A pair of new chromone enantiomers from Xylaria nigripes

Abstract A pair of new chromone derivative enantiomers, (+)-xylarichromone A (1a) and (−)-xylarichromone A (1b), were isolated from the solid fermentation of Xylaria nigripes. The planar structure of 1 was determined by extensive NMR spectroscopic data, and its absolute configuration was assigned by comparison the ECD spectra with the known chromone derivatives. Compound 1 was the first chromone derivative reported from this medicinal fungus. The neuroprotective effects of 1 against oxygen and glucose deprivation (OGD) induced pheochromocytoma-12 cells (PC12) injury was investigated. Graphical Abstract


Introduction
Xylaria nigripes belongs to the family of Xylariaceae, and is predominantly distributed in the around of the fungus combs of the odontotermes termite species (Rogers et al. 2005;Ju and Hsieh 2007).It is a high-value medicinal and edible fungus with a very special ecological type, and used as traditional Chinese medicine for the treatment of insomnia, depression, mental illness, and diseases of nervous system (Xu 1997;Wang et al. 2010;Zhao et al. 2014;Peng et al. 2015).The crude extracts of X. nigripes has been reported had the effects of antidepression, antianxiety, antioxidant, anti-inflammatory, and hematopoietic (Ma et al. 2009;Hung et al. 2015;Chang et al. 2017;Divate and Chung 2017;Chang et al. 2018).Wuling capsules (recorded in Chinese pharmacopoeia), a mycelial extracts of X. nigripes, has been used clinically for regulating the nervous systems and enhancing the immune system for a long time (Ma et al. 1999;Shi et al. 2009;Lin et al. 2013).However, X. nigripes has seldom been chemically investigated, although several sesquiterpenes, alkaloids, and steroids have been found (Yang et al. 2011;Li et al. 2015;Xiong et al. 2016;Chang et al. 2017;Hu and Li 2017).
In our previous research, five pairs of naphthalenone derivatives, including one pair of indole naphthalenones and four pairs of naphthalene-naphthalenone dimers, as well as four new resorcinol derivatives were isolated from X. nigripes (Li et al. 2021;2022).These results greatly inspired our interests to discover for more structurally unique scaffolds from this medicinal fungus, an ongoing phytochemical investigation of the extract of the solid fermentation of X. nigripes has been performed, which led to the isolation of a pair of new chromones (±1).Herein, we describe the isolation, structure and stereochemistry elucidation, and bioactivity evaluation of 1.
The double bonds D 9 was assigned as being in the E-form, based on the large coupling constants of J H-9, H-10 ¼ 15.4 Hz.There is a chiral carbon (C-2) in 1, but the ECD spectra of compound 1 did not show any apparent Cotton effects, and its optical rotation barely be detected, implying that 1 might be a racemic mixture.Subsequent chiral HPLC separation gave a pair of optically pure enantiomers (þ)-1a and (-)-1 b, respectively (Figure S5).For the two enantiomers, the experimental ECD curve of 1a showed first positive (331 nm), second negative (305 nm), and third positive (216 nm) Cotton effects, while the experimental ECD spectrum of 1 b showed an almost inversed ECD curve (Figure S2).It has been reported that the configuration at C-2 of the chromone derivatives can be clearly distinguished from the 200-400 nm region in ECD spectrum (Zhao et al. 2015).Therefore, the absolute configurations at C-2 in 1a and 1 b were determined by comparing their experimental ECD spectra with the reported data, and defined to be 2 R and 2S, respectively.

Neuroprotective activities
The effects of compound 1 on the cell viability of OGD-induced PC12 cells were evaluated by a method we reported recently (Li et al. 2021).The results show that compound 1 effectively increases the survival rate of OGD-induced PC12 cells at the concentrations from 0.01 to 10 lmol/L (Figure S3).As shown in Figure S4, pretreatment of PC12 cells with OGD significantly increased the percentage of apoptotic cells.In contrast, compound 1 reduced the percentage of apoptotic cells at the concentrations of 0.1 and 1 lmol/L, comparing to blank control.

General experimental procedures
UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer (Hitachi, Japan).IR spectra were obtained with a Shimadzu Fourier Transform Infrared Spectrometer using KBr pellets.Optical rotations were measured with a Rudoph AUTOPOL IV polarimeter.ECD spectra were recorded with an Applied Photophysics spectrometer.HRESIMS spectra were measured on a Q Exactive Obitrap mass spectrometer (ThermoFisher Scientific, USA). 1 D and 2 D NMR spectra were recorded on Avance III 600 MHz Bruker spectrometers (Bruker BioSpin, Rheinstetten, Germany) with TMS as an internal standard.Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People's Republic of China), Fuji Silysia Chemical Ltd.,Japan),Ltd.,Sweden).Medium Pressure Liquid Chromatography (MPLC) was performed on a Biotage SP1 System, and columns packed with RP-18 gel.Preparative high-performance liquid chromatography (prep-HPLC) were performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (Agilent, 5 lm, 9.4 mm Â 150 mm) and a DAD detector (Agilent Technologies, Santa Clara, CA, USA).Fractions were monitored by TLC (GF 254,Qingdao Haiyang Chemical Co.,Ltd. Qingdao,China), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.All solvents were analytical grade.

Fungal material
The strain of X. nigripes was collected from Ailao Moutain, Yunnan Province of China in 2013.The fungus was authenticated by Prof. Yu-Cheng Dai (Beijing Forestry University), a mushroom specialist.The strain of X. nigripes in this study was isolated from the fresh fruiting bodies and kept on potato, dextrose, and agar (PDA) culture medium.A voucher specimen (No.CGBWSHF00611) was deposited at the Mushroom Bioactive Natural Products Research Group in South-Central Minzu University, China.

Extraction and isolation
This strain of X. nigripes was cultured on potato dextrose agar medium at 25 C.After eight days, the agar plugs were cut into small pieces to incubate on solid rice medium (100 g rice and 100 mL tap water for each 500 mL Erlenmeyer flask, the total weight of rice was 10 kg) to culture for further 30 days at 25 C.The fermented rice medium was broken up into small pieces and extracted with 30 L ethyl acetate for 3 times.The extract was concentrated under vacuum with rotary evaporator to yield 95.5 g dark brown residue.

Cell viability assay
The cell viability was detected by the Cell Counting Kit 8 (CCK-8).In brief, PC12 cells (10 4 cells/well) were seeded onto 96-well plates and incubated for 3 h with hypoxia.Appropriate dilutions of the test compounds were added to the cultures and incubated for another 24 h under normal conditions.Afterwards, 10 lL of CCK-8 was added to each well and the plate were kept incubation for 3 h, then a microplate reader (Perkinelmer, Enspire) was used to detect the absorbance value at 450 nm.Each experiment was carried out in triplicate.Cell viability was expressed as a percentage of the control.

Apoptosis assay
PC12 cells (1 Â 10 4 cells/mL in 96-well plates) were treated with hypoxia for 3 h, then incubated with or without the test compounds for 24 h.The cells were collected and stained by Hoechst.The percentage of apoptotic cells was analyzed by laser scanning confocal microscope (Zesis, Germany).

Conclusions
In this study, (þ)-xylarichromone A (1a) and (À)-xylarichromone A (1 b), a pair of new chromone enantiomers were identified from the cultures of X. nigripes.The neuroprotective effects of 1 were studied in vitro.The results indicated that compound 1 significantly enhanced cell viability and inhibited apoptosis at the concentration of 0.1 and 1 lmol/L, which suggested that compound 1 had the potential in protecting PC12 cells from the OGD-induced injury.

13C
NMR and DEPT spectra showed resonances for two carbonyl groups including one ketone carbon (d C 194.1) and one carboxy group (d C 175.5), six aromatic carbon atoms