Note pubs.acs.org/jnp
Endophytic Diaporthe sp. LG23 Produces a Potent Antibacterial Tetracyclic Triterpenoid Gang Li,† Souvik Kusari,*,† Parijat Kusari,‡ Oliver Kayser,‡ and Michael Spiteller*,† †
Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, D-44221 Dortmund, Germany ‡ Department of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, TU Dortmund, Emil-Figge-Straße 66, D-44227 Dortmund, Germany S Supporting Information *
ABSTRACT: A new lanostanoid, 19-nor-lanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1), characterized by the presence of an aromatic B ring and hydroxylated at C-1, C-3, C-12, and C-22, was isolated from an endophytic fungus, Diaporthe sp. LG23, inhabiting leaves of the Chinese medicinal plant Mahonia fortunei. Six biosynthetically related known steroids were also isolated in parallel. Their structures were confirmed on the basis of detailed spectroscopic analysis in conjunction with the published data. Compound 1, an unusual fungus-derived 19-nor-lanostane tetracyclic triterpenoid with an aromatic B-ring system, exhibited pronounced antibacterial efficacy against both Gram-positive and -negative bacteria, especially the clinical isolates of Streptococcus pyogenes and Pseudomonas aeruginosa as well as a human pathogenic strain of Staphylococcus aureus. Our results reveal the potential of endophytes not only in conferring host fitness but also in contributing toward traditional host plant medicines.
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best of our knowledge, compound 1 is a rare fungal tetracyclic triterpenoid with an aromatic ring B system and is an unusual example in that the 19-methyl is “lost” during the aromatization of the B ring of the lanosterol skeleton. Similarly, the rearrangement for the 19-methyl moiety can be found in daedaleanic acid A and daedaleaside A, possessing a 4,5-secoring A.17 Furthermore, taking cues from the traditional use of M. fortunei in TCM,5 we evaluated the antibacterial efficacies of the isolated isoprenoid-derived compounds (1−7) against clinically important Gram-positive and -negative pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, as well as the soil-dwelling bacterium Bacillus subtilis. Compound 1 was isolated as a white powder, with the molecular formula C29H44O4 (eight double-bond equivalents) as determined from the ESIHRMS information ([M + H − H2O]+ at m/z 439.3207, [2M + H]+ at m/z 913.6559, and [3M + H]+ at m/z 1369.9796, Figure S7, Supporting Information). The mass spectrum (Figure S7, Supporting Information) shows the loss of four water molecules, revealing the possible presence of four hydroxy groups in 1, which was also supported by the proposed fragmentation pathway (Figure S8, Supporting
ndophytes are well known as a rich resource of functional biomolecules.1−3 The repertoire of their biosynthetic capacities encompassing a plethora of chemical scaffolds is a result of their evolutionary adaptation to their microenvironments inside host plants.2 In our continuing search for biologically active secondary metabolites from endophytic fungi harbored in traditionally used medicinal plants,4 we investigated the medicinal plant Mahonia fortunei (Berberidaceae) endemic to People’s Republic of China. M. fortunei (Chinese name “Shi da gong lao”) is a heat-tolerant, pestresistant plant that has been used in Traditional Chinese Medicine (TCM) for treating pneumoconiosis, psoriasis, and coughs, in addition to being a potent antimicrobial medicinal resource.5 We isolated an endophytic fungus, Diaporthe sp. LG23, inhabiting leaves of M. fortunei prospected from Shanghai, People’s Republic of China. Herein we report the isolation and structural elucidation of a new lanosterol derivative, 19-norlanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1), as well as six biosynthetically related known ergosterol derivatives,6−8 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (2),9 3β,5α,9α,14α-tetrahydroxy-(22E,24R)-ergosta-7,22-dien-6-one (3), 10 (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6α-triol (4),11,12 chaxine C (5),13 demethylincisterol A3 (6),14,15 and volemolide (7),16 from endophytic Diaporthe sp. LG23. To the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 20, 2015
A
DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Information) based on MS2 data.18,19 The 1H and 13C NMR spectra (Table 1) of 1 showed the presence of six tertiary Table 1. NMR Spectral Data for Compound 1 position 1 2 3 4 5 6 7 8 9 10 11α 11β 12 13 14 15α 15β 16α 16β 17 18 20 21 22 23 24 25 26 27 28 29 30
δC, mult.a
δH mult.b (J in Hz)
65.7, 37.0, 70.7, 39.7, 142.5, 124.5, 125.5, 144.1, 134.6, 132.2, 34.1,
CH CH2 CH qC qC CH CH qC qC qC CH2
5.04 t (2.5) 2.13 m 4.15 dd (4.0, 12.0)
2 1, 3 2
7.21 d (8.0) 7.01 d (8.0)
7 6
4, 8, 10 5, 9, 14
12
71.8, 49.6, 53.2, 32.4,
CH qC qC CH2
3.63 dd (8.0, 18.0) 2.69 dd (8.0, 18.0) 4.41 t (8.0)
8, 9, 12, 13 9, 10, 12 17, 18
28.3, CH2 44.6, 11.4, 41.0, 15.3, 75.0, 32.9, 120.4, 136.0, 18.1, 26.0, 27.1, 23.7, 28.0,
CH CH3 CH CH3 CH CH2 CH qC CH3 CH3 CH3 CH3 CH3
1.75 1.97 2.07 1.81 2.41 0.70 1.86 1.16 3.75 2.25 5.21
mc mc mc mc dt (6.5, 9.0) s m d (7.0) dt (5.0, 7.5) dd (7.5, 8.0) t (8.0)
1.70 1.78 1.46 1.14 1.10
s s s s s
COSY
11
HMBC
16α 16 15α, 17 17 16, 20 17, 20 20, 22, 23,
21, 22 23 24 26, 27
18 12, 13, 17, 21, 22, 26,
13, 17, 20, 24 24, 27
14, 17 21, 22 22
Figure 1. Chemical structures of compounds 1−7. 25
24, 25, 27 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28 8, 13, 14, 15
a
Recorded in CDCl3 at 125 MHz; 13C multiplicities were determined by HSQC experiment. bRecorded in CDCl3 at 500 MHz. cSignals overlapped.
Figure 2. Key HMBC correlations of compound 1.
9, and C-13. A benzene ring substructure formed in the tetracyclic skeleton of 1 was verified and located at the B ring by the HMBC correlations of H-6/C-8, H-6/C-10, H-7/C-5, H-7/C-9, and H-11/C-10 (Figure 2). HMBC correlations of H3-28 to C-3, C-4, C-5, and C-29, H3-29 to C-3, C-4, C-5, and C-28, and H-6 to C-4 coupled with the MS requirement indicated an A ring fusing to a B ring. Four hydroxy groups were located at C-1, C-3, C-12, and C-22, which were consistent with the chemical shifts of oxygenated methines and MS fragment information. The relative stereochemistry of 1 was characterized by analysis of the coupling constants and NOESY experiment (Table 1 and Figure 3). In the NOESY spectrum, the correlations of H3-28/H-3 and H3-29/H-2 and the coupling constants of H2-2/H-1 (J = 2.5) and H2-2/H-3 (J = 4.0, 12.0), as well as the absence of the NOESY correlation between H-1 and H-3, verified that two hydroxy groups at C-1 and C-3 in the A ring should be α and β configured, respectively.23 The strong NOESY correlations of H-1/H-11β (δH 2.69), H3-18/H-11β and H3-18/H-15β (δH 1.97), and H3-18/H-16β (δH 1.81) were indicative of their being in β-orientations. Moreover, H-12 and H3-30 were located on opposite sides based on their cross
methyls (δH 0.70, 1.10, 1.14, 1.46, 1.70, and 1.78), one secondary methyl (δH 1.16, d, J = 7.0 Hz), and four oxygenated methines (δC 65.7, 70.7, 71.8, and 75.0), as revealed by MS analysis. In addition to these structural characteristics, the 1D NMR data coupled with 2D NMR spectra determined five methylenes, two deoxygenated methines, three quaternary carbons, and eight aromatic/olefinic carbons including a cisdisubstituted double bond and a methine carbon. These data accounted for all 1H and 13C NMR resonances and indicated a tetracyclic triterpene skeleton of 1.20−22 Interpretation of 1 H−1H COSY NMR data identified the connection from C-1 to C-3, from C-6 to C-7, from C-11 to C-12, between C-15, 16, 17, 20, 22, 23, and 24, and from C-20 to C-21 (Figure 2). The detailed planar structure of 1 was further constructed by the HMBC correlations (Figure 2). In 1, the side chain with the connection to C-17 was confirmed from key HMBC correlations of H3-21 to C-17, C-20, and C-22, H3-26 to C24, C-25, and C-27, and H3-27 to C-24, C-25, and C-26. The C and D rings of the tetracyclic skeleton were deduced based on the HMBC correlations from H3-18 to C-12, C-13, C-14, and C-17, H3-30 to C-8, C-13, C-14, and C-15, and H-11 to C-8, CB
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cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol from a plant) with an aromatized B ring.24,28 Given the extensive use of the host plant M. fortunei in TCM,5 we embarked on a traditional knowledge-based dereplication of the functional roles of compounds 1−7 produced by the endophytic fungus Diaporthe sp. LG23 isolated from leaves of this plant. Our focus was to correlate the traditional usage of the plant in TCM to the bioactivity of the actual isolated compounds produced by the endophytic fungus LG23 residing inside the plant. Therefore, we evaluated the antibacterial efficacies of the biosynthetically related compounds 1−7 against the clinical risk-group 2 (RG2) human pathogens. We used both Gram-positive bacteria (S. aureus and S. pyogenes) and Gram-negative bacteria (E. coli and P. aeruginosa). We further included the Gram-positive soil bacterium B. subtilis. Compound 1 revealed pronounced antibacterial efficacy against all the tested organisms, especially against the clinical isolates S. pyogenes and P. aeruginosa (Table 2). Our results, therefore, indicate that 1 is a potent antibacterial lead compound. Compounds 2 and 5 were also active against B. subtilis, having a similar efficacy to that of the reference standard streptomycin. Our results provide further evidence that certain endophytes are inclined to demonstrate host fitness and biocontrol potency using their arsenal of bioactive secondary metabolites.29,30 Microorganisms rarely occur in axenic conditions in nature; endophytes are no exception. Endophytic microorganisms typically coevolve a plethora of traits in order to survive and function in their distinct ecological niches.3 These traits emerge as a result of multifaceted multispecies interaction of endophytes with associated organisms (other endophytes, invading generalist and specific phytopathogens, invading pests and parasites, as well as the host plant) and range from production of antimicrobial chemical defense compounds to triggers for activating cryptic biosynthetic pathways, production of precursors, quorum sensing molecules, epigenetic modulators, and even direct physical organismal interactions.30−32 All these chemical and molecular interactions among multiple organisms are difficult to study under in vitro conditions in the laboratory. For instance, in vitro culture conditions are mostly different from the in planta apoplast environment in which endophytes reside, and therefore, there is no direct means for quantitative estimation of the production of a compound by an endophyte in vitro and in planta.31 Furthermore, the in planta metabolic activity of an endophyte is dictated by interactions with coexisting organisms. Therefore, endophytes are known to reduce or stop production of certain compounds over repeated subculturing, mostly due to loss of suitable in planta triggers.3 Thus far, our present results demonstrate the potential of endophytic Diaporthe sp. LG23 in aiding host plant defense against invading pathogens by producing antibacterial com-
Figure 3. Key NOESY correlations of compound 1.
correlations. The key NOE correlations between H3-30/H-17, H-12/H-17, and H3-18/H-20 indicated the β-orientation of the side chain and the S configuration of C-20,24 which were consistent with general characteristics for the naturally occurring lanostane-type triterpenoids.20,21 From a biosynthetic standpoint, compounds having structural features similar to 1 (lanostane triterpenoids) are derived from lanosterol (Figure S10, Supporting Information).6,7,20 The conversion of lanosterol into steroid derivatives in fungi has been investigated and reviewed (Figure S11, Supporting Information),6,7 which shows their general configurations as shown in compounds 1−7. The 22S absolute configuration of 1 in the side chain was supported by the relatively small coupling constant of H-20/H-22 (J = 5.0) (details in Figure S12 in the Supporting Information),25 together with the significant differences of 13C NMR values of the same side chain between 1 and fungus-derived 22R inotodiol (Figure S13, Supporting Information).26,27 Therefore, compound 1 was assigned as 19-nor-lanosta-5(10),6,8,24tetraene-1α,3β,12β,22S-tetraol. Furthermore, we succeeded in purifying and identifying six biosynthetically related compounds, viz., 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien6-one (2),9 3β,5α,9α,14α-tetrahydroxy-(22E,24R)-ergosta-7,22dien-6-one (3), 10 (22E,24R)-ergosta-7,9(11),22-triene3β,5α,6α-triol (4),11,12 chaxine C (5),13 demethylincisterol A3 (6), and volemolide (7).14−16 Compound 1 is derived from the common biosynthetic intermediate lanosterol, which is biosynthesized via cyclization of 2,3-oxidosqualene in a chair−boat−chair conformation followed by sequential backbone rearrangement (two methyl migrations and hydride shifts).6,7,20 Subsequent aromatization of the B ring of the lanosterol skeleton may be responsible for the loss of Me-19, followed by hydroxylation at C-1, C-12, and C-22 to give rise to 1 (Figure S10, Supporting Information). It should be noted that compound 1 represents a rare fungusderived 19-nor tetracyclic triterpene with an aromatic B ring. Moreover, to the best of our knowledge, there is only one more reported natural lanostane/cucurbitane derivative (19-nor-
Table 2. Minimum Inhibitory Concentrations (MIC) of the Compounds 1−7 against Gram-Positive and Gram-Negative Bacteria Compared to Standard References (Streptomycin and Gentamicin)a
a
organism (DSMZ no.)
1
2
3
4
5
6
7
streptomycin
gentamicin
Staphylococcus aureus (DSM 799) Escherichia coli (DSM 682) Bacillus subtilis (DSM 1088) Pseudomonas aeruginosa (DSM 22644) Streptococcus pyogenes (DSM 11728)
5.0 5.0 2.0 2.0 0.1
>10 >10 5.0 >10 >10
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
1 >10 5.0 >10 >10
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
5.0 1.0 5.0 10.0 >10
1.0 1.0 1.0 1.0 10.0
All values are in μg/mL and derived from experiments in triplicate. C
DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
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in H2O/0.1% formic acid] λmax 222 nm; IR (solid) νmax 3352, 2929, 1638, 1454, 1413, 1379, 1100, 1033 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table 1; positive ESIHRMS m/z 439.3207 [M + H − H2O]+ (calcd for C29H43O3, 439.3207), [2M + H]+ at m/z 913.6559 (calcd for C58H89O8, 913.6552). Antibacterial Assay. The in vitro antibacterial activities of compounds 1−7 were tested against a panel of standard pathogenic control strains (obtained from Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using our previously described method.4
pounds. Admittedly, a further detailed investigation is warranted to examine the endophyte’s in planta metabolic processes to exemplify its true worth in host-plant-derived TCM.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were carried out on an A-Krüss Optronic polarimeter. IR spectra were measured using a Bruker IR spectrometer. NMR spectra were measured on a Bruker DRX-500 spectrometer operating at 500 (1H) and 125 (13C) MHz or a Bruker DRX-400 spectrometer operating at 400 (1H) and 100 (13C) MHz with tetramethylsilane (TMS) as internal standard. MS spectra were obtained with a LTQ-Orbitrap spectrometer (Thermo Fisher, USA) equipped with an ESI source. The spectrometer was equipped with an Agilent 1200 HPLC system. Preparative HPLC was performed on a Gynkotek pump equipped with a Dionex DG-1210 degasser, a Dionex UVD 340S detector, a Dionex Gina 50 autosampler, and a Gemini column (10 × 250 mm, 10 μm). Silica gel 60 (70−230 mesh; AppliChem, GmbH, Darmstadt, Germany) and Sephadex LH-20 (25−100 μm; Amersham Biosciences) were applied for column chromatography. Thin-layer chromatography (TLC) was carried out with glass precoated silica gel 60 plates (0.25 mm; Merck, Darmstadt, Germany). Fungal Material. The endophytic fungus Diaporthe sp. LG23 was isolated from the leaves of M. fortunei collected from Shanghai, People’s Republic of China, following a previously established procedure.33 For identification, the fungal strain was cultured on potato dextrose agar (PDA) at 28 ± 2 °C for 1 week in an incubator. The fungus was identified by ITS sequencing following our previously established method,34 suitably modified (see S15, Supporting Information). The ITS sequence of the identified endophytic fungus has been deposited at the EMBL-Bank (accession number LN552209). Finally, agar plugs from a week-old culture plate (grown on PDA at 28 ± 2 °C) were cut into small pieces under aseptic conditions, and 40 pieces were used to inoculate 20 flasks (1 L) each containing 80 g of rice, 120 mL of water, and 0.3% peptone. The cultures were incubated at room temperature for two months. Extraction and Isolation. The fermented material was extracted with ethyl acetate (EtOAc) by sonication at room temperature. The organic solvent was pooled and evaporated under reduced pressure to afford the crude extract (20 g). The extract was fractionated by column chromatography (CC) on silica gel, eluting with a gradient of cyclohexane−EtOAc from 100:0 to 0:100 (v/v). All the fractions were combined to give six major fractions (A−F) on the basis of TLC and LC-ESIHRMS analyses. While progressing with isolation, nearly all the subfractions were measured by LC-ESIHRMS to check their composition. Fraction B (1.2 g) was applied to the Sephadex LH-20 column (MeOH) to yield four subfractions (B1−B4). Subfraction B2 (310 mg) was purified by semipreparative HPLC (Venusil XBP C18 column, 10 × 250 mm; MeOH−H2O−0.1% FA, 90/10, 2.0 mL/min) to afford chaxine C (5, 1.7 mg, tR = 24.5 min), demethylincisterol A3 (6, 1.3 mg, tR = 18.0 min), and volemolide (7, 9.1 mg, tR = 29.5 min). After separation of fraction C (2.3 g) using a Sephadex LH-20 column (MeOH) to yield four subfractions (C1−C4), subfraction C2 was further separated by silica gel CC with cyclohexane−EtOAc into four subfractions (C21−C24). Subfraction C23 was fractioned by Sephadex LH-20 column chromatography using MeOH as eluent, followed by SPE column under vacuum to give the target subfraction C2322. This fraction was then separated by HPLC (MeOH−H2O, 90/10, 2.0 mL/ min) to obtain 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6one (2, 9.5 mg, tR = 17.5 min), 3β,5α,9α,14α-tetrahydroxy(22E,24R)-ergosta-7,22-dien-6-one (3, 1.1 mg, tR = 15.5 min), and (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6α-triol (4, 0.2 mg, tR = 23.0 min). Fraction F (1.5 g) was subjected to silica gel CC, followed by a Sephadex LH-20 column (MeOH), to give subfractions F21 and F33. Subfraction F21 (25 mg) was purified by HPLC (MeOH−H2O, 80/20, 2.0 mL/min) to afford 1 (2.4 mg, tR = 28.0 min). 19-Nor-lanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1): white powder; [α]20D +18.3 (c 0.24, MeOH); LC-UV [acetonitrile(aq)
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ASSOCIATED CONTENT
* Supporting Information S
Spectral data of compound 1, its proposed biosynthetic pathway, and protocol for identification of the endophytic fungus. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00170.
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49-(0)231-755-4086. Fax: +49-(0)231-755-4084. Email: [email protected]. *Tel: +49-(0)231-755-4080. Fax: +49-(0)231-755-4085. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the Ministry of Innovation, Science, Research and Technology of the State of North RhineWestphalia, Germany, and the German Research Foundation (DFG) for funding a high-resolution mass spectrometer. G.L. gratefully acknowledges the China Scholarship Council (CSC) for a doctoral fellowship. We thankfully acknowledge Dr. S. Zühlke (INFU, TU Dortmund) for discussions and critically reviewing our manuscript and Dr. F. M. Talontsi (INFU, TU Dortmund) for discussions. We thankfully acknowledge Dr. Wolf Hiller (Department of Chemistry and Chemical Biology, TU Dortmund) for realization of the NMR measurements.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
Endophytic Diaporthe sp. LG23 Produces a Potent Antibacterial Tetracyclic Triterpenoid Gang Li,† Souvik Kusari,*,† Parijat Kusari,‡ Oliver Kayser,‡ and Michael Spiteller*,† †
Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, D-44221 Dortmund, Germany ‡ Department of Biochemical and Chemical Engineering, Chair of Technical Biochemistry, TU Dortmund, Emil-Figge-Straße 66, D-44227 Dortmund, Germany S Supporting Information *
ABSTRACT: A new lanostanoid, 19-nor-lanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1), characterized by the presence of an aromatic B ring and hydroxylated at C-1, C-3, C-12, and C-22, was isolated from an endophytic fungus, Diaporthe sp. LG23, inhabiting leaves of the Chinese medicinal plant Mahonia fortunei. Six biosynthetically related known steroids were also isolated in parallel. Their structures were confirmed on the basis of detailed spectroscopic analysis in conjunction with the published data. Compound 1, an unusual fungus-derived 19-nor-lanostane tetracyclic triterpenoid with an aromatic B-ring system, exhibited pronounced antibacterial efficacy against both Gram-positive and -negative bacteria, especially the clinical isolates of Streptococcus pyogenes and Pseudomonas aeruginosa as well as a human pathogenic strain of Staphylococcus aureus. Our results reveal the potential of endophytes not only in conferring host fitness but also in contributing toward traditional host plant medicines.
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best of our knowledge, compound 1 is a rare fungal tetracyclic triterpenoid with an aromatic ring B system and is an unusual example in that the 19-methyl is “lost” during the aromatization of the B ring of the lanosterol skeleton. Similarly, the rearrangement for the 19-methyl moiety can be found in daedaleanic acid A and daedaleaside A, possessing a 4,5-secoring A.17 Furthermore, taking cues from the traditional use of M. fortunei in TCM,5 we evaluated the antibacterial efficacies of the isolated isoprenoid-derived compounds (1−7) against clinically important Gram-positive and -negative pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, as well as the soil-dwelling bacterium Bacillus subtilis. Compound 1 was isolated as a white powder, with the molecular formula C29H44O4 (eight double-bond equivalents) as determined from the ESIHRMS information ([M + H − H2O]+ at m/z 439.3207, [2M + H]+ at m/z 913.6559, and [3M + H]+ at m/z 1369.9796, Figure S7, Supporting Information). The mass spectrum (Figure S7, Supporting Information) shows the loss of four water molecules, revealing the possible presence of four hydroxy groups in 1, which was also supported by the proposed fragmentation pathway (Figure S8, Supporting
ndophytes are well known as a rich resource of functional biomolecules.1−3 The repertoire of their biosynthetic capacities encompassing a plethora of chemical scaffolds is a result of their evolutionary adaptation to their microenvironments inside host plants.2 In our continuing search for biologically active secondary metabolites from endophytic fungi harbored in traditionally used medicinal plants,4 we investigated the medicinal plant Mahonia fortunei (Berberidaceae) endemic to People’s Republic of China. M. fortunei (Chinese name “Shi da gong lao”) is a heat-tolerant, pestresistant plant that has been used in Traditional Chinese Medicine (TCM) for treating pneumoconiosis, psoriasis, and coughs, in addition to being a potent antimicrobial medicinal resource.5 We isolated an endophytic fungus, Diaporthe sp. LG23, inhabiting leaves of M. fortunei prospected from Shanghai, People’s Republic of China. Herein we report the isolation and structural elucidation of a new lanosterol derivative, 19-norlanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1), as well as six biosynthetically related known ergosterol derivatives,6−8 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (2),9 3β,5α,9α,14α-tetrahydroxy-(22E,24R)-ergosta-7,22-dien-6-one (3), 10 (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6α-triol (4),11,12 chaxine C (5),13 demethylincisterol A3 (6),14,15 and volemolide (7),16 from endophytic Diaporthe sp. LG23. To the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 20, 2015
A
DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Information) based on MS2 data.18,19 The 1H and 13C NMR spectra (Table 1) of 1 showed the presence of six tertiary Table 1. NMR Spectral Data for Compound 1 position 1 2 3 4 5 6 7 8 9 10 11α 11β 12 13 14 15α 15β 16α 16β 17 18 20 21 22 23 24 25 26 27 28 29 30
δC, mult.a
δH mult.b (J in Hz)
65.7, 37.0, 70.7, 39.7, 142.5, 124.5, 125.5, 144.1, 134.6, 132.2, 34.1,
CH CH2 CH qC qC CH CH qC qC qC CH2
5.04 t (2.5) 2.13 m 4.15 dd (4.0, 12.0)
2 1, 3 2
7.21 d (8.0) 7.01 d (8.0)
7 6
4, 8, 10 5, 9, 14
12
71.8, 49.6, 53.2, 32.4,
CH qC qC CH2
3.63 dd (8.0, 18.0) 2.69 dd (8.0, 18.0) 4.41 t (8.0)
8, 9, 12, 13 9, 10, 12 17, 18
28.3, CH2 44.6, 11.4, 41.0, 15.3, 75.0, 32.9, 120.4, 136.0, 18.1, 26.0, 27.1, 23.7, 28.0,
CH CH3 CH CH3 CH CH2 CH qC CH3 CH3 CH3 CH3 CH3
1.75 1.97 2.07 1.81 2.41 0.70 1.86 1.16 3.75 2.25 5.21
mc mc mc mc dt (6.5, 9.0) s m d (7.0) dt (5.0, 7.5) dd (7.5, 8.0) t (8.0)
1.70 1.78 1.46 1.14 1.10
s s s s s
COSY
11
HMBC
16α 16 15α, 17 17 16, 20 17, 20 20, 22, 23,
21, 22 23 24 26, 27
18 12, 13, 17, 21, 22, 26,
13, 17, 20, 24 24, 27
14, 17 21, 22 22
Figure 1. Chemical structures of compounds 1−7. 25
24, 25, 27 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28 8, 13, 14, 15
a
Recorded in CDCl3 at 125 MHz; 13C multiplicities were determined by HSQC experiment. bRecorded in CDCl3 at 500 MHz. cSignals overlapped.
Figure 2. Key HMBC correlations of compound 1.
9, and C-13. A benzene ring substructure formed in the tetracyclic skeleton of 1 was verified and located at the B ring by the HMBC correlations of H-6/C-8, H-6/C-10, H-7/C-5, H-7/C-9, and H-11/C-10 (Figure 2). HMBC correlations of H3-28 to C-3, C-4, C-5, and C-29, H3-29 to C-3, C-4, C-5, and C-28, and H-6 to C-4 coupled with the MS requirement indicated an A ring fusing to a B ring. Four hydroxy groups were located at C-1, C-3, C-12, and C-22, which were consistent with the chemical shifts of oxygenated methines and MS fragment information. The relative stereochemistry of 1 was characterized by analysis of the coupling constants and NOESY experiment (Table 1 and Figure 3). In the NOESY spectrum, the correlations of H3-28/H-3 and H3-29/H-2 and the coupling constants of H2-2/H-1 (J = 2.5) and H2-2/H-3 (J = 4.0, 12.0), as well as the absence of the NOESY correlation between H-1 and H-3, verified that two hydroxy groups at C-1 and C-3 in the A ring should be α and β configured, respectively.23 The strong NOESY correlations of H-1/H-11β (δH 2.69), H3-18/H-11β and H3-18/H-15β (δH 1.97), and H3-18/H-16β (δH 1.81) were indicative of their being in β-orientations. Moreover, H-12 and H3-30 were located on opposite sides based on their cross
methyls (δH 0.70, 1.10, 1.14, 1.46, 1.70, and 1.78), one secondary methyl (δH 1.16, d, J = 7.0 Hz), and four oxygenated methines (δC 65.7, 70.7, 71.8, and 75.0), as revealed by MS analysis. In addition to these structural characteristics, the 1D NMR data coupled with 2D NMR spectra determined five methylenes, two deoxygenated methines, three quaternary carbons, and eight aromatic/olefinic carbons including a cisdisubstituted double bond and a methine carbon. These data accounted for all 1H and 13C NMR resonances and indicated a tetracyclic triterpene skeleton of 1.20−22 Interpretation of 1 H−1H COSY NMR data identified the connection from C-1 to C-3, from C-6 to C-7, from C-11 to C-12, between C-15, 16, 17, 20, 22, 23, and 24, and from C-20 to C-21 (Figure 2). The detailed planar structure of 1 was further constructed by the HMBC correlations (Figure 2). In 1, the side chain with the connection to C-17 was confirmed from key HMBC correlations of H3-21 to C-17, C-20, and C-22, H3-26 to C24, C-25, and C-27, and H3-27 to C-24, C-25, and C-26. The C and D rings of the tetracyclic skeleton were deduced based on the HMBC correlations from H3-18 to C-12, C-13, C-14, and C-17, H3-30 to C-8, C-13, C-14, and C-15, and H-11 to C-8, CB
DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
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cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol from a plant) with an aromatized B ring.24,28 Given the extensive use of the host plant M. fortunei in TCM,5 we embarked on a traditional knowledge-based dereplication of the functional roles of compounds 1−7 produced by the endophytic fungus Diaporthe sp. LG23 isolated from leaves of this plant. Our focus was to correlate the traditional usage of the plant in TCM to the bioactivity of the actual isolated compounds produced by the endophytic fungus LG23 residing inside the plant. Therefore, we evaluated the antibacterial efficacies of the biosynthetically related compounds 1−7 against the clinical risk-group 2 (RG2) human pathogens. We used both Gram-positive bacteria (S. aureus and S. pyogenes) and Gram-negative bacteria (E. coli and P. aeruginosa). We further included the Gram-positive soil bacterium B. subtilis. Compound 1 revealed pronounced antibacterial efficacy against all the tested organisms, especially against the clinical isolates S. pyogenes and P. aeruginosa (Table 2). Our results, therefore, indicate that 1 is a potent antibacterial lead compound. Compounds 2 and 5 were also active against B. subtilis, having a similar efficacy to that of the reference standard streptomycin. Our results provide further evidence that certain endophytes are inclined to demonstrate host fitness and biocontrol potency using their arsenal of bioactive secondary metabolites.29,30 Microorganisms rarely occur in axenic conditions in nature; endophytes are no exception. Endophytic microorganisms typically coevolve a plethora of traits in order to survive and function in their distinct ecological niches.3 These traits emerge as a result of multifaceted multispecies interaction of endophytes with associated organisms (other endophytes, invading generalist and specific phytopathogens, invading pests and parasites, as well as the host plant) and range from production of antimicrobial chemical defense compounds to triggers for activating cryptic biosynthetic pathways, production of precursors, quorum sensing molecules, epigenetic modulators, and even direct physical organismal interactions.30−32 All these chemical and molecular interactions among multiple organisms are difficult to study under in vitro conditions in the laboratory. For instance, in vitro culture conditions are mostly different from the in planta apoplast environment in which endophytes reside, and therefore, there is no direct means for quantitative estimation of the production of a compound by an endophyte in vitro and in planta.31 Furthermore, the in planta metabolic activity of an endophyte is dictated by interactions with coexisting organisms. Therefore, endophytes are known to reduce or stop production of certain compounds over repeated subculturing, mostly due to loss of suitable in planta triggers.3 Thus far, our present results demonstrate the potential of endophytic Diaporthe sp. LG23 in aiding host plant defense against invading pathogens by producing antibacterial com-
Figure 3. Key NOESY correlations of compound 1.
correlations. The key NOE correlations between H3-30/H-17, H-12/H-17, and H3-18/H-20 indicated the β-orientation of the side chain and the S configuration of C-20,24 which were consistent with general characteristics for the naturally occurring lanostane-type triterpenoids.20,21 From a biosynthetic standpoint, compounds having structural features similar to 1 (lanostane triterpenoids) are derived from lanosterol (Figure S10, Supporting Information).6,7,20 The conversion of lanosterol into steroid derivatives in fungi has been investigated and reviewed (Figure S11, Supporting Information),6,7 which shows their general configurations as shown in compounds 1−7. The 22S absolute configuration of 1 in the side chain was supported by the relatively small coupling constant of H-20/H-22 (J = 5.0) (details in Figure S12 in the Supporting Information),25 together with the significant differences of 13C NMR values of the same side chain between 1 and fungus-derived 22R inotodiol (Figure S13, Supporting Information).26,27 Therefore, compound 1 was assigned as 19-nor-lanosta-5(10),6,8,24tetraene-1α,3β,12β,22S-tetraol. Furthermore, we succeeded in purifying and identifying six biosynthetically related compounds, viz., 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien6-one (2),9 3β,5α,9α,14α-tetrahydroxy-(22E,24R)-ergosta-7,22dien-6-one (3), 10 (22E,24R)-ergosta-7,9(11),22-triene3β,5α,6α-triol (4),11,12 chaxine C (5),13 demethylincisterol A3 (6), and volemolide (7).14−16 Compound 1 is derived from the common biosynthetic intermediate lanosterol, which is biosynthesized via cyclization of 2,3-oxidosqualene in a chair−boat−chair conformation followed by sequential backbone rearrangement (two methyl migrations and hydride shifts).6,7,20 Subsequent aromatization of the B ring of the lanosterol skeleton may be responsible for the loss of Me-19, followed by hydroxylation at C-1, C-12, and C-22 to give rise to 1 (Figure S10, Supporting Information). It should be noted that compound 1 represents a rare fungusderived 19-nor tetracyclic triterpene with an aromatic B ring. Moreover, to the best of our knowledge, there is only one more reported natural lanostane/cucurbitane derivative (19-nor-
Table 2. Minimum Inhibitory Concentrations (MIC) of the Compounds 1−7 against Gram-Positive and Gram-Negative Bacteria Compared to Standard References (Streptomycin and Gentamicin)a
a
organism (DSMZ no.)
1
2
3
4
5
6
7
streptomycin
gentamicin
Staphylococcus aureus (DSM 799) Escherichia coli (DSM 682) Bacillus subtilis (DSM 1088) Pseudomonas aeruginosa (DSM 22644) Streptococcus pyogenes (DSM 11728)
5.0 5.0 2.0 2.0 0.1
>10 >10 5.0 >10 >10
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
1 >10 5.0 >10 >10
>10 >10 >10 >10 >10
>10 >10 >10 >10 >10
5.0 1.0 5.0 10.0 >10
1.0 1.0 1.0 1.0 10.0
All values are in μg/mL and derived from experiments in triplicate. C
DOI: 10.1021/acs.jnatprod.5b00170 J. Nat. Prod. XXXX, XXX, XXX−XXX
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in H2O/0.1% formic acid] λmax 222 nm; IR (solid) νmax 3352, 2929, 1638, 1454, 1413, 1379, 1100, 1033 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table 1; positive ESIHRMS m/z 439.3207 [M + H − H2O]+ (calcd for C29H43O3, 439.3207), [2M + H]+ at m/z 913.6559 (calcd for C58H89O8, 913.6552). Antibacterial Assay. The in vitro antibacterial activities of compounds 1−7 were tested against a panel of standard pathogenic control strains (obtained from Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) using our previously described method.4
pounds. Admittedly, a further detailed investigation is warranted to examine the endophyte’s in planta metabolic processes to exemplify its true worth in host-plant-derived TCM.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were carried out on an A-Krüss Optronic polarimeter. IR spectra were measured using a Bruker IR spectrometer. NMR spectra were measured on a Bruker DRX-500 spectrometer operating at 500 (1H) and 125 (13C) MHz or a Bruker DRX-400 spectrometer operating at 400 (1H) and 100 (13C) MHz with tetramethylsilane (TMS) as internal standard. MS spectra were obtained with a LTQ-Orbitrap spectrometer (Thermo Fisher, USA) equipped with an ESI source. The spectrometer was equipped with an Agilent 1200 HPLC system. Preparative HPLC was performed on a Gynkotek pump equipped with a Dionex DG-1210 degasser, a Dionex UVD 340S detector, a Dionex Gina 50 autosampler, and a Gemini column (10 × 250 mm, 10 μm). Silica gel 60 (70−230 mesh; AppliChem, GmbH, Darmstadt, Germany) and Sephadex LH-20 (25−100 μm; Amersham Biosciences) were applied for column chromatography. Thin-layer chromatography (TLC) was carried out with glass precoated silica gel 60 plates (0.25 mm; Merck, Darmstadt, Germany). Fungal Material. The endophytic fungus Diaporthe sp. LG23 was isolated from the leaves of M. fortunei collected from Shanghai, People’s Republic of China, following a previously established procedure.33 For identification, the fungal strain was cultured on potato dextrose agar (PDA) at 28 ± 2 °C for 1 week in an incubator. The fungus was identified by ITS sequencing following our previously established method,34 suitably modified (see S15, Supporting Information). The ITS sequence of the identified endophytic fungus has been deposited at the EMBL-Bank (accession number LN552209). Finally, agar plugs from a week-old culture plate (grown on PDA at 28 ± 2 °C) were cut into small pieces under aseptic conditions, and 40 pieces were used to inoculate 20 flasks (1 L) each containing 80 g of rice, 120 mL of water, and 0.3% peptone. The cultures were incubated at room temperature for two months. Extraction and Isolation. The fermented material was extracted with ethyl acetate (EtOAc) by sonication at room temperature. The organic solvent was pooled and evaporated under reduced pressure to afford the crude extract (20 g). The extract was fractionated by column chromatography (CC) on silica gel, eluting with a gradient of cyclohexane−EtOAc from 100:0 to 0:100 (v/v). All the fractions were combined to give six major fractions (A−F) on the basis of TLC and LC-ESIHRMS analyses. While progressing with isolation, nearly all the subfractions were measured by LC-ESIHRMS to check their composition. Fraction B (1.2 g) was applied to the Sephadex LH-20 column (MeOH) to yield four subfractions (B1−B4). Subfraction B2 (310 mg) was purified by semipreparative HPLC (Venusil XBP C18 column, 10 × 250 mm; MeOH−H2O−0.1% FA, 90/10, 2.0 mL/min) to afford chaxine C (5, 1.7 mg, tR = 24.5 min), demethylincisterol A3 (6, 1.3 mg, tR = 18.0 min), and volemolide (7, 9.1 mg, tR = 29.5 min). After separation of fraction C (2.3 g) using a Sephadex LH-20 column (MeOH) to yield four subfractions (C1−C4), subfraction C2 was further separated by silica gel CC with cyclohexane−EtOAc into four subfractions (C21−C24). Subfraction C23 was fractioned by Sephadex LH-20 column chromatography using MeOH as eluent, followed by SPE column under vacuum to give the target subfraction C2322. This fraction was then separated by HPLC (MeOH−H2O, 90/10, 2.0 mL/ min) to obtain 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6one (2, 9.5 mg, tR = 17.5 min), 3β,5α,9α,14α-tetrahydroxy(22E,24R)-ergosta-7,22-dien-6-one (3, 1.1 mg, tR = 15.5 min), and (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6α-triol (4, 0.2 mg, tR = 23.0 min). Fraction F (1.5 g) was subjected to silica gel CC, followed by a Sephadex LH-20 column (MeOH), to give subfractions F21 and F33. Subfraction F21 (25 mg) was purified by HPLC (MeOH−H2O, 80/20, 2.0 mL/min) to afford 1 (2.4 mg, tR = 28.0 min). 19-Nor-lanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (1): white powder; [α]20D +18.3 (c 0.24, MeOH); LC-UV [acetonitrile(aq)
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ASSOCIATED CONTENT
* Supporting Information S
Spectral data of compound 1, its proposed biosynthetic pathway, and protocol for identification of the endophytic fungus. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jnatprod.5b00170.
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49-(0)231-755-4086. Fax: +49-(0)231-755-4084. Email: [email protected]. *Tel: +49-(0)231-755-4080. Fax: +49-(0)231-755-4085. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the Ministry of Innovation, Science, Research and Technology of the State of North RhineWestphalia, Germany, and the German Research Foundation (DFG) for funding a high-resolution mass spectrometer. G.L. gratefully acknowledges the China Scholarship Council (CSC) for a doctoral fellowship. We thankfully acknowledge Dr. S. Zühlke (INFU, TU Dortmund) for discussions and critically reviewing our manuscript and Dr. F. M. Talontsi (INFU, TU Dortmund) for discussions. We thankfully acknowledge Dr. Wolf Hiller (Department of Chemistry and Chemical Biology, TU Dortmund) for realization of the NMR measurements.
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REFERENCES
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