Asperflaloids A and B from Aspergillus flavipes DZ-3, an Endophytic Fungus of Eucommia ulmoides Oliver

The fungus strain DZ-3 was isolated from twigs of the well-known medicinal plant Eucommia ulmoides Oliver and identified as Aspergillus flavipes. Two new alkaloids, named asperflaloids A and B (1 and 2), together with 10 known compounds (3–12) were obtained from the EtOAc extract of the strain. Interestingly, the alkaloids 1–4 with different frameworks are characterized by the presence of the same anthranilic acid residue. The structures were established by detailed analyses of the spectroscopic data. The absolute configuration of asperflaloids A and B was resolved by quantum chemistry calculation. All compounds were screened for their inhibitions against α-glucosidase and the antioxidant capacities. The results were that compound 3 had an IC50 value of 750.8 μM toward α-glucosidase, and the phenol compounds 7 and 8 exhibited potent antioxidant capacities with IC50 values 14.4 and 27.1 μM respectively.


Introduction
Fungi are widespread in natural environments and have conquered almost all ecological niches. Moreover, fungi produce many structurally intriguing molecules, many of which exhibit promising pharmacological properties, including the famous antibiotics penicillin and cephalosporin C [1,2]. Aspergillus, a genus of filamentous fungi, is noted for its medical and commercial significance. Species in Aspergillus have been proved to be prolific and are considered as an important source of chemical leads with hopeful biological activities [3,4]. Among the Aspergillus species, Aspergillus flavipes was an outstanding member; recent chemical research of the species resulted in the production of plenty of molecules with new structures and obvious biological activities, such as structurally complex merocytochalasans [5][6][7], lumazine peptides possessing significant antibacterial and NF-κB inhibitory activities [8], unusual chlorinated PKS-NRPS hybrid metabolites bearing potent pancreatic lipase inhibitory activity [9], and rare prenylated phenylbutyrolactones [10].
In our continuous efforts to search for bioactive natural products from fungus [11][12][13][14][15], an endophytic fungus Aspergillus flavipes DZ-3 was isolated from the twigs of the medicinal plant Eucommia ulmoides Oliver. The 1 H-NMR spectrum and thin layer chromatography examination using the modified Dragendorff's reagent of the EtOAc extract indicated the presence of aromatic alkaloids. Extensive chromatographic separation of the largescale fermentation were carried out, which resulted in the isolation of 12 compounds 1-12 (Figure 1), including two new alkaloids. All compounds were screened for their inhibitions against α-glucosidase and the antioxidant capacities. Herein, the isolation, structure determination, and bioactivities of compounds 1-12 are described.
All compounds were tested for their α-glucosidase inhibitory activities and antioxidant capacities ( Table 2). We found that compound 3 inhibited α-glucosidase with an IC 50 value of 750.8 µM and was more active than the positive control acarbose (1.33 mM).

General Experimental Procedure
Specific rotations were measured by an SGW-1 automatic polarimeter (Shanghai Jing Ke Industrial Co., Ltd., Shanghai, China). ECD spectra were measured on an Aviv Model 420SF spectropolarimeter (Aviv Biomedical Inc., Lakewood, NJ, USA). The NMR spectra were recorded on a Bruker Avance III HD-400 NMR spectrometer. HRESIMS spectra were obtained on a Waters Xevo G2 Q-TOF spectrometer fitted with an ESI source. Semi-preparative high-performance liquid chromatography (HPLC) was undertaken on a Shimadzu LC-6AD pump (Shimadzu Co., Kyoto, Japan) using a UV detector, and a YMC-Pack ODS-A HPLC column (semipreparative, 250 × 10 mm, S-5 µm, 12 nm, YMC Co., Ltd., Kyoto, Japan) was used for separation.

Fungal Strain and Identification
Fungus DZ-3 was isolated from branches of Eucommia ulmoides Oliver. The strain was identified as Aspergillus flavipes based on microscopic examination and by internal transcribed spacer (ITS) sequencing. The ITS sequence has been deposited in GenBank (http://www.ncbi.nlm.nih.gov, accessed on 13 May 2021) with the accession number MZ148624. The strain DZ-3 (henuyxydz-3) was deposited at the School of pharmacy, Henan University.

α-Glucosidase Assay
The α-glucosidase inhibitory effect was assessed as described in our recently published paper [25].

Antioxidant Activity
The DPPH scavenging was assayed according to the reported method [26]. The DPPH radical scavenging test was performed in 96-well microplates. Samples (compounds 1-12) were added to 180 µL (150 µmol/L) DPPH solution in EtOH at 20 µL solutions of different concentrations between 10 and 500 µM. After 30 min of light avoidance, absorbance at 517 nm using a microplate reader (Tecan Trading AG, Männedorf, Switzerland) was measured and the percentage of activity was calculated. All assays were performed in three replicates, and vitamin C was used as a positive control.

Computational Details
ECD calculations. Conformational analyses were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 2.5 kcal/mol [27]. The results showed the three lowest energy conformers for R-1. The conformers were re-optimized using DFT at the b3lyp/6-31+g(d,p) level in methanol by the GAUSSIAN 09 program [28]. The energies, oscillator strengths, and rotational strengths (velocity) of the first 30 electronic excitations were calculated using the TDDFT methodology at the b3lyp/6-31+g(d,p) level in methanol. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, σ = 0.3, UV correction = −5 nm) [29]. To get the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G), theoretical ECD spectrum of the corresponding enantiomer S-1 was obtained by inverse of the ECD spectrum of R-1, respectively. By comparing the experiment spectrum with the calculated ECD spectra, the absolute configuration of the chiral center C-8 in 1 was resolved to be S.
Specific rotation calculations. Conformational analyses were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 2.0 kcal/mol. The results showed the three lowest energy conformers for R-2. The conformers were re-optimized using DFT at the b3lyp/6-31+g(d,p) level in methanol by the GAUSSIAN 09 program. The specific rotations for each conformer were calculated using the TDDFT methodology at the b3lyp/6-31+g(d) level in methanol. The specific rotations obtained for the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G) to give the specific rotation of R-2, the specific rotation of S-2 was theoretically determined to be the opposite value of R-2. By comparing the experiment data ([α] 20 D +270) with the calculated data (R-2: [α] 20 D −147; S-2: [α] 20 D +147), the absolute configuration of the chiral center C-2 in 2 was resolved to be S.