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A new aquatic pathogen inhibitor produced by the marine fungus Aspergillus sp. LS116
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A new aquatic pathogen inhibitor produced by the marine fungus Aspergillus sp. LS116 Peng Xu, Lijian Ding∗, Jiaxin Wei, Qiang Li, Minjie Gui, Xiaoping He, Dengquan Su, Shan He, Haixiao Jin∗∗ Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315832, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Marine fungi Vibrio harveyi Antibacterial substances Isolation
In this study, a fungal strain LS116 with antibacterial activity against aquatic pathogen Vibrio harveyi was screened out from sponge-associated fungi by Oxford cup method. According to its morphological characteristics and internal transcribed spacer (ITS) analysis, the active strain belonged to the genus Aspergillus. A bioactivityguided approach was conducted to identify the antibacterial substances in the culture extract of strain LS116, a new C23 steroid with bicyclo[4.4.1]A/B ring, named aspergillsteroid A (1), and a known C25 steroid with the same structure skeleton, neocyclocitrinol B (2), were isolated and purified by column chromatographs and HPLC (high-performance liquid chromatography) for the first time. Their chemical structures were determined by HRESIMS (high-resolution electrospray ionization mass spectroscopy) spectrometry, NMR (nuclear magnetic resonance) spectroscopy, and CD (circular dichroism). Among them, compound 1 displayed significant antibacterial activity against V. harveyi with a MIC value of 16 μg/mL. The study demonstrated that compound 1 can be considered as a new promising agent for the control of aquatic disease in the future.
1. Introduction China has become the world's largest aquaculture producer and exporter, contributing more than 60% to the global aquaculture industry, and its aquaculture industry has become one of the major players in global food security according to the recent report (FAO, 2014; Mo et al., 2017). For the time being, most domestic fish farmers use intensive farming in their fish farms, however, this type of fish farming has a series of problems. For example, with the increase in the density of fish farming, the overcrowding of farms, the lack of sanitary barriers between farms and failure to isolate fish farms from infected animals, these inappropriate farming practices can easily lead to rapid infection spread (Naylor et al., 2000; Naylor and Burke, 2005; Cabello, 2006). According to preliminary estimates, the yield loss caused by bacterial infection accounts for 15–20% of the total annual production, and more than 200 diseases have been found in cultured aquatic species in China (Wei, 2002). Among these bacteria, V. harveyi infection is a major cause of aquatic animals mortality (Austin and Zhang, 2006; Chari and Dubey, 2006; Won and Park, 2008). For a long time, we have used antibiotics to control V. harveyi
∗
infection. However, the inappropriate use of antibiotics in aquatic animals has resulted in the emergence of drug-resistant strains, which will be detrimental to the environment protection and human health (Leyton et al., 2011; Maneechote et al., 2017). For example, the sensitivity of five strains of V. harveyi from shrimp ponds and coastal areas to 15 antibiotics was analyzed by Stalin et al. (Stalin and Srinivasan, 2016). The results showed that 27% of coastal water sediment-derived strains and 53% of shrimp pond-derived strains were multi-drug resistant, compared with other antibiotics, resistant to ciprofloxacin, penicillin, and rifampicin and vancomycin strains have the highest frequency of occurrence. Therefore, it is urgent to discovery new antibacterial agents with unique modes of action and properties that are different from the currently used antimicrobials (Maneechote et al., 2017). Marine-derived fungi have proven to be a prolific resource of a plethora of biologically active and structurally diverse natural products for the discovery of new antibiotics (Lee et al., 2010; Cheung et al., 2014; Xu et al., 2015; Liu et al., 2017; Zhang et al., 2018). As a major dominant strain of marine fungi, Aspergillus fungi can produce many types of secondary metabolites. In addition, biologically active
Corresponding autho. College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315211, PR China. Corresponding author. E-mail addresses: [email protected] (L. Ding), [email protected] (H. Jin).
∗∗
https://doi.org/10.1016/j.aquaculture.2019.734670 Received 3 May 2019; Received in revised form 8 October 2019; Accepted 2 November 2019 Available online 04 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Peng Xu, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734670
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Fig. 1. Chemical structure of compounds 1 and 2.
and after the completion of the PCR reaction, the PCR product was detected by gel electrophoresis. Finally, the correct PCR product will be verified and submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. 25 μL of the PCR amplification system of the strain identification contained 8.5 μL sterilized ddH2O, 12.5 μL PCR premix buffer, 1.0 μL forward primer, 1.0 μL reverse primer and 2.0 μL template DNA. The PCR procedure for strain identification consisted of 30 thermal cycles of denaturation at 94 °C for 1 min, annealing for 1 min at 55 °C and extension for 1.5 min at 72 °C.
substances produced by the genus Aspergillus have been widely reported, such as antibiotic penicillin (Arnstein and Cook, 1947), anticholesterol drugs ovastatins (Szakács et al., 1998) and so on. These active substances have proven to be of great importance in industrial applications and medical treatments. In our study, we screened 16 fungal isolates from marine sponge Haliclona sp. and three of them showed antibacterial activity against V. harveyi KP635244 to some extent by Oxford cup method (Guo and Wang, 2017). One of the most active strains, Aspergillus sp. LS116, was cultured in large scale. Further bioassay-directed fractionation, a new antibacterial compound, named aspergillsteroid A (1), along with a structurally-related compound, neocyclocitrinol B (2) (Du et al., 2008; Xia et al., 2014), were isolated and characterized from the crude extract of the strain LS116. We report herein the isolation, structural identification, and biological activities of these active metabolites (1 and 2) (Fig. 1).
2.3. Fermentation and extraction The fungal strain was inoculated in 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth medium (PDB, 28 g of potatodextrose broth, 35 g of artificial sea salt, dissolved in 1 L of distilled H2O). Flask cultures were inoculated at 25 °C on a rotatory shaker at 180 rpm for 3 days, subsequently, the seed cultures was transferred to 100 × 1 L Erlenmeyer flasks containing rice media (80 g of rice, 35 g of artificial sea salt, 120 mL of distilled H2O in each flask), and 20 mL of the seed cultures was poured into each flask of rice media. Subsequently, the fermentation was conducted under static conditions for 40 days at 28 °C. The fermented material was extracted with ethyl acetate (EtOAc) three times. The organic layer was concentrated under reduced pressure to acquire 36 g of crude gum.
2. Materials and methods 2.1. General experimental procedures The optical rotation was determined with a JASCO P-2000 digital polarimeter. The UV spectrum was measured on an Evolution 201 UV–vis spectrophotometer. The IR spectrum was acquired with a Nicolet is5 spectrophotometer as KBr disks. The CD data was recorded on a JASCO J-1500 CD spectropolarimeter. The NMR spectra were obtained on a Varian 600 MHz spectrometer (Palo Alto, CA, USA), TMS was used as internal standard, recording chemical shifts as δ values. High-resolution mass spectroscopy were carried out an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, USA). Semipreparative HPLC was operated with a Waters HPLC instrument (Waters 600, Miford, MA, USA) equipped with a Waters 2996 detector and an ODS column (250 × 20 mm, 5 μm, YMC Co. Ltd., Tokyo, Japan). Medium-pressure liquid chromatography (MPLC) was performed on a FLEXA Purification System (Agela Technologies, Tianjin, China) using a ODS column. Column chromatography was performed using silica gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, PR China), and Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ, USA).
2.4. Isolation and purification The crude gum (36 g) was separated into seven fractions (Fr.1–Fr.7) on a silica gel VLC column using gradient elution with a mixture of petroleum ether/EtOAc (20:1, 10:1, 5:1, 5:2, 5:4, 1:1, 3:5, 1:5, 0:1, v/v) and then with EtOAc/MeOH (10:1, 5:2, 1:1, 0:1, v/v). Fraction 4 (3.9 g) was subjected to repeated chromatography on Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give four subfractions (Fr.4-1–Fr.4-4). Fr.4–2 (1.2 g) was further separated by MPLC with an ODS column, eluting with MeOH/H2O (20 to 80% MeOH, 150 min, 20 mL/min), and additionally purified by semipreparative reversed-phase HPLC (MeCN/ H2O, 31:69, 2.0 mL/min) to yield compound 1 (3.4 mg; tR, 53.2 min). Fraction 5 (4.7 g) was further isolated by Sephadex LH-20, with MeOH/ CH2Cl2 (1:1) as eluent, producing four subfractions (Fr.5-1–Fr.5–4). Fr.5–2 (2.4 g) was further subjected to MPLC separation on an ODS column eluting with MeOH/H2O (40 to 75% MeOH, 150 min, 20 mL/ min), and additionally separated by semipreparative reversed-phase HPLC (MeCN/H2O, 24:76, 4.0 mL/min) to yield compound 2 (8.8 mg; tR, 46.0 min).
2.2. Fungal material and identification Aspergillus sp. LS116 was isolated from marine sponge Haliclona sp. collected in the Linshui, Hannan province, China. It was identified using the ITS region sequencing analysis. The fungal strain LS116 was then received for deposit in China General Microbiological Culture Collection Center with the accession number CGMCC No. 3.15366. The ITS1-5.8S-ITS2 rDNA sequence and partial 18S and 28S rDNA sequences were amplified by PCR using the fungal ribosomal rDNA region universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 ( 5′-TCCTCCGCTTATTGATATGC-3′) on the basis of the previous reported protocol (White et al., 1990). 20 mL of 1% agarose gel was prepared,
2.5. Antibacterial assay Compounds 1 and 2 were tested against four V. harveyi ((KP635244, IFO15632, IFO15634 and LMG4044)) using broth microdilution in 96well microplates (Pierce et al., 2008). The indicated strain was cultured in a marine broth 2216 medium (MB, peptone 5 g, yeast extract 1 g, 2
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NaCl 19.45 g, MgCl2 12.6 g, MgSO4 6.64 g, CaCl2 1.8 g, KCl 0.55 g, NaHCO3 0.16 g, FeC6H5O7 0.1 g, SrCl2 57 mg, KBr 80 mg, H3BO3 22 mg, NaSiO3·9H2O 9.3 mg, NaF 2.4 mg, NH4NO3 2.4 mg, dissolved in 1 L of distilled H2O, PH 6.0–7.0), and the bacterial suspension were diluted (106 CFU per milliliter) for measuration, a series of different concentrations of the test compounds were dissolved in DMSO, placed on sterile 96-well microplates, and inoculated with V. harveyi at 28 °C for 24 h. Each well contained 49 μL of diluted bacterial suspension, 50 μL of MB medium and 1 μL of test compounds. In the sterile 96-well microplates, the final concentration of the test compounds and positive control were 128, 64, 32, 16, 8, 4, and 2 μg/mL. Chloramphenicol was used as a positive control and dimethyl sulfoxide (DMSO) was used a negative control. All the procedures in the assay were performed in triplicates. MIC values were used as the minimum inhibitory concentration that inhibited visible growth of pathogen. 3. Results 3.1. Screening of active strains Fig. 2. Antibacterial plate effect diagram of the crude extract of the strain LS116.
In this paper, 16 fungal isolates were screened out from marine sponge Haliclona sp. and three of them showed antibacterial activity against V. harveyi KP635244 to some extent by Oxford cup method (Guo and Wang, 2017). The diameter of the inhibition zone was measured with an electronic caliper, and the average value of the diameter of the inhibition zone was used as the antibacterial activity of the sample to be tested. 0.1 mg/mL streptomycin sulfate was used as the positive control. At this concentration, the inhibition zone diameter of streptomycin sulfate against V. harveyi KP635244 was 19.80 ± 0.99 mm, and the diameter of the inhibition zone was greater than or equal to a strain of 20.0 mm was regarded as an active strains. The antibacterial effect of 16 strains of fungi on V. harveyi KP635244 was shown in Table 1. The most active strain, Aspergillus sp. LS116 was selected and cultured on a large scale. Antibacterial plate effect diagram of the crude extract of strain LS116 was shown in Fig. 2. Strain LS116 was identified by morphological characteristics and internal transcribed spacer (ITS) analysis. The results showed that Strain LS116 exhibited morphological similarities to an Aspergillus sp. and correlated with the ITS sequence which had a 99% similarity with Aspergillus versicolor. (Genbank accession no. FJ864703). Therefore, it was identified as the genus Aspergillus. This sequence was compared to the sequences of other fungus from GenBank using MEGA7 (Kumar et al., 2016) to establish a phylogenetic tree (Fig. 3).
According to analysis of NMR and HRESIMS data (m/z [M+H]+ 357.2423) (Suppl. Fig. S8), the molecular formula was deduced as C23H32O3, revealing it possessed 8 degrees of unsaturation. The conjugated carbonyl group was determined by UV (Suppl. Fig. S9) absorbance at 243 nm and IR (Suppl. Fig. S10) absorbance at 1648 cm−1. The 1 H NMR spectrum (600 MHz, CD3OD) (Table 2 and Suppl. Fig. S1) displayed resonances for three signals of trisubstituted double bonds at δH 5.63 (1H, dd, 6.7, 8.1), 5.57 (1H, s) and 5.47 (1H, t, 6.5), one signal of oxymethine at δH 3.34 (1H, m), one signal of oxymethylene at δH 4.15 (2H, dd, J = 10.7, 6.5 Hz), two signals of methyl singlet at δH 0.62 (3H, s) and 1.72 (3H, s). The 13C NMR (Table 2 and Suppl. Fig. S2), together with DEPT NMR (Suppl. Fig. S3) and HSQC NMR (Fig. S5) data of 1 (150 MHz, CD3OD) revealed the presence of a carbonyl carbon at δC 207.7 (C-6), four quaternary carbons at δC 160.2 (C-8), 147.5 (C-10), 48.2 (C-13) and 137.9 (C-20), two methyls at δC 13.8 (C-19) and 18.0 (C-21), eight methylenes and eight methines. In combination with the remaining four degrees of unsaturation in the molecular formula speculated that 1 contained four rings. The cross peaks between H-1/ H2-2, H2-2/H-3, H-3/H2-4 H2-4/H-5, and H-5/H2-18 in the 1H–1H COSY spectrum (Fig. S4) and the correlations from H2-18 to C-1, C-4, C-5, C-6, C-9, and C-10, from H2-4 to C-6 and C-18, from H-1 to C-2, C-9, and C18, from H-9 to C-1, C-10, and C-18, and from H-5 to C-7 in the HMBC spectrum (Table 2 and Fig. S6) demonstrated the presence of the bicyclo[4.4.1] system of the A/B rings (Fig. 4). The HMBC signals from H3-19 to C-12, C-13, C-14 and C-17 and from H2-11, H-14 and H2-16 to C-13, as well as HMBC correlations from H-14 to C-7 and C-8 and from H-9 to C-10, C-1, C-18 and C-7 proved the linkage of the bicyclo [4.4.1]A/B ring and the other two rings in 1. In addition, a side chain was assigned by the COSY correlation from H2-23 to H-22 and the HMBC correlations from H3-21 to C-20 and C-22, from H2-23 to C-20. Finally, the planar structure (Fig. 4) of 1 was established by the key HMBC correlations from H-21 and H-22 to C-17 and from H-17 to C-20. On the basis of the above data analysis, compound 1 was determined as a C23 steroid with bicyclo[4.4.1]A/B ring. The relative configuration of aspergillsteroid A (1) was deduced by the NOESY spectrum (Fig. S7) and the comparison of NMR data with a known compound neocyclocitrinol B (2) (Du et al., 2008; Xia et al., 2014). The correlations between H3-21/H3-19, H3-19/H2-18 and H-9/ H-14 in the NOESY spectrum indicated the orientations of C-20 and C19 and C-18 on the same side of the A-D ring moiety and H-9, H-14 and H-17 on the other side of the ring system in 1. Moreover, 1H and 13C NMR chemical shifts of A/B ring moieties in 1 and 2 are almost similar, indicating that they possessed the same 3-C-OH. Furthermore, the
3.2. Structural elucidation Aspergillsteroid A (1) was obtained as a white, amorphous powder. Table 1 Antibacterial activity of 16 strains of fungi against V. harveyi KP635244. Number of strain
Inhibition zone (X ± SD, n = 3, mm)
LS101 LS102 LS103 LS104 LS105 LS106 LS107 LS108 LS109 LS110 LS111 LS112 LS113 LS114 LS115 LS116
14.36 13.66 16.88 17.05 15.69 16.12 16.45 17.24 15.24 14.58 15.88 20.08 15.92 20.82 17.02 22.12
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35 0.75 0.52 0.36 0.09 1.21 0.68 0.12 0.82 0.71 0.33 1.26 0.64 2.24 0.21 1.34
3
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Fig. 3. Phylogenetic tree of Aspergillus sp. LS116 based on 5.8S and ITS region sequences. Table 2 1 H, 13C, HMBC and COSY NMR data for 1 (600, 150 MHz, CD3OD). Position
δC
δH (J in Hz)
HMBC
COSY
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
123.1 36.8 65.3 42.2 50.0 207.7 125.3 160.2 55.5 147.5 29.0 38.9 48.2 56.4 23.8 25.3 60.5
5.63 2.21 3.34 1.66 2.77
C-2, 9, 18 C-1 C-1 C-6, 18 C-7
H-2 H-1, H-2, H-3, H-4,
18
28.5
2.62 (2H, m)
19
13.8
0.62 (3H, s)
20 21 22 23
137.9 18.0 126.9 59.6
(1H, dd, 6.7, 8.1) (1H, m) 2.48 (1H, m) (1H, m) (1H, m) 2.85 (1H, brd, 13.1) (1H, m)
3 4 5 18
5.57 (1H, s)
C-14
2.90 (1H, dd, 11.6, 5.7)
C-1, 7, 18
H-11
1.65 (1H, m) 1.90 (1H, m) 1.54 (1H, m) 1.92 (1H, m)
C-9, 13 C-19
H-9, 12
2.32 1.68 1.80 2.35
C-7, 15, 19 C-14 C-13, 15 C-19, 20, 21, 22 C-1, 4, 5, 6, 9, 10 C-12, 13, 14, 17
H-15 H-14, 16 H-15, 17 H-16
(1H, (2H, (1H, (1H,
brt, 8.4) m) m) 1.95 (1H, m) brt, 9.7)
1.72 (3H, s) 5.47 (1H, dd, 6.6, 6.2) 4.15 (1H, dd, 12.8, 6.2) 4.17 (1H, dd, 12.8, 6.6)
C-17, 20, 22 C-17, 23 C-20, 22
Fig. 5. CD spectra of compounds 1 and 2. Chemical characteristics of compound 1 are as follows.
the 20,22-double bond in 1 can be established on the basis of the NOESY correlation of H3-21 and H2-23. Based on the above analysis, the absolute configuration of 1 can be determined (Fig. 1). Neocyclocitrinol B (2) was isolated from the secondary metabolites of Aspergillus sp. LS116. Comparison of the spectroscopic data with reported known C25 steroid, compound 2 was determined as neocyclocitrinol B (Du et al., 2008; Xia et al., 2014). Aspergillsteroid A (1): White amorphous powder; C23H32O3; [α]D20 +37.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.23), 243 (3.69); CD (MeOH) λmax (Δε) 400 (+0.07), 364 (0), 318 (−1.45), 277 (+0.11), 245 (+2.88), 221 (+1.47), 203 (+7.51), 200 (+4.44); IR (KBr) νmax 2916, 2849, 2361, 2341, 1648, 1125, 1038, 668 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z [M+H]+ 357.2423 (calcd for C23H33O3, 357.2424).
H-5
H-23 H-22
3.3. Antibacterial activity Antibacterial activity against four bacterial V. harveyi strains effects of compounds 1 and 2 were evaluated using broth microdilution in 96well microplates. Remarkably, compound 1 exhibited significant antibacterial activity against V. harveyi KP635244 (Stalin and Srinivasan, 2016) with a MIC value of 16 μg/mL, however, compound 2 showed only weakly antibacterial activity against V. harveyi KP635244 with a MIC value of 128 μg/mL (Table 3). None of compounds 1 and 2 showed antibacterial activity against the other three V. harveyi strains at the tested concentrations up to 128 μg/mL (Nakayama et al., 2006).
Fig. 4. Key 1H–1H COSY, HMBC and NOESY correlations of 1.
circular dichroism (CD) spectrum (Fig. 5) of 1 displayed a negative Cotton effect at 318 (Δε −1.45) nm and a positive Cotton at 245 (Δε +2.88) nm, which was identical with 2. This suggested further that the absolute configuration of A-D ring moieties in 1 and 2 are the same (Du et al., 2008; Xia et al., 2014). At the same time, the E-configuration of
4. Discussion C25 steroids, represented by cyclocitrinol, are a large family of a 4
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Acknowledgments
Table 3 Antibacterial activity against V. harveyi KP635244 effects of compounds 1 and 2. Compounds
1 2 Chloramphenicola
This study was supported by the National Key Research and Development Program of China (2018YFC0310900), the National Natural Science Foundation of China (41776168, 41706167), Ningbo Public Service Platform for High-Value Utilization of Marine Biological Resources (NBHY-2017-P2), Zhejiang Provincial Public Welfare Technology Program (LGC19B020002), the Natural Science Foundation of Ningbo (2018A610303), Ningbo Sci. & Tech. Projects for Common Wealth (2017C10016), the National 111 Project of China (D16013), the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund, and the K.C. Wong Magna Fund in Ningbo University.
Concentration (μg/mL) 128
64
32
16
8
4
2
× + ×
+++ – ×
++ – ×
++ – ×
– – ×
– – ×
– – ×
− no antagonistic action, + presence antagonistic action, ++ moderate antagonistic action, +++ strong antagonistic action, × the growth was inhibited, a Chloramphenicol as positive control.
series of unusual steroids with bicyclo[4.4.1]A/B rings in their structures (Du et al., 2008; Xia et al., 2014; Lin et al., 2015). In the preliminary research, cyclocitrinol was shown to have good cytotoxic activity against HepG2 cell and CaSki cell (Deng et al., 2016) and generally weaker antitumor activity against several human cancer cell lines (Xia et al., 2014). To date, known C25 steroid isomers with bicyclo [4.4.1]A/B rings, were essentially isolated from the genus Penicillium, and their activity studies were mainly focused on cytotoxicity, while other bioactivities research were rarely reported (Du et al., 2008; Xia et al., 2014; Deng et al., 2016). However, to the best of our knowledge, steroids and their derivatives have good antibacterial activity against many pathogens, such as S. aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Salmonella typhimurium (Khan et al., 2008) and E. coli (Khan et al., 2007). Two cyclocitribol analogs, named aspergillsteroid A (1) and neocyclocitrinol B (2), were firstly isolated from a marine sponge-associated Aspergillus fungi. In this paper, our study firstly reported the antibacterial activity of compounds 1 and 2 against V. harvey KP635244. It's worth noting that compound 1 has significant antibacterial activity against V. harveyi KP635244 with a MIC value of 16 μg/mL, but compound 2 only showed weakly antibacterial activity against V. harveyi KP635244 with a MIC value of 128 μg/mL. Comparing the structures of these compounds, it can be found that the absence of the C-23 hydroxyl branch moiety in 1, however, according to previous reports in the literature, we know that modifying hydroxyl groups can improve the efficacy of most antibacterial drugs. In the preliminary study, the following four modifications to hydroxyl groups are generally carried out: acylation to amides or esters; alkylation or alkoxylation; substitution with halogens; remove hydroxyl groups directly (He and Li, 2006; Chen et al., 2013). Therefore, we can speculate that the absence of a C-23 hydroxyl branch moiety in 1 instead exerts a stronger antibacterial activity against V. harveyi KP635244 than 2. In addition, compounds 1 and 2 were also evaluated for the inhibition of the other three V. harveyi strains growth. This results displayed that compounds 1 and 2 showed no antibacterial activity at the tested concentrations up to 128 μg/mL. Antibiotics are a class of low molecular weight (< 3000 Da) natural organic molecules that have been isolated due to their ability to inhibit the activity of organisms (Ryan and Dow, 2008); in generally, they exert their antimicrobial action by combining with specific cellular targets. Therefore, we believe that compounds 1 and 2 may bind specific cellular targets of V. harveyi KP635244, thereby inhibiting growth of V. harveyi KP635244. At the same time, the other three V. harveyi strains lack specific celluar targets that bind to compounds 1 or 2, or specific celluar targets on the other three V. harveyi strains have a weaker ability to bind to compounds 1 or 2 than the V. harveyi KP635244. In conclusion, compound 1 maybe have potential in use as an antibiotic agent to control aquatic pathogens in the future.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734670. References Arnstein, H.R., Cook, A.H., 1947. The penicillin produced by Aspergillus parasiticus. Br. J. Exp. Pathol. 28, 94. Austin, B., Zhang, X.H., 2006. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett. Appl. Microbiol. 43, 119–124. Cabello, F.C., 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ. Microbiol. 8, 1137–1144. Chari, P.V.B., Dubey, S.K., 2006. Rapid and specific detection of luminous and non-luminous Vibrio harveyi isolates by PCR amplification. Curr. Sci. 90, 1105–1108. Chen, C., Wang, J., Guo, H., Hou, W., Yang, N., Ren, B., Liu, M., Dai, H., Liu, X., Song, F., Zhang, L., 2013. Three antimycobacterial metabolites identified from a marine-derived Streptomyces sp. MS100061. Appl. Microbiol. Biotechnol. 97, 3885–3892. Cheung, R.C.F., Wong, J.H., Pan, W.L., Chan, Y.S., Yin, C.M., Dan, X.L., Wang, H.X., Fang, E.F., Lam, S.K., Ngai, P.H.K., Xia, L.X., Liu, F., Ye, X.Y., Zhang, G.Q., Liu, Q.H., Sha, O., Lin, P., Ki, C., Bekhit, A.A., Bekhit, A.E.D., Wan, D.C.C., Ye, X.J., Xia, J., Ng, T.B., 2014. Antifungal and antiviral products of marine organisms. Appl. Microbiol. Biotechnol. 98, 3475–3494. Deng, Z., Zhou, J., Zhang, H., Guo, Z., Peng, Y., Zou, K., 2016. Unusual C25 steroids with bicyclo[4.4.1] skeleton at A/B rings from endophytic fungus Penicillium citrinum. Chem. Nat. Compd. 52, 545–547. Du, L., Zhu, T., Fang, Y., Gu, Q., Zhu, W., 2008. Unusual C25 steroid isomers with bicyclo [4.4.1]A/B rings from a volcano ash-derived fungus Penicillium citrinum. J. Nat. Prod. 71, 1343–1351. FAO, 2014. The State of World Fisheries and Aquaculture 2014. FAO, Rome. Guo, L., Wang, C., 2017. Optimized production and isolation of antibacterial agent from marine Aspergillus flavipes against Vibrio harveyi. 3. Biotech 7, 383. He, X., Li, Z., 2006. Progress in hydroxyl structure-activity relationship study of antibiotics. Chinese. J. Veteri. Drug. 40, 45–48. Khan, S.N., Kim, B.J., Kim, H.S., 2007. Synthesis and antimicrobial activity of 7-fluoro-3aminosteroids. Bioorg. Med. Chem. Lett. 17, 5139–5142. Khan, S.A., Singh, N., Saleem, K., 2008. Synthesis, characterization and in vitro antibacterial activity of thiourea and urea derivatives of steroids. Eur. J. Med. Chem. 43, 2272–2277. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Lee, Y.M., Li, H., Hong, J., Cho, H.Y., Bae, K.S., Kim, M.A., Kim, D.K., Jung, J.H., 2010. Bioactive metabolites from the sponge-derived fungus Aspergillus versicolor. Arch. Pharm. Res. 33, 231–235. Leyton, Y., Borquez, J., Darias, J., Cueto, M., Díaz-Marrero, A., Riquelme, C., 2011. Oleic acid produced by a marine Vibrio spp. acts as an anti-Vibrio parahaemolyticus agent. Mar. Drugs 9, 2155–2163. Lin, S., Chen, K., Fu, P., Ye, J., Su, Y., Yang, X., Zhang, Z., Shan, L., Li, H., Shen, Y., Liu, R., Xu, X., Zhang, W., 2015. Structure determination of two unusual C25 steroids with bicyclo[4.4.1]A/B rings from Penicillium decumbens by NMR spectroscopy. Magn. Reson. Chem. 53, 223–226. Liu, S., Wang, H., Su, M., Hwang, G.J., Hong, J., Jung, J.H., 2017. New metabolites from the sponge-derived fungus Aspergillus sydowii J05B-7F-4. Nat. Prod. Res. 31, 1682–1686. Maneechote, N., Yingyongnarongkul, B., Suksamran, A., Lumyong, S., 2017. Inhibition of Vibrio spp. by 2-hydroxyethyl-11-hydroxyhexadec-9-enoate of marine cyanobacterium Leptolyngbya sp. LT19. Aquac. Res. 48, 2088–2095. Mo, W.Y., Chen, Z.T., Leung, H.M., Leung, A.O.W., 2017. Application of veterinary antibiotics in China's aquaculture industry and their potential human health risks. Environ. Sci. Pollut. Res. 24, 8978–8989. Nakayama, T., Ito, E., Nomura, N., Nomura, N., Matsumura, M., 2006. Comparison of Vibrio harveyi strains isolated from shrimp farms and from culture collection in terms of toxicity and antibiotic resistance. FEMS Microbiol. Lett. 258, 194–199. Naylor, R., Burke, M., 2005. Aquaculture and ocean resources: raising tigers of the sea.
Declaration of competing interest The authors declare that they have no competing interests. 5
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Reantaso, M.B., MacRae, I.H. (Eds.), Primary Aquatic Animal Health Care in Rural, Small-scale, Aquaculture Development. FAO Fish. Tech. Pap. No. 406, Rome Italy (FAO). White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. Amplification and direct seqencing of fungal ribosomal RNA genes for phylogenetics. In: In: Innis, M.A., Gelfand, D.H., Sninsky, J.S., White, T.J. (Eds.), PCR Protoc: A Guide to Methods and Applications 38. Academic Press, San Diego, pp. 315–322. Won, K.M., Park, S.I., 2008. Pathogenicity of Vibrio harveyi to cultured marine fishes in korea. Aquaculture 285, 8–13. Xia, M., Cui, C., Li, C., Wu, C., 2014. Three new and eleven known unusual C25 steroids: activated production of silent metabolites in a marine-derived fungus by chemical mutagenesis strategy using diethyl sulphate. Mar. Drugs 12, 1545–1568. Xu, L., Meng, W., Cao, C., Wang, J., Shan, W., Wang, Q., 2015. Antibacterial and antifungal compounds from marine fungi. Mar. Drugs 13, 3479–3513. Zhang, X., Li, Z., Gao, J., 2018. Chemistry and biology of secondary metabolites from Aspergillus genus. Nat. Prod. J. 8, 275–304.
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A new aquatic pathogen inhibitor produced by the marine fungus Aspergillus sp. LS116 Peng Xu, Lijian Ding∗, Jiaxin Wei, Qiang Li, Minjie Gui, Xiaoping He, Dengquan Su, Shan He, Haixiao Jin∗∗ Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315832, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Marine fungi Vibrio harveyi Antibacterial substances Isolation
In this study, a fungal strain LS116 with antibacterial activity against aquatic pathogen Vibrio harveyi was screened out from sponge-associated fungi by Oxford cup method. According to its morphological characteristics and internal transcribed spacer (ITS) analysis, the active strain belonged to the genus Aspergillus. A bioactivityguided approach was conducted to identify the antibacterial substances in the culture extract of strain LS116, a new C23 steroid with bicyclo[4.4.1]A/B ring, named aspergillsteroid A (1), and a known C25 steroid with the same structure skeleton, neocyclocitrinol B (2), were isolated and purified by column chromatographs and HPLC (high-performance liquid chromatography) for the first time. Their chemical structures were determined by HRESIMS (high-resolution electrospray ionization mass spectroscopy) spectrometry, NMR (nuclear magnetic resonance) spectroscopy, and CD (circular dichroism). Among them, compound 1 displayed significant antibacterial activity against V. harveyi with a MIC value of 16 μg/mL. The study demonstrated that compound 1 can be considered as a new promising agent for the control of aquatic disease in the future.
1. Introduction China has become the world's largest aquaculture producer and exporter, contributing more than 60% to the global aquaculture industry, and its aquaculture industry has become one of the major players in global food security according to the recent report (FAO, 2014; Mo et al., 2017). For the time being, most domestic fish farmers use intensive farming in their fish farms, however, this type of fish farming has a series of problems. For example, with the increase in the density of fish farming, the overcrowding of farms, the lack of sanitary barriers between farms and failure to isolate fish farms from infected animals, these inappropriate farming practices can easily lead to rapid infection spread (Naylor et al., 2000; Naylor and Burke, 2005; Cabello, 2006). According to preliminary estimates, the yield loss caused by bacterial infection accounts for 15–20% of the total annual production, and more than 200 diseases have been found in cultured aquatic species in China (Wei, 2002). Among these bacteria, V. harveyi infection is a major cause of aquatic animals mortality (Austin and Zhang, 2006; Chari and Dubey, 2006; Won and Park, 2008). For a long time, we have used antibiotics to control V. harveyi
∗
infection. However, the inappropriate use of antibiotics in aquatic animals has resulted in the emergence of drug-resistant strains, which will be detrimental to the environment protection and human health (Leyton et al., 2011; Maneechote et al., 2017). For example, the sensitivity of five strains of V. harveyi from shrimp ponds and coastal areas to 15 antibiotics was analyzed by Stalin et al. (Stalin and Srinivasan, 2016). The results showed that 27% of coastal water sediment-derived strains and 53% of shrimp pond-derived strains were multi-drug resistant, compared with other antibiotics, resistant to ciprofloxacin, penicillin, and rifampicin and vancomycin strains have the highest frequency of occurrence. Therefore, it is urgent to discovery new antibacterial agents with unique modes of action and properties that are different from the currently used antimicrobials (Maneechote et al., 2017). Marine-derived fungi have proven to be a prolific resource of a plethora of biologically active and structurally diverse natural products for the discovery of new antibiotics (Lee et al., 2010; Cheung et al., 2014; Xu et al., 2015; Liu et al., 2017; Zhang et al., 2018). As a major dominant strain of marine fungi, Aspergillus fungi can produce many types of secondary metabolites. In addition, biologically active
Corresponding autho. College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, 315211, PR China. Corresponding author. E-mail addresses: [email protected] (L. Ding), [email protected] (H. Jin).
∗∗
https://doi.org/10.1016/j.aquaculture.2019.734670 Received 3 May 2019; Received in revised form 8 October 2019; Accepted 2 November 2019 Available online 04 November 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Peng Xu, et al., Aquaculture, https://doi.org/10.1016/j.aquaculture.2019.734670
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Fig. 1. Chemical structure of compounds 1 and 2.
and after the completion of the PCR reaction, the PCR product was detected by gel electrophoresis. Finally, the correct PCR product will be verified and submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. 25 μL of the PCR amplification system of the strain identification contained 8.5 μL sterilized ddH2O, 12.5 μL PCR premix buffer, 1.0 μL forward primer, 1.0 μL reverse primer and 2.0 μL template DNA. The PCR procedure for strain identification consisted of 30 thermal cycles of denaturation at 94 °C for 1 min, annealing for 1 min at 55 °C and extension for 1.5 min at 72 °C.
substances produced by the genus Aspergillus have been widely reported, such as antibiotic penicillin (Arnstein and Cook, 1947), anticholesterol drugs ovastatins (Szakács et al., 1998) and so on. These active substances have proven to be of great importance in industrial applications and medical treatments. In our study, we screened 16 fungal isolates from marine sponge Haliclona sp. and three of them showed antibacterial activity against V. harveyi KP635244 to some extent by Oxford cup method (Guo and Wang, 2017). One of the most active strains, Aspergillus sp. LS116, was cultured in large scale. Further bioassay-directed fractionation, a new antibacterial compound, named aspergillsteroid A (1), along with a structurally-related compound, neocyclocitrinol B (2) (Du et al., 2008; Xia et al., 2014), were isolated and characterized from the crude extract of the strain LS116. We report herein the isolation, structural identification, and biological activities of these active metabolites (1 and 2) (Fig. 1).
2.3. Fermentation and extraction The fungal strain was inoculated in 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth medium (PDB, 28 g of potatodextrose broth, 35 g of artificial sea salt, dissolved in 1 L of distilled H2O). Flask cultures were inoculated at 25 °C on a rotatory shaker at 180 rpm for 3 days, subsequently, the seed cultures was transferred to 100 × 1 L Erlenmeyer flasks containing rice media (80 g of rice, 35 g of artificial sea salt, 120 mL of distilled H2O in each flask), and 20 mL of the seed cultures was poured into each flask of rice media. Subsequently, the fermentation was conducted under static conditions for 40 days at 28 °C. The fermented material was extracted with ethyl acetate (EtOAc) three times. The organic layer was concentrated under reduced pressure to acquire 36 g of crude gum.
2. Materials and methods 2.1. General experimental procedures The optical rotation was determined with a JASCO P-2000 digital polarimeter. The UV spectrum was measured on an Evolution 201 UV–vis spectrophotometer. The IR spectrum was acquired with a Nicolet is5 spectrophotometer as KBr disks. The CD data was recorded on a JASCO J-1500 CD spectropolarimeter. The NMR spectra were obtained on a Varian 600 MHz spectrometer (Palo Alto, CA, USA), TMS was used as internal standard, recording chemical shifts as δ values. High-resolution mass spectroscopy were carried out an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, USA). Semipreparative HPLC was operated with a Waters HPLC instrument (Waters 600, Miford, MA, USA) equipped with a Waters 2996 detector and an ODS column (250 × 20 mm, 5 μm, YMC Co. Ltd., Tokyo, Japan). Medium-pressure liquid chromatography (MPLC) was performed on a FLEXA Purification System (Agela Technologies, Tianjin, China) using a ODS column. Column chromatography was performed using silica gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, PR China), and Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ, USA).
2.4. Isolation and purification The crude gum (36 g) was separated into seven fractions (Fr.1–Fr.7) on a silica gel VLC column using gradient elution with a mixture of petroleum ether/EtOAc (20:1, 10:1, 5:1, 5:2, 5:4, 1:1, 3:5, 1:5, 0:1, v/v) and then with EtOAc/MeOH (10:1, 5:2, 1:1, 0:1, v/v). Fraction 4 (3.9 g) was subjected to repeated chromatography on Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give four subfractions (Fr.4-1–Fr.4-4). Fr.4–2 (1.2 g) was further separated by MPLC with an ODS column, eluting with MeOH/H2O (20 to 80% MeOH, 150 min, 20 mL/min), and additionally purified by semipreparative reversed-phase HPLC (MeCN/ H2O, 31:69, 2.0 mL/min) to yield compound 1 (3.4 mg; tR, 53.2 min). Fraction 5 (4.7 g) was further isolated by Sephadex LH-20, with MeOH/ CH2Cl2 (1:1) as eluent, producing four subfractions (Fr.5-1–Fr.5–4). Fr.5–2 (2.4 g) was further subjected to MPLC separation on an ODS column eluting with MeOH/H2O (40 to 75% MeOH, 150 min, 20 mL/ min), and additionally separated by semipreparative reversed-phase HPLC (MeCN/H2O, 24:76, 4.0 mL/min) to yield compound 2 (8.8 mg; tR, 46.0 min).
2.2. Fungal material and identification Aspergillus sp. LS116 was isolated from marine sponge Haliclona sp. collected in the Linshui, Hannan province, China. It was identified using the ITS region sequencing analysis. The fungal strain LS116 was then received for deposit in China General Microbiological Culture Collection Center with the accession number CGMCC No. 3.15366. The ITS1-5.8S-ITS2 rDNA sequence and partial 18S and 28S rDNA sequences were amplified by PCR using the fungal ribosomal rDNA region universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 ( 5′-TCCTCCGCTTATTGATATGC-3′) on the basis of the previous reported protocol (White et al., 1990). 20 mL of 1% agarose gel was prepared,
2.5. Antibacterial assay Compounds 1 and 2 were tested against four V. harveyi ((KP635244, IFO15632, IFO15634 and LMG4044)) using broth microdilution in 96well microplates (Pierce et al., 2008). The indicated strain was cultured in a marine broth 2216 medium (MB, peptone 5 g, yeast extract 1 g, 2
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NaCl 19.45 g, MgCl2 12.6 g, MgSO4 6.64 g, CaCl2 1.8 g, KCl 0.55 g, NaHCO3 0.16 g, FeC6H5O7 0.1 g, SrCl2 57 mg, KBr 80 mg, H3BO3 22 mg, NaSiO3·9H2O 9.3 mg, NaF 2.4 mg, NH4NO3 2.4 mg, dissolved in 1 L of distilled H2O, PH 6.0–7.0), and the bacterial suspension were diluted (106 CFU per milliliter) for measuration, a series of different concentrations of the test compounds were dissolved in DMSO, placed on sterile 96-well microplates, and inoculated with V. harveyi at 28 °C for 24 h. Each well contained 49 μL of diluted bacterial suspension, 50 μL of MB medium and 1 μL of test compounds. In the sterile 96-well microplates, the final concentration of the test compounds and positive control were 128, 64, 32, 16, 8, 4, and 2 μg/mL. Chloramphenicol was used as a positive control and dimethyl sulfoxide (DMSO) was used a negative control. All the procedures in the assay were performed in triplicates. MIC values were used as the minimum inhibitory concentration that inhibited visible growth of pathogen. 3. Results 3.1. Screening of active strains Fig. 2. Antibacterial plate effect diagram of the crude extract of the strain LS116.
In this paper, 16 fungal isolates were screened out from marine sponge Haliclona sp. and three of them showed antibacterial activity against V. harveyi KP635244 to some extent by Oxford cup method (Guo and Wang, 2017). The diameter of the inhibition zone was measured with an electronic caliper, and the average value of the diameter of the inhibition zone was used as the antibacterial activity of the sample to be tested. 0.1 mg/mL streptomycin sulfate was used as the positive control. At this concentration, the inhibition zone diameter of streptomycin sulfate against V. harveyi KP635244 was 19.80 ± 0.99 mm, and the diameter of the inhibition zone was greater than or equal to a strain of 20.0 mm was regarded as an active strains. The antibacterial effect of 16 strains of fungi on V. harveyi KP635244 was shown in Table 1. The most active strain, Aspergillus sp. LS116 was selected and cultured on a large scale. Antibacterial plate effect diagram of the crude extract of strain LS116 was shown in Fig. 2. Strain LS116 was identified by morphological characteristics and internal transcribed spacer (ITS) analysis. The results showed that Strain LS116 exhibited morphological similarities to an Aspergillus sp. and correlated with the ITS sequence which had a 99% similarity with Aspergillus versicolor. (Genbank accession no. FJ864703). Therefore, it was identified as the genus Aspergillus. This sequence was compared to the sequences of other fungus from GenBank using MEGA7 (Kumar et al., 2016) to establish a phylogenetic tree (Fig. 3).
According to analysis of NMR and HRESIMS data (m/z [M+H]+ 357.2423) (Suppl. Fig. S8), the molecular formula was deduced as C23H32O3, revealing it possessed 8 degrees of unsaturation. The conjugated carbonyl group was determined by UV (Suppl. Fig. S9) absorbance at 243 nm and IR (Suppl. Fig. S10) absorbance at 1648 cm−1. The 1 H NMR spectrum (600 MHz, CD3OD) (Table 2 and Suppl. Fig. S1) displayed resonances for three signals of trisubstituted double bonds at δH 5.63 (1H, dd, 6.7, 8.1), 5.57 (1H, s) and 5.47 (1H, t, 6.5), one signal of oxymethine at δH 3.34 (1H, m), one signal of oxymethylene at δH 4.15 (2H, dd, J = 10.7, 6.5 Hz), two signals of methyl singlet at δH 0.62 (3H, s) and 1.72 (3H, s). The 13C NMR (Table 2 and Suppl. Fig. S2), together with DEPT NMR (Suppl. Fig. S3) and HSQC NMR (Fig. S5) data of 1 (150 MHz, CD3OD) revealed the presence of a carbonyl carbon at δC 207.7 (C-6), four quaternary carbons at δC 160.2 (C-8), 147.5 (C-10), 48.2 (C-13) and 137.9 (C-20), two methyls at δC 13.8 (C-19) and 18.0 (C-21), eight methylenes and eight methines. In combination with the remaining four degrees of unsaturation in the molecular formula speculated that 1 contained four rings. The cross peaks between H-1/ H2-2, H2-2/H-3, H-3/H2-4 H2-4/H-5, and H-5/H2-18 in the 1H–1H COSY spectrum (Fig. S4) and the correlations from H2-18 to C-1, C-4, C-5, C-6, C-9, and C-10, from H2-4 to C-6 and C-18, from H-1 to C-2, C-9, and C18, from H-9 to C-1, C-10, and C-18, and from H-5 to C-7 in the HMBC spectrum (Table 2 and Fig. S6) demonstrated the presence of the bicyclo[4.4.1] system of the A/B rings (Fig. 4). The HMBC signals from H3-19 to C-12, C-13, C-14 and C-17 and from H2-11, H-14 and H2-16 to C-13, as well as HMBC correlations from H-14 to C-7 and C-8 and from H-9 to C-10, C-1, C-18 and C-7 proved the linkage of the bicyclo [4.4.1]A/B ring and the other two rings in 1. In addition, a side chain was assigned by the COSY correlation from H2-23 to H-22 and the HMBC correlations from H3-21 to C-20 and C-22, from H2-23 to C-20. Finally, the planar structure (Fig. 4) of 1 was established by the key HMBC correlations from H-21 and H-22 to C-17 and from H-17 to C-20. On the basis of the above data analysis, compound 1 was determined as a C23 steroid with bicyclo[4.4.1]A/B ring. The relative configuration of aspergillsteroid A (1) was deduced by the NOESY spectrum (Fig. S7) and the comparison of NMR data with a known compound neocyclocitrinol B (2) (Du et al., 2008; Xia et al., 2014). The correlations between H3-21/H3-19, H3-19/H2-18 and H-9/ H-14 in the NOESY spectrum indicated the orientations of C-20 and C19 and C-18 on the same side of the A-D ring moiety and H-9, H-14 and H-17 on the other side of the ring system in 1. Moreover, 1H and 13C NMR chemical shifts of A/B ring moieties in 1 and 2 are almost similar, indicating that they possessed the same 3-C-OH. Furthermore, the
3.2. Structural elucidation Aspergillsteroid A (1) was obtained as a white, amorphous powder. Table 1 Antibacterial activity of 16 strains of fungi against V. harveyi KP635244. Number of strain
Inhibition zone (X ± SD, n = 3, mm)
LS101 LS102 LS103 LS104 LS105 LS106 LS107 LS108 LS109 LS110 LS111 LS112 LS113 LS114 LS115 LS116
14.36 13.66 16.88 17.05 15.69 16.12 16.45 17.24 15.24 14.58 15.88 20.08 15.92 20.82 17.02 22.12
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.35 0.75 0.52 0.36 0.09 1.21 0.68 0.12 0.82 0.71 0.33 1.26 0.64 2.24 0.21 1.34
3
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Fig. 3. Phylogenetic tree of Aspergillus sp. LS116 based on 5.8S and ITS region sequences. Table 2 1 H, 13C, HMBC and COSY NMR data for 1 (600, 150 MHz, CD3OD). Position
δC
δH (J in Hz)
HMBC
COSY
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
123.1 36.8 65.3 42.2 50.0 207.7 125.3 160.2 55.5 147.5 29.0 38.9 48.2 56.4 23.8 25.3 60.5
5.63 2.21 3.34 1.66 2.77
C-2, 9, 18 C-1 C-1 C-6, 18 C-7
H-2 H-1, H-2, H-3, H-4,
18
28.5
2.62 (2H, m)
19
13.8
0.62 (3H, s)
20 21 22 23
137.9 18.0 126.9 59.6
(1H, dd, 6.7, 8.1) (1H, m) 2.48 (1H, m) (1H, m) (1H, m) 2.85 (1H, brd, 13.1) (1H, m)
3 4 5 18
5.57 (1H, s)
C-14
2.90 (1H, dd, 11.6, 5.7)
C-1, 7, 18
H-11
1.65 (1H, m) 1.90 (1H, m) 1.54 (1H, m) 1.92 (1H, m)
C-9, 13 C-19
H-9, 12
2.32 1.68 1.80 2.35
C-7, 15, 19 C-14 C-13, 15 C-19, 20, 21, 22 C-1, 4, 5, 6, 9, 10 C-12, 13, 14, 17
H-15 H-14, 16 H-15, 17 H-16
(1H, (2H, (1H, (1H,
brt, 8.4) m) m) 1.95 (1H, m) brt, 9.7)
1.72 (3H, s) 5.47 (1H, dd, 6.6, 6.2) 4.15 (1H, dd, 12.8, 6.2) 4.17 (1H, dd, 12.8, 6.6)
C-17, 20, 22 C-17, 23 C-20, 22
Fig. 5. CD spectra of compounds 1 and 2. Chemical characteristics of compound 1 are as follows.
the 20,22-double bond in 1 can be established on the basis of the NOESY correlation of H3-21 and H2-23. Based on the above analysis, the absolute configuration of 1 can be determined (Fig. 1). Neocyclocitrinol B (2) was isolated from the secondary metabolites of Aspergillus sp. LS116. Comparison of the spectroscopic data with reported known C25 steroid, compound 2 was determined as neocyclocitrinol B (Du et al., 2008; Xia et al., 2014). Aspergillsteroid A (1): White amorphous powder; C23H32O3; [α]D20 +37.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.23), 243 (3.69); CD (MeOH) λmax (Δε) 400 (+0.07), 364 (0), 318 (−1.45), 277 (+0.11), 245 (+2.88), 221 (+1.47), 203 (+7.51), 200 (+4.44); IR (KBr) νmax 2916, 2849, 2361, 2341, 1648, 1125, 1038, 668 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z [M+H]+ 357.2423 (calcd for C23H33O3, 357.2424).
H-5
H-23 H-22
3.3. Antibacterial activity Antibacterial activity against four bacterial V. harveyi strains effects of compounds 1 and 2 were evaluated using broth microdilution in 96well microplates. Remarkably, compound 1 exhibited significant antibacterial activity against V. harveyi KP635244 (Stalin and Srinivasan, 2016) with a MIC value of 16 μg/mL, however, compound 2 showed only weakly antibacterial activity against V. harveyi KP635244 with a MIC value of 128 μg/mL (Table 3). None of compounds 1 and 2 showed antibacterial activity against the other three V. harveyi strains at the tested concentrations up to 128 μg/mL (Nakayama et al., 2006).
Fig. 4. Key 1H–1H COSY, HMBC and NOESY correlations of 1.
circular dichroism (CD) spectrum (Fig. 5) of 1 displayed a negative Cotton effect at 318 (Δε −1.45) nm and a positive Cotton at 245 (Δε +2.88) nm, which was identical with 2. This suggested further that the absolute configuration of A-D ring moieties in 1 and 2 are the same (Du et al., 2008; Xia et al., 2014). At the same time, the E-configuration of
4. Discussion C25 steroids, represented by cyclocitrinol, are a large family of a 4
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Acknowledgments
Table 3 Antibacterial activity against V. harveyi KP635244 effects of compounds 1 and 2. Compounds
1 2 Chloramphenicola
This study was supported by the National Key Research and Development Program of China (2018YFC0310900), the National Natural Science Foundation of China (41776168, 41706167), Ningbo Public Service Platform for High-Value Utilization of Marine Biological Resources (NBHY-2017-P2), Zhejiang Provincial Public Welfare Technology Program (LGC19B020002), the Natural Science Foundation of Ningbo (2018A610303), Ningbo Sci. & Tech. Projects for Common Wealth (2017C10016), the National 111 Project of China (D16013), the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund, and the K.C. Wong Magna Fund in Ningbo University.
Concentration (μg/mL) 128
64
32
16
8
4
2
× + ×
+++ – ×
++ – ×
++ – ×
– – ×
– – ×
– – ×
− no antagonistic action, + presence antagonistic action, ++ moderate antagonistic action, +++ strong antagonistic action, × the growth was inhibited, a Chloramphenicol as positive control.
series of unusual steroids with bicyclo[4.4.1]A/B rings in their structures (Du et al., 2008; Xia et al., 2014; Lin et al., 2015). In the preliminary research, cyclocitrinol was shown to have good cytotoxic activity against HepG2 cell and CaSki cell (Deng et al., 2016) and generally weaker antitumor activity against several human cancer cell lines (Xia et al., 2014). To date, known C25 steroid isomers with bicyclo [4.4.1]A/B rings, were essentially isolated from the genus Penicillium, and their activity studies were mainly focused on cytotoxicity, while other bioactivities research were rarely reported (Du et al., 2008; Xia et al., 2014; Deng et al., 2016). However, to the best of our knowledge, steroids and their derivatives have good antibacterial activity against many pathogens, such as S. aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Salmonella typhimurium (Khan et al., 2008) and E. coli (Khan et al., 2007). Two cyclocitribol analogs, named aspergillsteroid A (1) and neocyclocitrinol B (2), were firstly isolated from a marine sponge-associated Aspergillus fungi. In this paper, our study firstly reported the antibacterial activity of compounds 1 and 2 against V. harvey KP635244. It's worth noting that compound 1 has significant antibacterial activity against V. harveyi KP635244 with a MIC value of 16 μg/mL, but compound 2 only showed weakly antibacterial activity against V. harveyi KP635244 with a MIC value of 128 μg/mL. Comparing the structures of these compounds, it can be found that the absence of the C-23 hydroxyl branch moiety in 1, however, according to previous reports in the literature, we know that modifying hydroxyl groups can improve the efficacy of most antibacterial drugs. In the preliminary study, the following four modifications to hydroxyl groups are generally carried out: acylation to amides or esters; alkylation or alkoxylation; substitution with halogens; remove hydroxyl groups directly (He and Li, 2006; Chen et al., 2013). Therefore, we can speculate that the absence of a C-23 hydroxyl branch moiety in 1 instead exerts a stronger antibacterial activity against V. harveyi KP635244 than 2. In addition, compounds 1 and 2 were also evaluated for the inhibition of the other three V. harveyi strains growth. This results displayed that compounds 1 and 2 showed no antibacterial activity at the tested concentrations up to 128 μg/mL. Antibiotics are a class of low molecular weight (< 3000 Da) natural organic molecules that have been isolated due to their ability to inhibit the activity of organisms (Ryan and Dow, 2008); in generally, they exert their antimicrobial action by combining with specific cellular targets. Therefore, we believe that compounds 1 and 2 may bind specific cellular targets of V. harveyi KP635244, thereby inhibiting growth of V. harveyi KP635244. At the same time, the other three V. harveyi strains lack specific celluar targets that bind to compounds 1 or 2, or specific celluar targets on the other three V. harveyi strains have a weaker ability to bind to compounds 1 or 2 than the V. harveyi KP635244. In conclusion, compound 1 maybe have potential in use as an antibiotic agent to control aquatic pathogens in the future.
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Declaration of competing interest The authors declare that they have no competing interests. 5
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