Isochromenes from the Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities

Abstract Three new isochromenes, (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol (1), 3-(3-(hydroxymethyl)-5-methoxy-1H-isochromen-7-yl)propan-1-ol (2), and (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3), along with three known analogues (4–6) were isolated from the fermentation products of a Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor. Their structures were elucidated by spectroscopic methods, including extensive 1 D and 2 D NMR techniques. Compounds 1–3 and 6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activity with inhibition rates of 28.6, 30.5, and 26.2%, respectively. The IC50 of compounds 1–3 and 6 were also tested, and showed IC50 values of 49.3, 22.4, 42.2, and 54.1 µM, respectively. Graphical Abstract


Introduction
Endophytic fungi are considered an important and promising source of bioactive metabolites for drug discovery in the research field of natural products (Xu et al. 2019;Gupta et al. 2020). Among the most known species of endophytic fungi, those belonging to the Aspergillus genus, family Trichocomaceae, are highly recognized containing economically important species, as well as pathogenic species of animals and plants (Bongomin et al. 2018;Ibrahim et al. 2022), and they can produce a number of structurally complicated molecules with various biological activities (Choi et al. 2019;Bouz and Dolezal 2021;Zhao et al. 2022). In our previous work, some bioactive metabolites, such as, diterpenoids (Wang et al. 2019), alkaloids (Zhou et al. 2014;He et al. 2020), butyrolactones (Ye et al. 2014;Zhou et al. 2015;Zhou et al. 2016a), isocoumarins and isochromenes Zhou et al. 2016b;Zhou et al. 2017), and the like, had been isolated from this fungus.
The isocoumarins, an important class of naturally occurring coumarins isomers with a reversed lactone moiety, are abundantly distributed in natural sources and are being extracted from different plants, molds, lichens, fungi and bacteria strains (Shabir et al. 2021). The isochromenes belong to the isocoumarin skeleton, and it is a benzopyran with a methylene in the first position instead of a carbonyl (Clementina and Artur 2012). As one of the characteristic components of Aspergillus fungi, isocoumarins and isochromenes also attract much attention from chemists and biologists due to their diverse structures and biological properties (Clementina and Artur 2012;Noor et al. 2020). With the aim of continuously explore bioactive metabolites from Aspergillus fungi, the chemical investigations of the ethyl acetate extract of fermentation broth of A. versicolor YNCA2023 obtained from Nicotiana tabacum L. (family Solanaceae) were carried out. As a result, we discovered three new isochromenes (1-3), as well as three known compounds (4-6). Herein, details of the isolation, structure determination, and anti-TMV activities of above compounds are presented.
Compound 1 was obtained as a pale yellow gum. Its molecular formula was determined as C 16 H 20 O 3 by positive HRESIMS at m/z 283.1316 [M þ Na] þ (calcd for C 16 H 20 O 3 Na þ , 283.1310), indicating seven degrees of unsaturation. The IR spectrum showed the absorption bands for hydroxyl (3398 cm À1 ) and aromatic group (1612, 1548, and 1476 cm À1 ). The UV spectrum exhibited absorption bands at 215, 274, and 340 nm. The above-mentioned evidence suggested the existence of aromatic chromophore. The 1 H and 13 C NMR spectra of 1 (Table S1) showed the presence of a 1,2,3,5-tetrasubstituted benzene ring (C-5-C-10, H-6, and H-8), a prenyl group (C-2 0 -C-6 0 , H 2 -2 0 , H-3 0 , H 3 -5 0 , and H 3 -6 0 ) ), a pair of double bonds (C-3, C-4, and H-4), an oxidized methylene carbon (C-1 and H 2 -1), a hydroxymethyl group (C-1 0 and H 2 -1 0 ), and a methoxy group (d C 55.9 q and d H 3.74 s). According to above NMR data, the double bonds and oxidized methylene carbon should be incorporated with benzene ring to form an isochromene ring to support the seven degrees of unsaturation, and the above signals can also be attributed to a prenylated isochromene closely related to oryzaein D (Zhou et al. 2016b), except for the substituents variation on the benzene ring. In addition, the existence of isochromene core was supported by the HMBC correlations ( Figure S1) from H 2 -1 to C-3, C-8, C-9, and C-10, H-4 to C-3, C-5, C-9, and C-10, H-8 to C-1, C-6 and C-10, together with H-6 to C-8 and C-10.
Since the isochromene skeleton was determined, the positions of substituents (prenyl, hydroxymethyl, and methoxy groups) also can be determined by further analysis of the HMBC data ( Figure S1). The HMBC correlations from the H 2 -1 0 to C-3, C-4, together with H-4 and active proton 1 0 -OH (d H 4.94 br s) to C-1 0 established that the hydroxymethyl group was located at C-3. The methoxy group located at C-5 was clearly indicated by the HMBC correlations from methoxy proton (d H 3.74) to C-5. Furthermore, the prenyl located at C-7 can also be determined by the HMBC correlations of H 2 -2 0 with C-6, C-7, C-8, H-3 0 with C-7, H-6 and H-8 with C-2 0 . On the basis of above evidence, the structure of 1 was established, and gave the systematic name of (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol.
Compound 3 was assigned the molecular formula of C 12 H 14 O 3 according to HRESIMS at m/z 229.0847 [M þ Na] þ (calcd for C 12 H 14 O 3 Na þ , 229.0841), 44 mass units lower than that of 2, same indices of hydrogen deficiency. The NMR spectra of both compounds were very similar, except that the signal for 3-hydroxypropyl group in 2 appeared as a methyl group in 3 (Table S1). The methyl group attached to C-7 was supported by the HMBC correlations from H 3 -2 0 to C-6, C-7, and C-8. Thus, the structure of (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3) was determined.
Since certain of isochromenes and its derivatives exhibit potential anti-TMV activities Zhou et al. 2016b;Hu et al. 2017). Compounds 1-3 and 6 were evaluated for their anti-TMV activities. The anti-TMV activities were tested by half-leaf method (Hu et al. 2013;Zhou et al. 2014), using ningnanmycin (C 16 H 25 N 7 O 8 , CAS#: 156410-09-2, a commercial new cytosine nucleoside peptide antibiotics for plant viral diseases in China, with inhibition rate of 34.2%) as a positive control. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activities with inhibition rates of 28.6, 30.5, and 26.2%, respectively. In addition, the IC 50 values of compounds 1-3, and 6 were also tested, and the results ( In this study, compounds 1, 3, 6 showed potential anti-TMV activities, and compound 2 exhibited high anti-TMV activity. These results may reveal that the isochromenes substructure is fundamental for anti-TMV activity and the oxygen-containing substituent groups also increase the inhibitory activity. This study of structure-activity relationship is helpful to find new anti-TMV activity inhibitors.

Fungal material
The strain A. versicolor YNCA2023 was isolated from the leave of cultivated tobacco (N. tabacum L., family Solanaceae) collected in Gengma County (23 40 0 N, 98 50 0 E), Lincang Prefecture, Yunnan Province, in 2020. The fresh asymptomatic leaves were collected in a plastic case and stored at 4 C. Leaves were washed under running tap water before surface-sterilized. The surfaces of the leaves were sterilized with 75% ethanol for 30 sec, 1.5% sodium hypochlorite for 3 min, and finally rinsed three times in fresh sterilized distilled water. And then leaves were cut into small pieces (approximately 0.5 Â 0.5 cm). Some of pieces were put into potato dextrose agar (PDA) medium (Land Bridge, Beijing, China), pH was not adjusted. Fungal strains were grown out from small leaf tissues after three to seven days' incubation. The fungal strain was picked up and purified with repeated streak cultivation. The strain YNCA2023 was identified by one of authors (Dr. Qi-Li Mi) based on the analysis of the ITS sequence (Genbank Accession number: MT549144). The strain YNCA2023 was grown on PDA plates at 28 C for 5 days. Agar plugs were inoculated into 50 mL of seed medium consisting of potato dextrose broth (pH was not adjusted) in a 250 mL Erlenmeyer flasks incubated for 4 days at 28 C on a rotary shaker (200 rpm). A 15.0 mL portion of the seed culture was transferred to a 1000 mL Fernbach flasks containing 300 mL of the production medium consisting of glucose 5%, peptone 0.15%, yeast 0.5%, KH 2 PO 4 0.05%, MgSO 4 0.05% in 1 L of deionized water, adjusted to pH 6.5 before sterilization, and the fermentation was carried out on a rotary shaker (200 rpm) at 28 C for 20 days.

Extraction and isolation
The whole culture broth of A. versicolor was extracted with ethyl acetate (10 L Â 4) at room temperature and filtered. The crude extract (122 g) was applied to silica gel

Anti-TMV assays
The anti-TMV activities were conducted according to previous reports using the halfleaf method (Hu et al. 2013;Zhou et al. 2014), and ningnanmycin (2% water solution) was used as a positive control. Briefly, the virus was inhibited by mixing with the solution of tested compounds. After 30 min, the mixture was inoculated on the left side of the leaves of Nicotiana glutinosa, whereas the right side of the leaves was inoculated with the mixture of DMSO solution and the virus as control. The local lesion numbers were recorded 3-4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula: Where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment. Ningnanmycin was used as a positive control.

Conclusions
In the course of our ongoing search for bioactive metabolites from natural resources, three new isochromenes (1-3), along with three known ones (4-6) were isolated from the fermentation products of the A. versicolor YNCA2023. Compounds 1-3 and 6 were evaluated for their anti-TMV activities. All compounds displayed anti-tobacco mosaic virus activity, and compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. The successful isolation and structure identification of isochromenes provide the valuable materials for the screening of anti-TMV activity inhibitors, and contribute to the development and utilization of N. tabacum-derived microorganisms.

Authors contributions
FXY conducted most of the practical work. JMD and FXY elucidated the structures, JW did IRspectroscopy, HYL and QLM sequenced the fungus, JDZ planned and performed the bioassays, XML, WGW, MZ, YKL, FXY and QFH wrote the manuscript.

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