The antifungal properties of terpenoids from the endophytic fungus Bipolaris eleusines

A series of terpenoids (1–17), comprising six new compounds designated bipolariterpenes A-F (1–6) and eleven recognized compounds (7–17), were isolated from the wheat culture of the potato endophytic fungus Bipolaris eleusines. Their structures and stereochemistry were clarified by HRESIMS, NMR, DP4 + probability analyses, and computations for electronic circular dichroism (ECD). All compounds are made up of six meroterpenoids, four sesterterpenes and seven sesquiterpenes. Among them, four sesterterpenes (4, 5, 10, 11) were investigated for their antifungal, antibacterial and cytotoxic properties, and six meroterpenoids (1–3, 7–9) were evaluated for their antifungal properties. The compounds 7, 9, and 10 had substantial antifungal activity against Epidermophyton floccosum at a concentration of 100 µM. No antibacterial and cytotoxic activities were observed. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-023-00407-x.


Introduction
Terpenoids, possessed several hundred initial terpene scaffolds, are very important secondary metabolites [1].Terpenoids have a basic unifying trait that all terpenoids are derived from the five carbon precursor molecules dimethylallyl diphosphate and isopentenyl diphosphate [2].According to their chemical skeletons, terpenoids are further categorized into monoterpenes, sesquiterpenes, diterpenes, sesterterpenes and triterpenes as well as meroterpenoids derived from the hybridization of polyketide and terpenoid [3].Terpenoids offer a wide spectrum of biological functions, such as the cancerfighting monoterpene carvacrol [4], the sesquiterpene atractylenolide V with anti-inflammatory effects [5], and the diterpene 7-deoxynimbidiol with antioxidant activity [6].One of the most important sources of terpenoids is endophytic fungi, which have been widely reported to have specific biosynthetic capabilities [7,8].As a result, the metabolites of endophytic fungi deserve to be further investigated.
Bipolaris is a genus of fungi belonging to the family Pleosporaceae [9,14], and many metabolites have been reported from several species of this genus, such as terpenes, polyketides, quinones, peptides and alkaloids [10,11,14].The majority of them have a wide spectrum of beneficial biological qualities, such as antifungal, antibacterial, and cytotoxic properties [12][13][14].In our previous study on B. eleusines, a series of meroterpenoids, sesquiterpenes and chromones have been characterized [14][15][16].Among them, bipolarithizole A, a new phenylthiazole-sativene merosesquiterpenoid having a MIC value of 16 μg/mL, inhibited Rhizoctonia solani [14].To find more potent biological agents, a chemical study on B. eleusines with a large-scale fermentation was carried out.As a result, seventeen terpenoids, including six undescribed compounds, were isolated from cultures of the fungus B. eleusines.The details of its isolation, structural elucidation, and antifungal, antibacterial as well as cytotoxic activities are reported in this paper (Fig. 1).

Results and discussion
Compound 1 was purified as a colorless solid.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 29 H 41 NO 6 S (10 indices of hydrogen deficiency).The 1 H NMR spectrum of 1 (Table 1) revealed indications for seven methyl groups [δ H 0.85 (t, J = 7.4 Hz), 1.20 (s), 1.19 (s), 1.11 (s), 1.36 (s), 1.44 (d, J = 7.1 Hz) and 0.76 (d, J = 6.6 Hz)], two oxymethine protons [δ H 3.29 (m), and 3.26 (m)], as well as a number of aliphatic methylene multiplets.In connection with the DEPT experiment, the 13 C NMR spectrum (Table 1) revealed 29 carbon resonances traceable to seven methyls, five methylenes, seven methines and ten quaternary carbons.The one-dimensional (1D) NMR data of 1 were quite similar to those of known substance 19-dehydroxyl-3-epi-arthripenoid A [17], which is a meroterpenoid with sesquiterpenoid and polyketide components.This was followed by more research into the HMBC and    to C-13 and C-14 (Fig. 2).The polyketide unit (C-1-C-11, C-27 and C-28) was clarified using the 1 H-1 H COSY correlations of H 3 -1/ H 2 -2/H-3, as well as HMBC cross-peaks from H 3 -28 to C-2, C-3, and C-4 and from H-27 to C-4, C-5, and C-6 (Fig. 2).Following that, the two units were linked using the HMBC cross-peaks from H 2 -12 to C-8, C-9, C-10, C-13, C-  5.49) proposed that H-21, H-17, and H-13 were α-oriented, whereas H 3 -26, H 3 -25 and H-12 were β-oriented in the sesquiterpenoid section (Fig. 3).Based on prior discoveries from single crystal   X-ray diffraction and the stereoselectivity of enzymes in biosynthesis, the relative configuration of C-5 is known to be fixed (5S-configuration) [18,19].The absolute configuration of C-3 can be determined using the diagnostic chemical shift of C-2.The 13 C chemical shift values at the C-2 location are substantially downshifted (δ C 26.8 for 3S and δ C 23.6 for 3R), as reported for the 3S-conformation [18].Therefore, the chemical shifts of C-2 at δ C 27.3 in 1 revealed that C-3 is S-configured.The proposed structure of 1 was further confirmed by the quantum chemical calculations of the NMR data (qccNMR) of two diastereoisomers (3S-1 and 3R-1).The two epimers were submitted to a thorough conformational screening approach, and the NMR chemical shifts were estimated using the PCM solvent model in methanol at the mPW1PW91/6-31 + G(d,p)//M06-2X/def2-SVP level of theory.As a result, the calculated 13 C NMR data for 3S-1 agreed well with its experimental values and DP4 + analysis also identified 3S-1 as the most instructive structure of 1 with 100% DP4 + chance for all data (Fig. 4 and Additional file 1: Fig. S78).Therefore, compound 1 was defined as (3S,5S,12S,13S,14R,17R,18R,21R)-1.
Compound 2 was purified as a purple solid.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 28 H 37 NO 5 (11 indices of hydrogen deficiency).The 1 H NMR spectrum of 2 (Table 1) revealed indications for seven methyl groups [δ H 0.85 (t, J = 7.4 Hz), 1.20 (s), 1.19 (s), 1.12 (s), 1.53 (s), 2.25 (s) and 1.27 (d, J = 7.0 Hz)], two oxymethine protons [δ H 3.21 (m) and 3.19 (m)], as well as a number of aliphatic methylene multiplets.In connection with the DEPT experiment, the 13 C NMR spectrum (Table 1) revealed 28 carbon resonances traceable to seven methyls, five methylenes, four methines (two oxygenated), and twelve quaternary carbons (three oxygenated carbons and two carbonyls).The 1 H and 13 C NMR data of 2 were structurally similar to those of cochlioquinone G [17] except for the absence of a hydroxyl group at C-12 and the presence of an additional double bond at δ C 111.1 (C-12) and 145.2 (C-13).This hypothesiswas validated by HMBC correlations from H-12 (δ H 6.38) to C-10 (δ C 175.3), C-9 (δ C 116.3), C-8 (δ C 151.0) and C-14 (δ C 81.2) (Fig. 2).The ROESY correlations of H-21/H-17 indicated that H-21 and H-17 were on the same face and were given as α-orientation (Fig. 3).The correlations of CH 3 -25 and CH 3 -26 established the β-orientation of H 3 -25 and H 3 -26, which was similar to those of cochlioquinone G. Similar to 1, the absolute configuration of C-3 was calculated utilizing chemical shifts of C-2 (δ C 29.8) and the quantum chemical calculations of the NMR data as well as DP4 + analysis (Fig. 4 and Additional file 1: Fig. S79).Therefore, the absolute configuration of 2 was defined as (3S,14R,17R,18R,21R)-2.Compound 3 was purified as a yellow oil.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 28 H 39 NO 5 (10 indices of hydrogen deficiency).The NMR data (Table 1) showed that compound 3 was comparable to compound 2 except for the lack of a double bond group at C-13 (δ C 145.2) and C-12 (δ C 111.1) and the presence of two alkyl carbons of C-13 (δ C 48.5) and C-12 (δ C 17.1).This difference indicated that the double bond of C-13 and C-12 were replaced by a single bond, which was confirmed by HMBC correlations from H-12 (δ H 2.35) to C-10 (δ C 182.9), C-8 (δ C 159.2) and C-14 (δ C 83.1) and 1 H-1 H COSY correlations of H-13/H-12 (Fig. 2).Based on the ROESY profile (Fig. 3) and its likely biosynthesis origin from 2, the conformation of 3 was determined as the 3S,13R,14R,17R,18R,21R conformation, which should be the same as 2.
Compound 4 was purified as a colorless powder.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 27 H 40 O 6 (8 indices of hydrogen deficiency).Its 13 C NMR spectra indicated 27 signals generated by six methyl carbons, seven sp 3 methylene carbons, four sp 3 tertiary carbons, two sp 2 methine carbons, two sp 3 quaternary carbons, four sp 2 nonprotonated carbons and two carbonyl carbons.The 1 H and 13 C NMR data of 4 (Table 2) were they were assigned as α-orientation (Fig. 3).Furthermore, quantum chemical calculations of the nuclear magnetic resonance data (qccNMR) of two diastereoisomers, (12S,13S)-4 and (12R,13R)-4, were undertaken to identify the configuration of the epoxy ring on C-12 and C-13.The two epimers were rigorously conformationally screened, and NMR chemical shifts were estimated in methanol using the mPW1PW91/6-31 + G(d,p)// M06-2X/def2-SVP level of theory and the PCM solvent model.With a DP4 + probability of 100%, the DP4 + analysis also revealed (12R,13R)-4 as the most plausible structure for 4. (all data) (Fig. 4), which was compatible with the ROESY study results presented above.Therefore, the absolute configuration of 4 was specified as (1S,11S,12R, 13R,15R,23S)-4.
Compound 5 was purified as a colorless powder.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 25 H 38 O 5 (7 indices of hydrogen deficiency).Its 13 C NMR spectra revealed 25 signals from five methyl carbons, seven sp 3 methylene carbons, four sp 3 tertiary carbons, two sp 2 methine carbons, two sp 3 quaternary carbons, four sp 2 nonprotonated carbons and a carbonyl carbon.The NMR spectra of 5 indicated that it is closely linked to 4, except that 4 lacks an acetyl group and has an additional hydroxyl group at C-24, which is corroborated by the 42 Da drop in molecular weight of 5 when compared to 4. Based on ROESY profile (Fig. 3) and its potential biosynthetic origin from 4, the configuration of 5 would be equivalent to 4 for (1S,11S,12R,13R,15R,23S)-5.
Compound 6 was purified as a colorless powder.The protonated molecule signal in the HRESIMS was used to calculate its chemical formula, which was C 15 H 24 O 2 (4 indices of hydrogen deficiency).The 1 H and 13 C NMR spectra (Table 2) revealed 15 signals from three methyl carbons, three sp 3 methylene carbons, one sp 2 methylene carbon, five sp 3 tertiary carbon, two sp 3 quaternary carbons, and one sp 2 nonprotonated carbon.According to the preceding data, the structural properties of 6 were comparable to those of longi-β-nozigiku alcohol [21], with the exception of an extra hydroxyl group in 6.Chemical shifts at the C-5 (δ C 67.2) level have increased relative, together with the critical HMBC correlation from H-5 (δ H 3.87) to C-3 and C-9, suggesting the hydroxyl group was annexed to C-5 (Fig. 2).The ROESY correlations of H-15/H-7and H-13/H-7 indicated that H-15, H-13 and H-7 are on the identical side and belong to the α-orientation (Fig. 3).The correlations of H-1/H-10 determined the β-orientation of H-1.Meanwhile, the correlations of H-5/H-11, H-11/H-13 and H-8/H-5 indicated that H-13, H-11, H-8 and H-5 are on the identical side and belong to the α-orientation (Fig. 3).Hence, the hydroxyl groups that exist at C-5 is β-configuration.The absolute configurations of 6 was determined by comparing the experimental ECD spectra with calculated ECD spectra (Fig. 5).Therefore, the absolute configuration of 6 was defined as (1R,3R,5R,7S,13S,15R)-6.
Since terpenoids are known to exhibit a variety of biological activities, the four sesterterpenes (4, 5, 10 and 11) were tested for their antibacterial, antifungal and cytotoxic properties, and six meroterpenoids (1-3, 7-9) were evaluated for their antifungal activities.The antibacterial activity was evaluated against the Escherichia coli, Staphylococcus aureus subsp.Aureus, Salmonella enterica subsp.Enterica and Pseudomonas aeruginosa [29], but all tested compounds did not show any promising activity up to the highest tested concentration of 100 µM (Additional file 1: Table S7).Likewise, no cytotoxic activity against the human cancer cell lines (HL-60, A549, SMMC-7721, MDA-MB-231 and SW480) could be detected (at 40 μM) (Additional file 1: Table S8).The antifungal activity was evaluated against the Epidermophyton floccosum, Trichophyton rubrum, Microsporum gypseum and Candida albicans.The compounds 7, 9, and 10 had good antifungal activity against E. floccosum at a concentration of 100 µM, and compounds 1, 4, 5 and 10 exhibited moderate or weak antifungal activity against M. gypseum (Table 3).

Conclusions
In conclusion, seventeen terpenoids, including six undescribed compounds, were identified from the endophytic fungus B. eleusines.Among these isolates, no compounds exhibited antibacterial and cytotoxic activities.However, the compounds 7, 9 and 10 had significant antifungal activity against E. floccosum.Our initial findings indicate that the endophytic fungus B. eleusines is a plentiful supply of different and bioactive terpenoids.

General experimental procedures
A Bruker spectrometer (Bruker, Germany, model AM600) was used to obtain 1D and 2D spectra.A Q Exactive HF mass spectrometer (Thermo Fisher Scientific, USA) was used to collect HRESIMS date.Applied Circular dichroism spectrometer (Chirascan, New Haven, USA) was used to record CD spectra.Column chromatography (CC) was performed on silica gel (Qingdao Marine Chemical Ltd., Qingdao, China), RP-18 gel (Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Sweden) [30].Semipreparative HPLC experiments were carried out on an Agilent 1260 HPLC with an Agilent Zorbax SB-C 18 column (5 μm, 250 × 9.8 mm).Thin layer chromatography (GF 254) was used to monitor fractions, and spots were seen by heating silica gel plates coated with vanillin and 10% H 2 SO 4 in ethanol.

Culture and fermentation of fungal material
This fungus was identified by PCR amplification using fungal 18S universal primers ITS1 and ITS4.The sequenced 18S rDNA ITS sequences were subjected to homology sequence alignment analysis, which, using BLAST, showed 99% maximum similarity to Bipolaris eleusines with the gene bank registration number (KY909768.1).Therefore, it was identified as B. eleusines, which was preserved in South-Central Minzu University, China.The strains were inoculated into PDA agar medium and incubated for 3-5 days at a constant temperature of 25 °C.The medium was then cut into several small pieces and placed into a solid wheat medium (50 g wheat and 50 mL water in 500 mL culture flasks, autoclaved at 120 °C for 30 min, for a total of 212 flasks) and fermented at 25 °C for 28 days.

Quantum chemical calculations 3.4.1 13 C NMR calculation
The NMR chemical shift calculations were carried out in Gaussian 16 using density functional theory (DFT) [31].Spartan'14 software was used to conduct the preliminary conformational distribution search.GaussView 6.0 was used to view conformational structures and modify computation input files for calculation.At the M062X/def-2svp level, all ground-state geometries were optimized, and the stable conformations obtained at that level were then used in magnetic shielding constants at the mpw1pw91/6-31 + g(d,p) level.GaussView 6.0 was used to display the calculated chemical shift of each atom in each conformer, and the final chemical shift of each atom was calculated from the Boltzmann distribution of each conformer.The stereochemistry was assigned using the DP4 + probability analysis of calculated and experimental chemical shifts.

ECD calculations
The absolute conformations of the compounds were determined using Gaussian 16 software.Briefly, the relative conformations of the compounds were first determined by ROESY spectroscopy, followed by a preliminary conformational analysis based on Spartan'14software.The obtained conformations were optimized at the M062X/def2svp level of density functional theory (TDDFT), and then ECD calculations were performed by M062X/def2svp.The ECD curves were generated by Origin software.

Antifungal assay
The endophytic fungus used in this assay was Bipolaris eleusines, and the samples to be tested were diluted in 96-well plates, and the fungal solution was added to each well.C. albicans was nurtured at 37 °C for 24 h, and Filamentous fungi were nurtured at 25 °C for 5 days, and the absorbance value at 625 nm was measured by an enzyme marker.The experiment was set up with a medium blank control, a fungal control [32], and positive drug controls for amphotericin B and terbinafine hydrochloride.

Antibacterial assay
The samples were evaluated for their antibacterial activity against Escherichia coli, Staphylococcus aureus subsp.Aureus, Salmonella enterica subsp.Enterica and Pseudomonas aeruginosa.Dissolve the compounds to be tested to a concentration range of 100 μM and add it to a 96-well culture plate.Each well received a bacteria solution until the final concentration reached 5 × 10 5 CFU/ ml.It was then cultivated at 27 ℃ for 24 h, and the inhibition rate was determined by the microplate reader at OD 600 nm.The medium blank control was used in the experiment [12].Sodium penicillin G and ceftazidime were used as the positive control.

Cytotoxicity assay
Five human cancer cell lines including HL-60 (promyelocytic leukemia), A549 (lung epithelial cancer), SMMC-7721 (liver cancer), MDA-MB-231 (breast cancer), and SW480 (colon cancer) were used for cytotoxicity assays by MTS method [12].In a word, cells were cultured in DMEM media supplemented with 10% fetal bovine serum before being injected in 96-well culture plates and treated with different concentrations of compounds.After 24 h, each well received 20 μL of MTS, and the OD at 492 nm was determined using a microplate reader.

Fig. 5
Fig. 5 Experimental and calculated ECD curves of compound 6 1 H − 1 H COSY spectra, which confirmed the planar structure of 1.The sesquiterpenoid unit Fan et al.Natural Products and Bioprospecting (2023) 13:43 Fig.1The chemical structures of compounds 1-17

δ C , type δ H (J in Hz) δ C , type δ H (J in Hz) δ C , type δ H (J in Hz)
1 H (600 MHz) and 13 C (150 MHz) NMR data of compounds 1-3 Measured in CD 3 OD, b Measured in CDCl 3 a

Table 2 1
H (600 MHz) and 13 C (150 MHz) NMR data of compounds 4-6 Measured in CD 3 OD, b Measured in CDCl 3 a

Table 3
Inhibitory effect of compounds on four strains of fungi