Two new cytochalasins from the endophytic fungus Xylaria sp. GDGJ-77B

Abstract Two new open-chain cytochalasins, xylarchalasins A and B (1 and 2), together with six known analogues (3-8), were isolated from the endophytic fungus Xylaria sp. GDGJ-77B from the Chinese medicinal plant Sophora tonkinensis. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. Compound 2 displayed moderate antibacterial activities against Bacillus subtilis and Escherichia coli with MIC values of 25 and 12.5 μg/mL, respectively. Graphical Abstract


Introduction
Endophytic fungi could be isolated from all plant tissues, such as roots, stems, leaves, flowers and fruits.Over the past 20 years, endophytic fungi attracted considerable attention due to their ability to produce bioactive compounds, which are often structurally unique and exhibit various biological activities.Therefore, endophytic fungi are utilized for medicinal, pharmaceutical, and agricultural applications (Manganyi and Ateba 2020).
Sophora tonkinensis is a traditional Chinese medicinal plant, which is often used to treat throat inflammation and fever (Wei et al. 2020;Qin et al. 2022).In our continuing search for bioactive compounds from its associated fungi (Xu et al. 2017;Zheng et al. 2018;Liang et al. 2019;Mo et al. 2021), we found that the EtOAc extract of the fungus Xylaria sp.GDGJ-77B exhibited antibacterial activities against Staphylococcus aureus and Escherichia coli.Chemical investigation of this fungus led to the isolation of two new cytochalasins, xylarchalasins A and B (1 and 2), along with six known compounds (3-8).Herein, we report the details of the isolation, structure determination and antibacterial activities of these compounds.
Previous studies suggested that the essential elements of most cytochalasins' skeletons have the same stereochemistry because they should share the same plausible biosynthetic pathway, such as cis-stereochemistry across the 5/6 ring junction and the trans-stereochemistry of the macrocyclic ring (Liu et al. 2008), and the S configuration of C-8, C-9, and C-16 in these open-chain cytochalasins (Wang et al. 2011;Zhang et al. 2014;Wu et al. 2019;Xin et al. 2019).The chemical shifts of 1 were similar to those of cytochalasin Z 18 (3), suggesting that 1 and 3 might share the same configuration.It was supported by the NOESY correlations (Figure S3).The NOE correlations between H-3/H 3 -11, H 3 -11/H 3 -12, and H 3 -12/H-7 of 1 indicated A-orientation of H-3, H 3 -11, H 3 -12, and H-7.In contrast, the NOE correlations of H-5/H-8 suggested that H-5 and H-8 were assigned as b-orientation. 1 was tentatively regarded having the same absolute configuration about their open chains as 3.And then, the R-configuration at C-18 was further confirmed by the comparison of NMR data and the experimental CD spectra (Figure S2) to the known compound cytochalasin Z 18 (3) (Lin et al. 2009;Miao et al. 2022).The E-geometry for the double bond at C-13 was deduced by the large coupling constant (J ¼ 15.3 Hz) for the olefinic protons H-13 and H-14.Thus, the absolute configuration of 1 was deduced to be 3S, 4S, 5S, 6 R, 7S, 8S, 9S, 16S, 18 R on the base of the experimental CD spectrum, which was in good accordance with 3. Hence, compound 1's absolute configuration was established and was named as xylarchalasin A.
The antibacterial activities of all the isolated compounds against Gram-positive bacteria Bacillus anthracis, Bacillus megaterium, Bacillus subtilis, Bacillus paratyphosus B, and Staphylococcus aureus, and Gram-negative bacteria Escherichia coli, and Shigella dysenteriae, were evaluated.Compounds 1, 3, 4, and 6 showed antibacterial activities against.B. megaterium, B. anthracis, E. coli and B. subtilis, respectively, with MIC value of 25 lg/mL (Table S2).Compound 2 showed antibacterial activities against B. subtilis and E. coli with MIC values of 25 and 12.5 lg/mL, respectively, while compound 5 displayed antibacterial activities against B. anthracis and S. aureus with MIC values of 25 and 12.5 lg/mL.Some other cytochalasans, such as cytochalasins A, H and J (Betina et al. 1972;Jouda et al. 2016), 18metoxycytochalasin J, phomopsichalasin (Horn et al. 1995), diaporthalasin (Khamthong et al. 2014), and alternariasin A (Guo et al. 2021), were reported to possess antibacterial properties.It suggested that cytochalasins were potential antimicrobial agents.

Fungal material and fermentation
The strain GDGJ-77B was isolated from the leaves of Sophorae tonkinensis, which was collected from Baise, Guangxi Province, China in 2017.The fungal strain was identified as Xylaria sp.based on the sequence of its internal transcribed spacer region (ITS) and morphology.ITS-rDNA of GDGJ-77B was submitted to GenBank and the accession number is OK 483375.The strain is preserved at the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University.

Antibacterial assay
The antibacterial activities of Gram-positive bacteria B. anthracis, B. megaterium, B. subtilis, B. paratyphosus B, and S. aureus, and Gram-negative bacteria E. coli and S. dysenteriae were evaluated by using the broth micro-dilution method in 96-well plates (Pierce et al. 2008;Tian et al. 2017;Qin et al. 2022).The respective compounds were dissolved in DMSO and diluted to the following final concentrations: 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 lg/mL.Ampicillin was used as a positive control.The MIC was determined as the lowest concentration at which no growth was observed.

Conclusions
In summary, two new open-chain cytochalasins, xylarchalasins A and B (1 and 2), together with six known analogues were isolated from the extract of the endophytic fungus Xylaria sp.GDGJ-77B.Their structures were determined by spectroscopic analysis.All of the isolated cytochalasins 1-8 showed selective antibacterial activities from 12.5-50 mgÁmL À1 .Especially, compound 2 showed antibacterial activities against B. subtilis and E. coli with MIC values of 25 and 12.5 lg/mL, respectively.

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