Two new sesquiterpene derivatives, dendocarbin B and bisaborosaol C with antifungal activity from the endophytic fungus Nigrospora chinensis GGY-3

Abstract Two novel sesquiterpene derivatives, dendocarbin B (1), bisaborosaol C (2), and nine known compounds (3-11), were isolated from Nigrospora chinensis GGY-3 derived from Ilex cornuta. The structures of new compounds were elucidated using HR-ESI-MS, 1 D and 2 D NMR spectra, X-ray diffraction analysis as well as ECD calculation and comparison. Compound 1 showed moderate antifungal activities against Rhizoctonia solani and Botrytis cinerea. Compounds 5 and 6 exhibited significant inhibitory activity against Phytophthora capsici, Magnaporthe oryzae and R. solani with EC50 values ranging from 13.91 to 29.49 μg/mL. Compounds 10 and 11 displayed moderate antibacterial effects on Bacillus subtilis and Xanthomonas oryzae pv. oryzae (Xoo), with MIC values of 16-64 μg/mL. Particularly, 11 presented strong antibacterial activity against Staphylococcus aureus with an MIC value of 4 μg/mL (2 μg/mL for streptomycin sulfate). In addition, compound 11 also possessed DPPH radical scavenging capability with an IC50 value of 14.80 μg/mL. Graphical Abstract


Introduction
There is an urgent need to search for new chemical substances with novel structure and excellent activity, given the increasing incidence of resistance in pathogens and potential environmental and mammalian toxicities (Kumar and Kaushik 2012;Wang et al. 2013).The natural products from endophytes have obtained much attention in the research field due to their wide structural diversities and potential biological applications (Gunatilaka 2006;Revathi et al. 2013;Silva-Hughes et al. 2015;Li et al. 2018;Wang et al. 2020).Endophytes, organisms that live inside plant tissues without causing any infections during their entire life cycle (Saikkonen et al. 1998), are a potential source of novel natural products for pharmaceutical, agricultural and industrial development.
The genus of Nigrospora, widely present in a variety of hosts (Ruiz-Sanchez et al. 2010;Xu et al. 2016;Wu et al. 2018), has previously been reported to produce a range of structurally unique chemicals, such as sesquiterpenoids (Chen et al. 2016;Wang et al. 2022), cyclopentenone (Wu et al. 2018), cyclodepsipeptides (Ding et al. 2016), and diketopiperazines (Huang et al. 2022).Drimane-type sesquiterpenes are a type of C 15 isoprenoids, mainly synthesised from farnesyl diphosphate through the mevalonate pathway in the cytosol (Dudareva et al. 2005;Sallaud et al. 2009), some of which show various bioactivities including antibacterial (Neuhaus and Loesgen 2021), antifungal (Paz et al. 2020), cytotoxic (Allouche et al. 2009) and anti-inflammatory (Chen et al. 2019) properties, indicating their promising applications in the development of medicinal or agricultural drugs.
In this study, the chemical investigation of the fermentation extract of the endophytic fungus Nigrospora chinensis GGY-3 derived from Ilex cornuta was conducted, leading to the isolation of two new sesquiterpene derivatives, dendocarbin B (1) and bisaborosaol C (2), together with nine known compounds (Figure 1).Herein, we report their isolation, structural elucidation and biological activities in detail.

Results and discussion
Dendocarbin B (1) was isolated as colorless crystals with the molecular formula of C 16 H 24 O 4 inferred by HR-ESI-MS at m/z 281.1747 1747), suggesting five degrees of unsaturation. 1 H NMR data (Table S3) of 1 revealed the existence of one methoxyl group (d H 3.54), three methyl groups (d H 0.85, 0.89 and 1.01) and one olefinic atom (d H 6.76).The HMBC correlations from H-1 to C-2, C-3, C-4 and C-10, and from H-5 to C3, C-4, and C-10, along with 1 H-1 H COSY correlations of H-1/ H-2 and H-2/H-3, could deduce a cyclohexane ring (Figure S1).In addition, Key HMBC correlations of H 3 -13/H 3 -14 to C-4 and H 3 -15 to C-10 indicated that three methyl groups were located at C-4 and C-10, respectively.Another cyclohexene ring was determined by HMBC correlations from H-9 to C-5, C-7, C-8, and C-10, as well as 1 H-1 H COSY correlations of H-5/H-6 and H-6/H-7.These structure features of 1 showed high similarity to those of 3b-OH-dendocarbin A (3) (Chen et al. 2016; Table S3), except that 1 contained one more methoxyl group (d H 3.54 and d C 57.6), which was confirmed by the HMBC correlations from H-16 to C-11.Considering its absolute configuration, a single X-ray diffraction analysis (Figure S2) was carried to define it as 3S,5R,9R,10S,11R.Compound 3 was first reported by Chen et al. (Chen et al. 2016) with its absolute configuration undetermined.A close comparison of the specific optical rotation value and ECD spectrum of 3 with those of the co-isolated compound 1 strongly suggested that compounds 1 and 3 share the same absolute configuration (Figure S3a).S3), three methyl groups (d H 1.03, 1.03 and 1.05), one olefinic proton (d H 6.87), one carboxyl group (d H 12.09), one hydroxyl group (d H 3.98) and two alkene carbon atoms (d C 130.2 and 138.8) were observed.A cyclohexene ring was identified by the HMBC correlations of H-2 to C-1, C-3 and C-5 and 1 H-1 H COSY correlations of H-2/H-3, H-3/H-4, H-4/H-5 and H-5/H-6, where a carboxyl group was located at C-1 via the HMBC correlations from H-2 to C-7 and H-6 to C-7 (Figure S1). were associated to C-13 as seen in HMBC correlation spectrum (Figure S1).However, the relative conformation of 2 was difficult to determine based on the NOESY spectrum, which led to eight possible isomers, including four pairs of enantiomers.Herein, 2 most likely possesses the 4S,8S,12R configuration by the comparison of the calculated and experimental ECD spectra (Figure S3b).
All isolated compounds except 7 and 9-11 (less than 10 mg) were evaluated for antifungal activity.As shown in Table S4, 1 and 2 displayed different levels of antifungal activity against Phytophthora capsici, Rhizoctonia solani and Botrytis cinerea at the concentration of 50 lg/mL, with the inhibition rates ranging from 18.18% to 44.91%.The inhibitory effects of 5 against P. capsici and R. solani reached 88.57% and 83.72%, and the EC 50 values were calculated as 21.94 and 26.10 lg/mL, respectively (Tables S4  and S5).Compound 6 also exhibited good antifungal activities against Magnaporthe oryzae and R. solani with EC 50 values of 13.91 and 29.49lg/mL, respectively (Table S5).The other compounds have no significant antifungal effects against the test fungi.
Apart from the above, all compounds were investigated for antibacterial and antioxidant activities.As a result (Table S6), compounds 10 and 11 were active against the Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and the Gramnegative bacterium (Xanthomonas oryzae pv.oryzae, Xoo) with MIC values of 4-64 lg/ mL.Notably, 11 had a significant antibacterial inhibition on S. aureus with an MIC value of 4 lg/mL, compared to positive control (streptomycin sulfate, 2 lg/mL).In the antioxidant assay (Table S7), only 11 showed weak DPPH radical scavenging capability with an IC 50 value of 14.80 lg/mL (Figure S4).

Microbial material
The endophytic fungus GGY-3 was isolated from the leaves of Ilex cornuta collected in October 2018 from Nanjing Botanical Garden, Nanjing, Jiangsu Province, China, and further identified as Nigrospora chinensis (GenBank accession number MK371770.1)(Wang et al. 2022).The test strains for bioactivity evaluations, including six pathogenic fungi and eight bacteria (two Gram-positive bacteria and six Gram-negative bacteria) (Table S1), were supplied by the Lab of Natural Products and Pesticide Chemistry, Nanjing Agricultural University, Nanjing, Jiangsu Province, China.

ECD calculation
The absolute configuration of 2 was determined by comparing the experimental and calculated ECD spectra.The Gaussian 09 software package was used to conduct ECD computation as previously described (Yang et al. 2020).Briefly, the stereoisomers' lowest energy conformations were identified via a conformational search, and their geometric optimisations were performed using the density functional theory (DFT) method at the B3LYP/6-31 G(d) level.The ECD calculations at the B3LYP/6311 þ G (2d, p) level were then applied to the optimised structures for each conformer.Finally, Boltzmann statistics were used to simulate the ECD calculated spectra for each stereoisomer.

Antifungal and antibacterial evaluations
The isolated compounds were tested for antifungal activity in vitro according to the previous report (Li et al. 2016) with slight modifications.Compounds were dissolved in dimethyl sulfoxide (DMSO) to prepare 10,000 mg/mL stock solution and adjusted to a working concentration of 50 mg/mL for initial screening.The half effect concentration (EC 50 ) was used to evaluate the precision antifungal activity.The tebuconazole was used as the positive control.The above experiments were repeated three times, and the inhibition rate was calculated by the following equation: where D 0 and D 1 represented the colony diameter of the blank control (DMSO) and treatments, respectively.The in vitro antibacterial assay was performed in sterile 96-well microplates by MTT method (Zhao et al. 2020) as previously reported.

Antioxidant assay
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capability was used to investigate the antioxidant activity of the isolates (Xiao et al. 2014).The reaction mixture (total 1.2 mL), composed of 0.3 mL of 0.2 mM DPPH ethanol solution and 0.9 mL of samples ethanol solution, was mixed evenly and incubated in darkness for 30 min.After that, the absorbance of 517 nm was measured using the enzyme-labeled instrument.Meanwhile, vitamin C was used as a positive control.The experiment was performed three times with three replicates.The DPPH radical scavenging rate was calculated as follows: where A 0 , A 1 and A 2 represent the 517 nm absorbance of the DPPH ethanol solution, reaction mixture and samples ethanol solution, respectively.

Conclusions
In summary, we identified eleven metabolites from the endophytic fungus Nigrospora chinensis GGY-3 including two new structures (1 and 2).The absolute configuration of known compound 3 was also determined by ECD comparison.The antimicrobial screening revealed that 1 displayed moderate antifungal activities against R. solani and B. cinerea at the concentration of 50 lg/mL.Regarding the drimane sesquiterpenes, 5 and 6 could effectively suppress mycelial growth of P. capsici, M. orazae and R. solani at 50 lg/ mL, while their derivatives 1, 3 and 4 showed moderate antifungal activities.These results could be attributed to their 3-OH group, which might weaken the antifungal activity in their structure-activity relationship.Moreover, 11 exhibited strong antibacterial activity against S. aureus and moderate DPPH radical scavenging capability.

Disclosure statement
No potential conflict of interest was reported by the authors.
Agricultural University (NAUSY-ZD04), Key Project of Natural Science Foundation of the Higher Education Institution of Jiangsu Province (19KJA430013).
Bisaborosaol C (2), isolated as a yellow oil, had a molecular formula of C 16 H 26 O 4 determined by HR-ESI-MS (m/z 267.1599 [M-H] -, calcd for C 16 H 25 O 4 -, 267.1602), indicating four degrees of unsaturation.In 1 D NMR spectra (Table The HMBC correlations from H-11 to C-8, C-9, C-12, combined with the chemical shift of C-8 (d C 84.0) and C-12 (d C 86.0) and the remaining degree of unsaturation, proved the existence of a five-membered O-heterocycle, which was connected to the cyclohexene ring via C-4/C-8 evidenced by the HMBC correlations from H-4 to C-8, and H 3 -10 to C-8 and C-4.Further, HMBC correlations of H 3 -10 to C-4, C-8 and C-9 showed the presence of singlet methyl group (d H 1.05) at C-8, and the other two methyl groups (d H 1.03 and d H 1.03) and one hydroxyl group (d H 3.98)