A New Alkaloid from the Endophytic Fungus of Crocus sativus L., Aspergillus fumigatus Y0107

A new alkaloid (compound 1), together with 8 known alkaloids (compounds 2-9), were isolated from Aspergillus fumigatus Y0107 extracts from the lateral buds of Crocus sativus Linn (saffron). The structure of compound 1 was elucidated with NMR spectroscopy, High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and compared with its analogues in the literatures. The antibacterial activities of the crude ethyl acetate (EA) extract (CEE) and isolated compounds (compounds 1, 2, 6, 8) were evaluated against plant pathogenic bacteria. Compound 2 and the CEE demonstrated moderate inhibitory activity with MICs of 100 μg/mL against Erwinia sp.


General Experimental Procedures
The optical rotation was measured by a Jasco P-2000 digital polarimeter. The NMR spectra (including 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC, and HMBC) were recorded on a Bruker AVANCE Ⅲ-400 spectrometer. The 77.160 ppm and 7.260 ppm resonances of CDCl3 and the 206.260 ppm and 2.050 ppm resonances of acetone-d6, were used as internal references to ascertain 13 C and 1 H chemical shifts, respectively. A Waters Synapt G2 type UHPLC-q-TOF mass spectrometer was used to obtain HR-ESI-MS data. Column chromatography (CC) was performed with silica gel (Qingdao Marine Chemical Co., Ltd, China) and Sephadex LH-20 (Pharmacia, Sweden), and thin layer chromatography was performed using silica gel 60 GF 254 (TLC, Qingdao Marine Chemical Co., Ltd, China). A reversed-phased C 18 column (5 μm, 10 mm × 250 mm, YMC-Pack ODS-A column) was used to carry out on an Agilent 1260 Infinity Ⅱ HPLC (Aligent Technologies Inc., Germany).

Fungal Material
The endophytic fungus strain was isolated from lateral buds of Crocus sativus L. (purchased from Jiande Sandu Saffron Professional Cooperative) on May 7 th , 2020, and stored in a -80 ℃ cryopreservation box (ThermoFisher Scientific (China) Co. Ltd). Morphology analysis and 18S rRNA gene sequencing were performed to assign the strain as Aspergillus fumigatus (GenBank accession no. MZ854147) and named as Y0107.

Extraction and Isolation
The seed culture with Aspergillus fumigatus Y0107 strains was cut into small pieces and inoculated into a solid medium (rice 120 g of 150 mL ultrapure water in 1 L erlenmeyer flask, 140 bottles) for stationary fermentation at 26 ℃ for 21 days. After cultivation, the crude extract (182.1 g) of the fermented rice medium was obtained by soaking ethyl acetate (EA, 18 + 4 × 10 L) overnight. The crude extract was dispersed into 2.5 L ultrapure water, and then extracted with 2 L petroleum ether (PE), EA, and n-butanol three times in turn. Then EA layers were collected and concentrated by vacuum to yield a crude EA extract (CEE, 49.3 g).

Antibacterial Bioassays
The antibacterial activities of compounds 1, 2, 6 and 8, together with CEE, were determined based on a microbroth dilution method in 96-well culture plates according to the National Committee for Clinical Laboratory Standards [15][16]. The plant pathogenic bacteria, Agrobacterium tumefaciens, Pantoea agglomerans, Ralstonia solanacearum, Erwinia sp. were purchased from Ningbo testobio Co., Ltd, Zhejiang, China, and cultured in the NA nutrient medium (1 g yeast extract, 3 g beef extract, 5 g peptone, and 5 g glucose in 1 L sterile water, the pH was adjusted to 7.2 with NaOH) [17]. Streptomycin (USP Grade, Sangon Biotech Co., Ltd, Shanghai, China), an antibacterial drug, was used as the positive control. The tested bacteria were placed in a constant temperature oscillation incubator (100 rpm) for 12 h at 30 ℃. The NA nutrient broth was used to adjust the bacterial concentration to 1×10 5 -1×10 6 cfu/mL by observing the OD600 values of each bacterial dilution. The concentration of monomers, positive control and CEE initial solution in DMSO/H2O was 200, 200 and 400 μg/mL, respectively. First of all, the diluted bacterial solution was added into each well of a 96-well culture plate (50 μL per well). Then, the initial solution (50 μL) was added into the first well and mixed evenly, from which 50 μL of solution was transferred into the second well and mixed well following the two fold dilution method. Serial dilutions were repeated from the third to the twelfth well in triplicate. The minimal inhibitory concentrations (MIC) were determined after 24 h of incubation.

Structure Elucidation
Compound 1 was isolated as a white solid and characterized as a dark spot under UV light at 254 nm and a brown spot after spraying with 10% H2SO4 in EtOH with heating. Its molecular formula was determined to be C24H27NO9 from the [M + Na] + ion at m/z 496.1582 (calculated 496.1564) in HR-ESI-MS, requiring 12 degrees of unsaturation. The 13 C NMR/HSQC spectrum (Table 1), showed 24 carbon resonances, assigned to four electrophilic groups (two carbonyls at δc 197.8 (C-4) and 194.5 (C-17), an ester group at δc 170.5 (C-24) and an acylamino at δc 165.2 (C-6)), and four aromatic chemical shifts. Ten and four carbon signals were ascribed to sp 3 and sp 2 hybridized carbon atoms, respectively. The 1 H NMR spectroscopy (Table 1)  and H-23, indicating that a carbonyl was connected to a mono substituted benzene. Furthermore, C-5 exhibited correlations to H-9 and NH-7, while C-9 also correlated to NH-7, suggesting that there was a N-containing fivemembered heterocyclic ring in compound 1. The correlations between H-16 and C-2, C-3 and C-4 uncovered the position of CH3-16, while protons of oxymethyl that correlated to C-8, revealed the position of the oxymethyl group. Moreover, H-25 exhibited correlations with C-24 and C-11, while H-11 was correlated to C-24, which indicated the position C-24 and CH3-25. And the correlation between H-10 and C-2 displayed the connection of C-2 and C-10. The presence of a cis double bond was confirmed by the typical J coupling constant of 10 Hz at H-12. The above data further established the planar structure of compound 1, possessing a 1-oxa-7-azaspiro [4.4]non-2-ene-4,6-dione skeleton.

Antibacterial Activity
Several isolated compounds and CEE were tested for antibacterial activity against plant pathogenic bacteria. As shown in Table 2, compound 2 (MIC 100 μg/mL) and CEE (MIC 100 μg/mL) exhibited moderate antibacterial activity against Erwinia sp., which was weaker than the positive control (streptomycin, MIC 25 μg/mL), while compounds 1, 6 and 8 showed no activity against this strain when their concentration range was within 100 μg/mL. Unfortunately, none of the tested samples displayed inhibition against the strains, A. tumefaciens, P. agglomerans and R. solanacearum when their concentration range was within 100 μg/mL, except the positive control.

Conclusions
In this work, a new alkaloid compound, namely 11-acetyl-pseurotin A2 (1) together with eight known alkaloids were isolated from CEE of Aspergillus fumigatus Y0107, an endophytic fungus of the lateral buds of Crocus sativus Linn. The structure of compound 1 was elucidated with various spectroscopies, HR-ESI-MS analysis and comparison with its analogues in the literatures. Among them, compound 2 and CEE displayed antimicrobial activities against Erwinia sp., while no significant effects against the other three plant pathogenic bacteria were observed when their concentration range was within 100 μg/mL. Additionally, compounds 1, 6 and 8 showed no markedly inhibition against the abovementioned plant pathogenic bacteria when their concentration range was within 100 μg/mL.