New sesquiterpenoids with anti-inflammatory effects from phytopathogenic fungus Bipolaris sorokiniana 11134
Abstract
Sesquiterpenoids represent a structurally diverse class of natural products widely recognized for their ecological significance and pharmacological potential, including antimicrobial, anti-inflammatory, and anticancer properties. As part of our efforts to explore bioactive secondary metabolites from phytopathogenic fungi, we conducted a molecular networking-based analysis of Bipolaris sorokiniana isolate BS11134, which was fermented on a rice medium. This analysis led to the identification of three new seco-sativene-type sesquiterpenoids (1–3) and seven known analogues (4–10), with the NMR data of compound 4 being reported for the first time. The structures of these compounds were elucidated using HR-ESI-MS and extensive spectroscopic data analysis. Notably, compound 9 significantly inhibited nitrous oxide expression in lipopolysaccharide (LPS)-treated RAW264.7 cells in vitro (inhibition rate: 84.7 ± 1.7% at 10 μM), while compound 1 (10 μM) showed a weak inhibitory effect (inhibition rate = 28.0 ± 2.4%). Additionally, we proposed a biosynthetic pathway for these compounds. This study not only expands the chemical space of the helminthoporene class of molecules but also underscores the untapped potential of phytopathogenic fungi as promising sources of structurally unique and biologically active natural products.Graphical Abstract
Keywords
Phytopathogenic fungi Bipolaris sorokiniana 11134 Sesquiterpenoid Anti-inflammatory1 Introduction
Inflammation in animal tissues is a complex reaction regulated by several inflammatory mediators, including nitric oxide (NO), prostaglandins E2, cytokines, and growth factors [1]. Inflammation is commonly associated with various pathophysiological conditions, such as arthritis, tumours, Alzheimer, or cardiovascular diseases [2–4]. Macrophages, pivotal in host defence, release inflammatory mediators upon activation by lipopolysaccharides (LPS), contributing to pathogenesis. Thus, inhibition of macrophage-produced mediators may attenuate the development of related diseases.
Sesquiterpenes play significant roles in biology and ecology [5], displaying a range of activities in fighting against tumors, viruses, fungi, HIV, biotic pressures, immunity issues, and pests [6–13].
Fungi, especially those interacting with plants, are a rich source of novel sesquiterpenes [14, 15]. Based on cross-talk between plants and phytopathogens, phytopathogenic fungi can co-evolve with host plants, which leads to the production of unique natural products. Phytopathogenic fungi are a source for the discovery of new active compounds.
Traditionally, the isolation of secondary metabolite (SMs) has relied on bioactivity-directed isolation or via screening targets with distinct spectroscopic features. Recently, Molecular Networking (MN) has revolutionized the visualization of chemical diversity of extracts through the spectral alignment of tandem mass spectrometry (MSb) data, which captures in-source fragmentation and ionization patterns [16, 17]. MN is particularly adept at identifying unclassified molecules that may hold biological significance or represent novel metabolic pathways, especially useful in comparative studies of different states, such as time points or genetic variant [18]. To enhance the capabilities of MN, Feature-based molecular networking (FBMN) was introduced in 2020, offering enhanced functionality by incorporating chromatographic feature detection into spectral alignment [19].
Bipolaris sorokiniana, a well-known phytopathogenic fungus, causes diseases of barley and wheat, such as root rots, leaf spots, seedling blight, and head blight [20]. Known for producing a range of phytotoxic substance [21, 22], this fungus has been linked to approximately 20 sesquiterpenoids related to helminthosporol across different Bipolaris species. In this study, we report the discovery of ten sesquiterpenoids (1–10) from B. sorokiniana with a helminthoporene skeleton. This included the identification of three new seco-sativene type compounds (1–3) through Global Natural Product Social (GNPS) molecular networking and LC–MS/MS analysis. Additionally, we assessed the anti-inflammatory potential of these isolated sesquiterpenoids in vitro, focusing on their capacity to inhibit NO production induced by LPS in the cell lines of RAW264.7.
2 Results
2.1 Sesquiterpenoids gene clusters exploration from BS11134 based on genome analysis
BS11134 was classified as Bipolaris sorokiniana based on morphology and sequence of the internal transcribed spacer (ITS) region (GenBank accession number KU297882) [23]. We used the Hidden Markov Models file (Terpene_syn_C_2) classified in the Pfam database [24] to examine the BS11134 genome obtained in our previous study [23] for potential sesquiterpene cyclase (STC) genes, resulting in four homologous sequences of known STC proteins with DDXXD metal binding motifs. Further annotation of these four STC-encoding biosynthetic gene clusters using antiSMAS [25] and 2ndfinder online software revealed various post-modification enzymes, including cytochrome P450, acetyltransferase, decarboxylase, etc. (Fig. 1). Notably, the length of STC8 gene cluster was only 15 kb (Fig. 1), consisting of a three-gene cassette very similar to the recently identified seco-sativene gene cluste [26].
Organization of the sesquiterpenoids biosynthetic gene cluster in BS11134
2.2 FBMN guided detection of sesquiterpenoids
We used rice medium to ferment the fungus because this medium gave the most induced secondary metabolites compared with other six media used [23]. A previously reported seco-sativene type sesquiterpenoid helminthosporol (7) was characterized from the crude extract of BS11134 based on its UV spectrum and HR-MS data (Fig. 2B and Fig. S7a). Further analysis using GNPS FBMN identified new seco-sativene type sesquiterpenoids using helminthosporol (7) as the seed compound (Fig. 2A), the precursor m/z of 7 was found to be within Cluster I, comprising 97 nodes with similar MS/MS patterns.
FBMN-guided characterization of new helminthosporol analogs from BS11134 crude extract. A Clustering of sesquiterpenoids analogs. Helminthosporol (7) was used as seed compound (yellow shaded square) in cluster analysis, and 26 analogs (Purple circles: compounds isolated in this study; Blue circles: other potential new sesquiterpenoids identified by HRMS annotation) were identified within the network. B HPLC–UV chromatogram of sesquiterpenoids fraction RD3 examined at 254 nm, and the UV spectrum of helminthosporol. C Structures of compounds 1–10 isolated from strain BS11134
Detailed HRESIMS data annotation (Table 1) revealed 27 potential sesquiterpenoids, ten of which showed similar UV spectra to helminthosporol in the HPLC chromatogram of subfraction RD3. Among these, five nodes exhibited previously unreported m/z values (291.1571, 291.1574, 321.1682, 301.1782, 253.1804), guiding the isolation of new sesquiterpenoids from several subfractions. Three new sesquiterpenoids (1–3) with a helminthoporene skeleton as well as seven known compounds (4–10) were identified (Fig. 2C).
Detailed annotation for sesquiterpnoid related nodes in cluster I from the BS11134 featured based molecular networking
2.3 Structure elucidation of isolated sesquiterpenoids
The molecular formula of compound 1 was C15H24O4 confirmed by its HRESIMS data ([M+Na]+ m/z 291.1567, calcd. for C15H24O4Na+ 291.1571) (Fig. S1a). Analysis of 1H NMR, 13C NMR, and HSQC spectra (Table 2 and Figs. S1b-S1 d) revealed a carboxyl cluster [δC 167.5], two olefinic carbons [δC 157.7, 128.5], four methylene (two of which were substituted with the hydroxyl groups [δC 55.6, 59.9]), four methine and three methyl groups, as well as one sp3 quaternary carbon, outlining a seco-sativene sesquiterpene skeleton. The 1H-1H COSY (Fig. S1e) revealed a continuous spin system extending from H2−4 to H3−10/H2−11. In addition, H-6 was further coupled with H-7, which also coupled with H-13, and H-13 coupled with H2−14 (Fig. 3 and Table S1). The two hydroxyl groups at C-12 (δC 55.6) and C-14 (δC 59.9) could be further determined by the HMBC (Fig. S1f) correlations of oxymethene protons at δH 4.54 (d, J = 13.2 Hz) and 4.12 (d, J = 13.2 Hz) with C-1 (δC 128.5), C-2 (δC 157.7) and C-3 (δC 49.2), and oxymethene protons at δH 3.44 (dd, J = 10.3, 4.9 Hz), and 3.05 (dd, J = 10.3, 9.4 Hz) with C-3, C-7 (δC 43.2) and C-13 (δC 62.0). A six-membered ring was formed on C-13 was connected to C-4 through C-3, according to the HMBC correlations between H3−8 and C-3, C-4, and C-13. The presence of a methyl group (Me-8) at C-3 was inferred as well. The olefinic carbons C-1 and C-2 were connected to C-7 and C-3, respectively, according to the HMBC correlations of H-7/C-15, H-7/C-1, H-7/C-2, H2−12/C-2, and H2−12/C-3. These observations suggested the structure of compound 1 closely resembled helminthosporic acid [27], with the notable substitution of a methyl group for a hydroxylmethylene group. The relative configurations of 1 could be established according to the ROESY (Fig. S1 g) correlations of H-7/H3−11, H-7/H-13, H3−8/H-13, and H-13/H-6, as well as comparison with literature [27] (Fig. 3). To figure out the absolute configurations of 1, the electronic circle dichromatography (ECD) calculation of two epimers 3R,6R,7S,13S-1/3S,6S,7R,13R-1 was performed, resulting in the 3R,6R,7S,13S configurations of 1 (Fig. 4A). Therefore, the structure of 1 was established (Fig. 2C) and it was named 12-hydroxyhelminthosporic acid.
1H and 13C NMR Data of compounds 1–5
2D NMR correlations of compounds 1–5. A Key 1H-1H COSY and 1H-13C HMBC correlations. B Key ROESY correlations
Experimental and computed ECD of compounds 1 (A), 2 (B), and 5 (C)
Compound 2 has a molecular formula of C15H24O4, as confirmed by HRESIMS data (Fig. S2a). Comparative analysis of 1H and 13C NMR data (Table 2 and Fig. S2b–d) between compounds 1 and 2 revealed that two secondary methyl groups [δC/δH 20.8/0.76 (d, J = 6.3 Hz), δC/δH 21.8/0.97 (d, J = 6.3 Hz)] were replaced by two methyl groups at lower field [δC/δH 28.0/1.13 (s), δC/δH 28.5/1.04 (s)]. The appearance of an oxygenated quaternary carbon (δC 70.5) suggested the presence of one hydroxyl group at C-9. HMBC (Fig. 3 and Supplementary Fig. S2e, f) further confirmed this hypothesis with correlations of H3−10/C-6, H3−10/C-9, H3−10/C-11. Resonances at δH 1.88 (s) showed correlations with carbons at δC 49.5(C-3), δC 127.6(C-1), and δC 156.1(C-2), establishing the methyl group location of C-12. The relative configurations of 2 were consistent with those of 1 by detailed analysis of their ROESY (correlations of H-7/H3−11, H-7/H-13, H3−8/H-13, and H-13/H-6) (Fig. 3, Fig. S2 g, and Table S2). The absolute configurations were assigned as 3R,6S,7R,13S through ECD calculation. Thus, the structure of 2 was established (Fig. 2C), and this compound was named 9-hydroxyhelminthosporic acid.
The molecular formula of compound 3 was deduced as C18H24O5 based on HRESIMS (Fig. S3a) (m/z 343.1514 [M+Na]+, calcd. for 343.1516) with 7 degrees of unsaturation. 1H, 13C and 2D NMR data (Figs. S3b–f) revealed a similar structural skeleton with compound 1, except for resonances of two additional carboxyls (δC 164.9, 166.8) and one olefinic bond (δC 145.6, 131.2). HMBC correlations from H-14 (δH 6.51 d, J = 10.9 Hz) to C-3, C-7, C-16, C17, and C-18 revealed that two carboxyl groups were connected with C-14 through non-protonated carbon C-16. The relative configurations of 3 were consistent with 1 based on key ROESY correlations of H-7/H3−11, H-7/H-13, H3−8/H-13, and H-13/H-6 (Fig. 3, Fig. S3 g, and Table S3). Given the shared biosynthetic pathway of compounds 1 and 3 through the seco-sativene sesquiterpene skeleton, the absolute configurations of 3 were deduced to align with that of 1, and was designated 3R,6R,7S,13S. This alignment was supported by its specific optical rotation ([α]−20) in contrast to compound 1 ([α]−38) and to bipolarisorokin H ([α]−137) [28]. Thus, 3 was a new compound (Fig. 2C) and named bipolarisorokin I.
HRESIMS of 4 displayed a molecular ion peak at m/z 301.1775 for [M+Na]+ (calcd. for 301.1774) (Fig. S4a) and indicating a molecular formula of C17H26O3. 1D and 2D NMR data (Figs. S4b–f) showed that compound 4 shared a structural framework with helminthosporol (7) [29], distinguished by an additional acetyl group [δC/δH 20.7/1.98 (s), δC 170.4]. HMBC correlations from H3−17 and H2−14 to C-16 confirmed compound 4 as a 14-OH acetylated analogue of 7. Through ROESY analysis, the crosspeaks of H-7/H3−11, H-7/H-13, H3−8/H-13, and H-13/H-6 revealed that the relative configurations of 4 were consistent with those of 1 (Fig. 3, Fig. S4 g, and Table S4). Its absolute configurations were deduced as 3R,6R,7S,13S considering its biosynthetic lineage from seco-sativene sesquiterpene, and comparison of specific rotation values (4, [α]+11.0) with literature (bipolenin H, [α]+17.5) [30]. Accordingly, compound 4 was definitively identified and named bipolarisorokin J, with a defined structure (Fig. 2C). The NMR data of compound 4 is reported here for the first time.
Compound 5 possessed a molecular formula of C15H24O3 (four degrees of unsaturation) determined by HRESIMS data (Fig S5a). NMR analysis (1H and 13C, Table 2 and Figs. S5b-S5 d) revealed a structural resemblance to helminthosporol (7), except that the methyl group (Me-11) was replaced by a hydroxylmethylene group. The presence of a hydroxyl group at C-11 could be assigned by HMBC (Fig. 3 and Fig. S5e, f) correlations of the downfield shifted signals at δH 3.87 (dd, J = 11.1, 3.9 Hz) and 3.62 (dd, J = 11.1, 4.7 Hz) with C-6, C-9 and C-10. Detailed analysis of ROESY afforded the 3R*,6S*,7S*,13S* relative configuration in 5 (Fig. 4B, Fig. S5 g, Table S5). The relative configurations between C-6 and C-9 in 5 was determined to be 6S*,9S* by 13C NMR calculations and DP4+ analyses (Fig. 5, Table S9) [31]. ECD calculations confirmed the absolute configurations of 5 as 3R,6S,7S,9S,13S, leading to the identification of compound 5 as 11-hydroxyhelminthosporol (Fig. 2) [32].
13C NMR calculation results of two plausible isomers 5a/5b at the B3LYP/6–311++G(2 d,p) level. A Linear correlation plots of the calculated and experimental 13C NMR values. B Relative errors between the calculated and recorded 13C values. C DP4+ probability
Compounds 6–10 were identified as helminthosporic acid derivative (6) [33], helminthosporol (7) [29], helminthosporic acid (8) [27], sorokinianin (9) [34], and secolongifolene diol (10) [29], by comparing their spectroscopic data with those in previous literature.
2.4 Proposed biosynthetic pathway of the isolated sesquiterpenoids
Sativene could be synthesized from FPP by sesquiterpene synthase (Fig. 6), by a mechanism involving ionization and successive rearrangement [35]. Compounds 1–9 belong to seco-sativene type irregular terpenoids, while compound 10 belongs to seco-longifolene type sesquiterpenoids [29]. The plausible biosynthetic routes of compounds 1–10 were described based on recent research [26, 36] (Fig. 6).
Organization of the seco-sativene biosynthetic gene cluster in B. sorokiniana ND90Pr and BS11134 (A), and proposed biosynthetic pathway for isolated sesquiterpenoids 1–10 (B)
Intermediate IM2 was generated from sativene through successive oxidation by P450 and a spontaneous shift of the Δ2,12 olefinic bond. Subsequent reduction of the C-14 aldehyde, catalysed by a Aldo–keto reductase (AKR) yielded compound 7. Compounds 4, 5, and 8 can be derived from Compound 7 through acetylation at 14-OH, hydroxylation at C-11, and carboxylation of C-15, respectively. Hydroxylation at C-9 or C-12 of compound 8 yielded compounds 1 and 2, respectively. Compound 6 resulted from the acetylation at 14-OH of Compound 8. The γ-butyrolactone moiety in Compound 9 was proposed to derive from the TCA cycle intermediate 2-oxosuccinic acid via an aldol condensation reaction, as supported by previous isotope labelling studies [37]. A similar process occurred with compound 3, where the 2-methylenemalonic acid moiety was incorporated using malonic acid as intermediate through an aldol condensation reaction with C-14 aldehyde of IM2 (Fig. 6B). seco-Longifolene type sesquiterpenoid 10 was postulated to possess a divergent cyclization route, generating an end product with a seven-membered ring [27, 38].
2.5 In vitro anti-inflammatory assay
All isolated sesquiterpenoids from strain BS11134 were screened for their anti-inflammatory activity (Table 3) by evaluating inhibition effects on the NO production induced by LPS in RAW264.7, a mouse macrophage cell line. Compounds 1 and 9 exhibited anti-inflammatory effects in vitro, with inflammation inhibition rates of 28.0 ± 2.4% and 84.7 ± 1.7%, respectively, at a concentration of 10 μM, compared to the positive control, indomethacin, which showed a 51.2 ± 8.2% inhibition rate.
NO inhibitory activities of compounds
3 Discussion and conclusion
The fungal kingdom is a primary source of new natural products, yet the capabilities of only a few species have been uncovered [39]. Terpenoids represent a crucial class of natural products derived from filamentous fungi. Fungal sesquiterpenoids, in particular, are significant targets for medical drug design because of their potential bioactivity [14, 40]. Our research uncovered ten sesquiterpenoids with a sativene architecture from the phytopathogenic fungus B. sorokiniana 11134.
Several structural classes related to our isolated compounds have been previously reported from related members of the family Pleosporaceae including species of Bipolaris, Cochliobolus, and Drechslera; such compounds include sativene, seco-sativene, isosativene, longifolene, and seco-longifolene. We expand the reported seco-sativene repertoire with three new congeners 1–3. seco-sativenes sesquiterpenoids, characterized by a bicyclo[3.2.1]octane structure (6–5 ring system) [41], are biosynthetically generated from sativene through the C14–C15 bond cleavage, and undergo various post-modifications such as isomerization, ketone reduction, acetylation, carboxylation, and glycosylation [41].
Recently, Zhang et al. revealed the biosynthetic gene cluster of seco-sativenes in B. sorokiniana ND90Pr through heterologous expression of a three-gene cassette in Aspergillus nidulans. The terpene cyclase (SatA) responsible for forming the 6-5-5 ring sativene skeleton, the cytochrome P450 (SatB) involved in C14–C15 bond cleavage, and the aldo–keto reductase (SatC) for the regioselective reduction of C14 aldehyde have been characterized [26]. We detected a similar three-gene cassette in strain BS11134, responsible for the biosynthesis of compounds 1–10, showing high similarity to the sat cluster (Fig. 6A). The proposed biosynthetic pathway for compounds 1–10 suggested that additional, yet unidentified enzymes (acetylase, oxidoreductase, double-bond translocase, etc.) are required for transforming the key intermediate IM1 into these sesquiterpenoids (Fig. 6B).
Currently, more than 40 seco-sativenes featuring the bicyclo[3.2.1]octane structure have been isolated from fungi, exhibiting diverse bioactivities such as phytotoxicity, growth promotion, anti-NO production, ACAT inhibition, and antifungal effects [41]. In this study, we reported the NO production inhibition activity of sorokinianin (84.7 ± 1.7% at 10 μM) for the first time, which has been recognized as phytotoxin for decades [34]. Previously, only a few seco-sativene sesquiterpenoids were known for their anti-inflammatory potential. For instance, bipolenins G and 9-hydroxyhelminthosporol demonstrated anti-NO production activities with IC50 values of 23.8 and 17.5 μM, respectively [30].
In summary, feature-based molecular networking generated from GNPS confirmed the cluster of sesquiterpenoids, and HRMS-oriented isolation led to the characterization of ten sesquiterpenoids with a helminthoporene skeleton, including three new compounds (1–3), alongside the first detailed NMR data for compound 4. The structures of these compounds were unequivocally determined through comprehensive analyses of MS and NMR data. This research extends the known helminthoporene class of molecules and underscores the untapped potential of phytopathogenic fungi as sources of novel compounds.
4 Materials and methods
4.1 General experimental section
NMR spectra were recorded using a Bruker Advance DRX600 spectrometer and Bruker Advance Ⅲ HDX 800 MHz spectrometer. Deuterated solvents (CDCl3 and DMSO-d6) were purchased from Cambridge Isotope Laboratories (CIL). A Bruker Maxis Ⅱ ETD QTOF mass spectrometer was used to run HRESIMS analysis. Column chromatography was performed with Sephadex LH-20 (GE Healthcare BioSciences AB) and ODS-A (YMC, Japan). Semi-preparative HPLC was carried out equipped with Phenomenex Luna C18 (5 μm, 9.4 × 250 mm), Eclipse XDB-C3 (5 μm, 9.4 × 250 mm), and ZORBAX RX-C8 (9.4 × 250 mm) columns. HPLC analysis were carried out with an Agilent 1100 Series separation module coupled with DAD detector. The UV–vis spectra were measured using a Cary 50 spectrophotometer. The optical rotation was recorded on a Perkin-Elmer Model 343 polarimeter. Biological media, reagents, and chemicals were obtained from standard commercial sources.
4.2 Characterization and identification of pathogenic fungus BS11134
The original culture of BS11134 was obtained from a leaf of Poa pratensis collected from Sujiatun (GPS 40.21432, 116.53574), Chaoyang District, Beijing, in July 2013. It was characterized as Bipolaris sorokiniana by ITS DNA gene sequence (accession no. KU297882) and morphology [42], and has been deposited in the China General Microbiological Culture Collection Center (accession No. 3.18317), which is a member of the World Data Centre for Microorganisms (WDCM 550).
4.3 GNPS directed dereplication and prediction of new sesquiterpenoids
Samples were dissolved in MeOH to make 1 mg/mL solution and eluted with a gradient of H2O (1‰ HCOOH) and CH3CN with a gradient method as follows: 5% CH3CN gradient elution to 99% in 9 min, 99% CH3CN kept for 3 min, 99% CH3CN changed to 5% in 0.1 min, and 5% CH3CN kept for 3 min with the flow rate of 0.35 mL/min. LC–MS/MS data was acquired on Bruker Maxis Ⅱ ETD QTOF mass spectrometer coupled with Thermo scientific UltiMate 3000 HPLC system. The raw data was converted to the mzXML file format using the Bruker Data Analysis software. The.mzML file was processed using the MZmine (version 2.53). The mass detections were realized keeping the noise level at 1.0E3 for MS1 and 1.0E1 for MS2, respectively. The ADAP chromatogram building used a minimum time span of 0.1 min, m/z tolerance of 0.02 (or 5 ppm), and minimum height of 1.0E3. Deisotop was carried out utilizing isotopic peaks grouper algorithm, with a RT tolerance of 0.1 min and m/z tolerance of 0.001 (or 5 ppm). Duplicate peaks were filtered with filter mode of old average, m/z tolerance of 0.001 (or 5 ppm), and retention time (RT) range = 1 min. A feature-based molecular networking was created using the web-based workflow (version release_28.2) at GNPS (https://gnps.ucsd.edu/) [17, 19]. Cytoscape (version 3.7.2) was used to visualize the corresponding output FBMN data.
4.4 Scale-up fermentation and secondary metabolites purification
The strain BS11134 was cultured on potato dextrose agar (PDA) at 28 ℃ for 10 days. The well-grown agar cultures were cut into small cubes (0.5 × 0.5 × 0.5 cm3) and used to inoculate 250 mL Erlenmeyer flasks containing 50 mL PDB medium, which were further incubated on a rotary shaker at 28 ℃, 170 rpm for 5 days to generate the seed culture. The scale-up fermentation was carried out with twenty 1000 mL Erlenmeyer flasks, each containing 160 g of rice and 240 mL of distilled water, soaked overnight, followed by being autoclaved at 15 psi for 30 min. Then each flask was inoculated with 12 mL of the seed culture, and grown at 28 ℃ for 40 days.
The fermentation products were extracted exhaustively with EtOAc and were concentrated in vacuo. The crude extracts (27.7 g) were separated by silica gel, Sephadex LH-20 column chromatography and ODS-MPLC [43], to obtain sub-fractions containing the sesquiterpenoids, guided by the molecular networking and LC–MS results. Among these sub-fractions, E1 C7D (89 mg) was further separated by preparative-HPLC running with Eclipse XDB-C8 (9.4 × 250 mm) column at a flow rate of 3.0 mL/min eluting with the following gradient: 0 min, 50% CH3CN-H2O; 30 min 50% CH3CN; 60 min 70% CH3CN to obtain 7 (1.5 mg, tR = 13.9 min). E6B was seperated by ODS-MPLC using gradient elution from 50 to 100% CH3OH-H2O for 170 min to obtain eleven fractions (E6B1–E6B11). E6B9 was purified on a Sephadex LH-20 column using CH3OH to obtain compound 4 (1.0 mg). E6B7-2 (100 mg) was purified by preparative RP-HPLC using an Eclipse XDB-C8 (9.4 × 250 mm) column eluting at a flow rate of 3.0 mL/min using a gradient elution: 0 min, 20% CH3CN–H2O; 30 min 20% CH3CN–H2O; 42 min 23% CH3CN–H2O to obtain 1 (9.5 mg, tR = 40.1 min). E6B7–10 (145 mg) was purified by preparative RP-HPLC using an ZORBAX RX-C8 (9.4 × 250 mm) column eluting at a flow rate of 3.0 mL/min using a gradient elution: 0 min, 25% CH3CN–H2O; 26 min 34% CH3CN-H2O to obtain 2 (22.0 mg, tR = 21.4 min). E6B7–8 (142 mg) was purified by preparative RP-HPLC using an ZORBAX RX-C8 (9.4 × 250 mm) column eluting at a flow rate of 3.0 mL/min using a gradient elution: 0 min, 20% CH3CN–H2O; 24 min 36% CH3CN–H2O to obtain 3 (26.2 mg, tR = 16.6 min). E2 C6 (470 mg) was purified by preparative RP-HPLC using an Phenomenex Luna C18 column (5 μm, 9.4 × 250 mm) eluting at a flow rate of 4.0 mL/min using a gradient elution: 0 min, 60% CH3OH–H2O; 25 min 95% CH3OH–H2O to obtain 5 (1.0 mg, tR = 18.3 min). E2D3 (349 mg) was purified by preparative RP-HPLC using an Eclipse XDB-C3 column (5 μm, 9.4 × 250 mm) eluting at a flow rate of 3.0 mL/min using a gradient elution: 0 min, 40% CH3CN–H2O; 25 min 60% CH3OH–H2O to obtain 6 (2.1 mg, tR = 21.3 min). Fraction E3 was fractionated on a Sephadex LH-20 column using CH2Cl2–CH3OH (1:1) to give 33 fractions (1–33). Sub-fraction E3–24 (150 mg) was purified by preparative RP-HPLC using an ZORBAX RX-C8 (9.4 × 250 mm) column eluting at a flow rate of 3.0 mL/min using a gradient elution: 0 min, 35% CH3CN–H2O; 50 min 45% CH3CN–H2O to obtain 8 (9.9 mg, tR = 30.1 min) and 9 (4.4 mg, tR = 40.0 min). E3D4 (190 mg) was purified by preparative RP-HPLC using an Phenomenex Luna C18 column (5 μm, 9.4 × 250 mm) eluting at a flow rate of 4.0 mL/min using a gradient elution: 0 min, 50% CH3OH–H2O; 25 min 80% CH3OH-H2O to obtain 10 (2.2 mg, tR = 20.0 min).
4.5 Quantum chemical computation details of ECD and 13C NMR spectra
Calculations were performed using the density functional theory (DFT) as carried out in Gaussian 03 [44], with the methods described in previous study [42, 45].
4.6 In vitro anti-inflammatory assay
The in vitro anti-inflammatory assay followed the methods of Yang et al. [46].
Notes
Acknowledgements
We are grateful for the funding support from the National Key Research and Development Program of China (2019YFA0906200 to X.L., and 2020YFA0907200 to J.Z.); the the Shanghai Sci-Tech Inno Center for Infection & Immunity (Grant No. SSIII-2024 A02 to G.Z.); the National Natural Science Foundation of China (31430002, 31720103901, and 32121005 to L.Z.; 21977029 to X.L.); Shanghai Rising-Star Program (20QA1402800) to J.Z.; the Open Project Funding of the State Key Laboratory of Bioreactor Engineering, the 111 Project (B18022); Shanghai Science and Technology Commission (18 JC1411900); and the Shandong Taishan Scholar Program of China to L.Z.; and the Natural Science and Engineering Research Council of Canada to T. H.
Author contributions
Lixin Zhang and Xueting Liu designed this study. Jingyu Zhang, Ronald J Quinn, Guoliang Zhu and Jianying Han arranged the research consortium. Qiang Yin, Jianying Han, Jingyu Zhang, Keke Zou, Kangjie Lv, Zexu Lin, Zhijun Song and Guixiang Yang performed fermentation, compound purification, and structural elucidation. Tom Hsiang sequenced and assembled genomic data and revised the manuscript. Lei Ma and Miaomiao Liu analyzed the biological data. Jianying Han, Jingyu Zhang, Guoliang Zhu and Xueting Liu drafted this manuscript. All authors read and approved the final manuscript.
Funding
This study was funded by National Key Research and Development Program of China (2019YFA0906200, 2020YFA0907200); National Natural Science Foundation of China (31430002, 31720103901, 32121005, 21977029); Shanghai Rising-Star Program (20QA1402800); Open Project Funding of the State Key Laboratory of Bioreactor Engineering (B18022); Shanghai Science and Technology Commission (18 JC1411900); Shanghai Sci-Tech Inno Center for Infection & Immunity (SSIII-2024 A02).
Availability of data and materials
Data will be made available on request.
Declarations
Competing interests
All authors declare no competing financial interests.
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