Two new sesquiterpenoids from plant endophytic fungus Flammulina velutipes

Abstract Two new sesquiterpenoids, flammupin A (1) and flammupin B (2), along with two known compounds, enokipodin C (3) and 5,5'-dibuthoxy-2,2'-bifuran (4) were obtained from Flammulina velutipes, an endophytic fungus isolated from the roots of Caulophyllum robustum Maxim. The structures were elucidated by the combination of HR-ESI-MS, NMR, and ECD analyses. Compound 3 exhibited moderate to potent cytotoxicity against A549, HeLa, and SMMC-7721 cells with IC50 values ranged from 3.69 to 11.84 μM. Graphical Abstract


Introduction
Plant endophytic fungi are the microorganisms that inhabited in the interior of host plants without any apparent pathogenic symptom, could produce plentiful structurally novel bioactive secondary metabolites, and contributed greatly to the search for new drugs [1,2]. Fungi of Flammulina velutipes are a fresh edible mushroom frequently consumed in China and Japan, and belong to the family of Tricolomataceae [3][4][5]. Previous chemical investigation on the mycelial culture and fruiting body of F. velutipes resulted in the discovery of various compounds consisting of polysaccharides, sterols, and sesquiterpenoids with potent bioactivities, such as antiviral, antitumor, antimicrobial, and immunomodulatory properties [6][7][8]. In the course of our ongoing screening for biologically meaningful and structurally unique natural products from plant endophytes [9][10][11][12][13], chemical constitutents of F. velutipes, which was isolated from the fresh healthy roots of traditional Chinese medicinal plant Caulophyllum robustum Maxim, were investigated. Two new sesquiterpenoids flammupin A (1) and flammupin B (2), together with two known compounds enokipodin C (3) and 5,5'dibuthoxy-2,2'-bifuran (4) were obtained ( Figure 1). The isolation, structure elucidation, and cytotoxicity are presented herein.

Results and discussion
Flammupin A (1) was obtained as a red amorphous solid. Its molecular formula was deduced to be C 15 -5)]. The other signals were attributed to four methyl groups, one methylene and two quaternary carbons. The above data revealed that the structure of 1 is similar to enokipodin D [5]. The main difference is that one proton at C-2 was substituted by an amino group in 1. Observed 1 H-1 H COSY correlation ( Figure 2) of H-8 with H-9, along with HMBC correlations from H-8 to C-7, C-9, and C-14, from H-9 to C-7 and C-11, confirmed the cyclopentanone moiety. The HMBC correlations from H-5 to C-1 and C-3, constructed the benzoquinone moiety. The downfield chemical shift of C-8 confirmed the existence of hydroxyl group. In addition, the existence of primary amino group at C-2 was inferred from the molecular weight, and further supported by the downfield chemical shift of C-2. The cyclopentanone moiety was connected with the benzoquinone moiety, as determined by HMBC correlations of H-8 and H 3 -14 to C-6 and of H-5 to C-7. These analyses further revealed that 1 shares the similar structure with enokipodin D. The relative configuration of 1 was assigned by analysis of NOESY    13 C NMR (100 MHz) spectral data of compounds 1 (in CDCl 3 ) and 2 (in CD 3 OD). 1 2   Flammupin B (2) was isolated as a yellow amorphous solid, with the molecular formula C 15 (Table 1) spectrum of 2 delineated the existence of 12 carbons, while in the HMBC spectrum, the cross-peaks of four more carbon signals at d C 122.9, 168.7, 179.6, and 202.2 were observed and the cross-peak of the carbon signal at d C 111.4 was not observed. Therefore, the 13 C NMR data together with DEPT, HSQC, and HMBC implied that the signal of the carbon (d C 111.4) was impurity signal and 2 possessed 15 carbon atoms. Detailed 2D NMR data ( Figure 2) indicated that the planar structure of 2 was almost the same as illinitone B [14], with the main difference in that the methoxyl at C-4 in illintone B was substituted by a hydroxyl group in 2. The relative configuration of 2 was shown to be as presented in Figure 3, H-13 and OCH 3 -4 were on the opposite side of the molecule. The absolute configuration of the stereo genic carbon C-4 was determined to be S based on ECD calculation ( Figure 5). Thus, the structure of 2 was identified and named as flammupin B.
All of the isolates were tested for cytotoxic activities against HeLa, A549 and SMMC-7721 cell lines using the MTT assay. Compound 3 exhibited potent cytotoxicity against A549 cells with IC 50 value of 3.69 lM, which was stronger than the positive control etoposide (IC 50 ¼ 11.63 lM). 3 also showed moderate cytotoxicity against HeLa and SMMC-7721 cells with IC 50 values of 11.84 and 7.49 lM, respectively. However, the other compounds were inactive at the concentration of 100 lM.

General experimental procedures
NMR spectra were measured on an Agilent DD2 400-MR NMR spectrometer (Agilent Technologies Inc., Santa Clara, USA), using TMS as an internal standard. HR-ESI-MS spectra were provided by an AB SCIEX Triple TOFV R 4600 system (AB SCIEX, Framingham, USA). IR spectra were obtained using a Fourier Transform Infrared Spectrometer (PerkinElmer Inc., Massachusetts, USA). UV spectra were recorded on a UV-2550 UV/V spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Optical rotations were measured on a MCP 300 polarimeter (Anton Paar GmbH, Graz, Austria). Preparative HPLC was conducted using an Agilent 1200 series instrument (Agilent Technologies Inc., Santa Clara, USA) equipped with a 1260 pump, a 1260 diode array detector and a Waters XBridge C18 column (19 mm Â 250 mm, 5 lm, 15 mL/min). Semi-Prep HPLC was performed on a Waters HPLC system coupled with a W1525 Binary pump, a W2998 photodiode array detector, a W1500-CH column compartment, and a W2707 auto-sampler, using a Waters XBridge C18 column (10 mm Â 250 mm, 5 lm, 3 mL/min) (Waters Corporation, Milford, USA). MPLC was carried out using a Buchi SepacoreV R Chromatography with a dual-pump gradient system, a UV detector (Switzerland). Silica gel (200-300, 300-400, and 230-400 mesh) used for column chromatography (CC) were from Qingdao Haiyang Chemical Company Ltd., Qingdao, China, and Merck, Darmstadt, Germany, respectively. Sephadex LH-20 (GE Healthcare Life Sciences, Pittsburgh, USA).

Fungal material
The fungal strain Flammulina velutipes was isolated from fresh healthy roots of Caulophyllum robustum Maxim, which was collected in Qinling Mountains, Shaanxi Province, China. The fungus was identified on the basis of the phylogenetic taxonomy with sequence alignment of ITS and had a genetic closeness of 99% to the F. velutipes strain by Beijing Sunbiotech Co., Ltd. The strain was deposited at Shaanxi Key Laboratory of Phytochemistry, Baoji University of Arts and Sciences, Baoji, China.

Fermentation, extraction, and isolation
F. velutipes was cultured on slants of PDA medium at 28 C for 5 days. Plugs of agar supporting mycelium growth were cut and transferred into 50 mL Erlenmeyer flasks, each containing 30 mL of PD liquid medium. Seven flasks of the seed cultures were incubated at 28 C on a rotary shaker at 120 rpm for 5 days. Eight hundred microlitre of the seed culture was transferred aseptically to 500 mL Erlenmeyer flasks, each containing 40 g rice and 60 mL distilled water, a total of 220 flasks. The flasks were incubated at 28 C for 30 days. After the completion of the fermentation, the fermented rice substrate was extracted three times by ultrasonic with MeOH. The MeOH extract was concentrated in vacuo to yield 10 L aqueous solution, which was, then, degreased with petroleum ether. Then, the aqueous solution was extracted three times with EtOAc, and the EtOAc extract was combined and evaporated under reduced pressure to give the crude extract (28.0 g).

Disclosure statement
No potential conflict of interest was reported by the authors.