PE, 3 Â 500 ml).The aqueous layer was concentrated under reduced pressure to remove MeOH, and the resultant mixture was extracted with EtOAc (3 Â 400 ml) to obtain the crude extract (90 g), which was subjected to silica gel CC

Cholinesterase inhibitory assay
The inhibitory activities of compounds 1-12 against AChE and BuChE were evaluated by a modified Ellman method.20 ml of sample solution (with different concentrations in DMSO) and 20 ml of AChE or BuChE solution (0.2 units/ml in saline) were added to a 96-well plate containing 140 ml of phosphate buffer (pH 7.0).After incubating at 37 C for 15 min, 10 ml of the substrate solution (acetylcholine or butylcholine, 1.0 mg/ml, dissolved in physiological saline) was added to initiate the enzyme reaction.The resultant mixture was kept at 37 C for another 20 min.Then, the absorbance was measured at 412 nm on a microplate reader.Tacrine was used as a positive control.All tests were performed in triplicate.The IC 50 values of two compounds, viz., 4 and 5, of which the BuChE inhibition rates were greater than 50% at the initial concentration of 10 lM, were calculated using GraphPad Prism 9 software according to the data of the cholinesterase inhibition experiment.

Disclosure statement
No potential conflict of interest was reported by the author(s).
Compound 2 was isolated as an amorphous powder.Its molecular formula C 26 H 36 O 7 with nine degrees of unsaturation was deduced from a positive HR-ESIMS ion at m/z 478.2799 [M þ NH 4 ] þ .The 1 H NMR spectroscopic data of 2 revealed the

Figure 4 .
Figure 4. Experimental and calculated ECD curves for 1 and 2.