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p-Terphenyl alcohols from a marine sponge-derived fungus, Aspergillus candidus OUCMDZ-1051

  • Corresponding author: Weiming Zhu, weimingzhu@ouc.edu.cn
  • Received Date: 2019-12-23
    Accepted Date: 2020-02-24
    Published online: 2020-04-02
  • Edited by Chengchao Chen.
  • Dongyang Wang and Peng Qu made equal contributions to this work.
  • Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42995-020-00039-x) contains supplementary material, which is available to authorized users.
  • In order to discover structurally new and bioactive compounds from marine life,we studied the secondary metabolites of the marine-derived fungi associated with a marine sponge (XS-3) from the Xisha islands. As a result,4-O-methylcandidusin A (1),a new p-terphenyl alcohol,along with nine known analogs (2-10),were isolated and identified from the marine sponge-derived fungus Aspergillus candidus OUCMDZ-1051. The structures of these compounds were determined by analyzing spectroscopic data,especially 1D and 2D NMR. The new compound 1 selectively inhibited the growth of the MDA-MB-468,BT474 and A431 human cancer cell lines with the IC50 values of 1.84,6.05 and 0.98 μmol/L,respectively. Compound 1 also displayed a selective antimicrobial activity against Escherichia coli with an MIC value of 27.3 μmol/L. The results indicated 4-O-methylcandidusin A (1) as a potential lead in the new drug discovery for triple negative breast cancer,invasive ductal breast cancer and epidermoid cancer. The antimicrobial metabolites also evidenced a clue for chemical defense of sponges by their associated microorganisms.
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p-Terphenyl alcohols from a marine sponge-derived fungus, Aspergillus candidus OUCMDZ-1051

    Corresponding author: Weiming Zhu, weimingzhu@ouc.edu.cn
  • 1. Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
  • 2. Open Studio for Druggability Research of Marine Natural Products, Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266003, China
  • 3. State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China

Abstract: In order to discover structurally new and bioactive compounds from marine life,we studied the secondary metabolites of the marine-derived fungi associated with a marine sponge (XS-3) from the Xisha islands. As a result,4-O-methylcandidusin A (1),a new p-terphenyl alcohol,along with nine known analogs (2-10),were isolated and identified from the marine sponge-derived fungus Aspergillus candidus OUCMDZ-1051. The structures of these compounds were determined by analyzing spectroscopic data,especially 1D and 2D NMR. The new compound 1 selectively inhibited the growth of the MDA-MB-468,BT474 and A431 human cancer cell lines with the IC50 values of 1.84,6.05 and 0.98 μmol/L,respectively. Compound 1 also displayed a selective antimicrobial activity against Escherichia coli with an MIC value of 27.3 μmol/L. The results indicated 4-O-methylcandidusin A (1) as a potential lead in the new drug discovery for triple negative breast cancer,invasive ductal breast cancer and epidermoid cancer. The antimicrobial metabolites also evidenced a clue for chemical defense of sponges by their associated microorganisms.

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Introduction
  • Marine sponges and their associated secondary metabolites have played a vital role in drug studies (Anjum et al. 2016) featuring compounds such as Cytarabine, Vidarabine, and Eribulin Mesylate. More and more data suggest that the real producers of the natural compounds from marine invertebrates are the symbiotic microorganisms which comprise about 40% by volume of the sponge, including strains that are cultivable and uncultivable (Wilson et al. 2014). Compared with the non-reproducible sponges, the renewable and inexhaustible microorganisms around them seem an eco-friendly source to discovery novel molecules bearing pharmaceutical potential (Brinkmann et al. 2017).

    Statistically, symbiotic microorganisms within sponges accounted for 19% of new and bioactive marine natural products (MNPs) from marine fungi between 1951 and 2014 (Wang et al. 2019a; Zhu et al. 2015). Our research is also interested in the discovery of bioactive new compounds from marine sponge-associated fungi (Huang et al. 2019; Jia et al. 2019; Kong et al. 2015; Liu et al. 2005, 2019; Xin et al. 2005, 2007; Zhu et al. 2018). In our ongoing research, we obtained 47 fungi from an unidentified marine sponge (XS-3) from the Xisha islands, South China Sea. One of these fungal strains, Aspergillus candidus OUCMDZ-1051, produced bioactive metabolites which were cytotoxic to the murine lymphocytic leukemia cell line (P388) at 10 μg/ml and antibacterial to Escherichia coli at 10 μg/disk. Chemical isolation led to the identification of a new p-terphenyl alcohol, 4-O-methylcandidusin A (1) and nine known analogs, 4, 5-di-O-methylcandidusin A (2) (Guo et al. 2012), candidusin A (3) (Kobayashi et al. 1982; Liu et al. 2012), prenylcandidusins B (4) and C (5) (Cai et al. 2011), candidusin C (6) (Rahbaek et al. 2000), 3-hydroxyterphenyllin (7) (Yen et al. 2001), terphenyllin (8) (Kamigauchi et al. 1998), 4″-deoxy-3-hydroxyterphenyllin (9) (Guo et al. 2012) and prenylterphenyllin B (10) (Cai et al. 2011) (Fig. 1). Compounds 1-3 and 8 exhibited antimicrobial activity against E. coli, Enterobacter aerogenes, Candida albicans and Pseudomonas aeruginosa with MIC (minimal inhibitory concentration) values of 27.3, 33.2, 37.2 and 71.7 μmol/L, respectively. The new compound 1 showed a broad-spectrum cytotoxic activity against 21 cancer cell lines among 26 human cancer cell lines tested with IC50 (half maximal inhibitory concentration) values between 0.98 and 19.1 μmol/L.

    Figure 1.  Structures of compounds 1-10

Results
  • Compound 1, a white powder, was established to have the molecule formula C21H18O6 from the HRESIMS peak at m/z 367.1166 [M+H]+ (Supplementary Fig. S1) that is 14 Da more than candidusin A (3). 1H-NMR (Table 1 and Supplementary Table S1) and 1H-1H-COSY (Fig. 2 and Supplementary Fig.S5) showed the structure containing one 1, 2, 4, 5-tetrasubstituted benzene ring [δH-3 7.37(s), δH-6 7.41(s)], one 1, 4-disubstituted benzene ring [δH-2″/6″ 7.42 (d, J=8.8 Hz), δH-3″/5″ 6.86 (d, J=8.8 Hz)], one penta-substituted benzene ring [δH-5′ 6.72 (s)], and three methoxy groups [δH-6′-OCH3 3.97(s), δH-4-OCH3 3.87(s), δH-3′-OCH3 3.77(s)]. 13C-NMR (Table 1, Supplementary Figs. S3, S4 and S6) gave 18 aromatic carbons between 96.2 and 156.8 ppm including 11 aromatic nonprotonated carbons and seven aromatic methine carbons. These NMR data featured molecule 1 as a p-terphenyl skeleton fused a furan moiety between rings B and C that is very closely related to candidusin A (3) (Liu et al. 2012). The major difference was presented in benzene ring C (Table 1, Supplementary Tables S1 and S2). Except for an additional methoxy signal at δH/C 3.87/56.1, C-2-C-4 and H-3 had obvious shifts and the shift values were +1.3, +2.9, -1.6 and -0.26 ppm, respectively. These data indicated a 4-O-methylation that was further supported by the following 2D NMR experiment. The key HMBC correlations (Fig. 2, Supplementary Figs. S7 and S8) of H-5′ (δH 6.72) to C-1″ (δC 128.6), H-3″/5″ (δH 6.86) to C-1″ and H-2″/6″ (δH 7.42) to C-4′ (δC 131.0) indicated a connection of benzene rings A and B by a C-1″-C-4′ single bond. The key HMBC correlations of H-5′ to C-1′ (δC 113.9), H-6 (δH 7.41) to C-1′ and H-3 (δH 7.37) to C-1 (δC 114.6) indicated a connection of benzene rings B and C by a C-1′-C-1 single bond. The HMBC correlations of 3′-OCH3 (δH 3.77) to C-3′ (δC 136.0), 6′-OCH3 (δH 3.97) to C-6′ (δC 149.6) and 4-OCH3 (δH 3.87) to C-4 (δC 148.0) located the positions of these three methoxy groups. Thus, compound 1 was established as 4-O-methylcandidusin A. The known compounds 2-10 were identified by comparing their NMR data (Supplementary Tables S1-S3) and Physical properties (see Physical properties of compounds 2-10 in Supporting material).

    No. δH (J in Hz) δC, type
    1 114.6, C
    2 148.7, C
    3 7.37, s 96.2, CH
    4 148.0, C
    5 143.6, C
    6 7.41, s 107.0, CH
    4-OMe 3.87, s 56.1, CH3
    1′ 113.9, C
    2′ 149.3, C
    3′ 136.0, C
    4′ 131.0, C
    5′ 6.72, s 105.7, CH
    6′ 149.6, C
    3′-OMe 3.77, s 60.6, CH3
    6′-OMe 3.97, s 55.9, CH3
    1″ 128.6, C
    2″ 7.42, d (8.8) 130.4, CH
    3″ 6.86, d (8.8) 115.1, CH
    4″ 156.8, C
    5″ 6.86, d (8.8) 115.1, CH
    6″ 7.42, d (8.8) 130.4, CH

    Table 1.  The 1H (600 MHz) and 13C (150 MHz) NMR data for compound 1 in DMSO-d6

    Figure 2.  The key 2D NMR correlations of compound 1

    The cytotoxic activity of new compound 1 against 26 human tumor cell lines (Table 2 and Bioassay Protocol in Supporting material) were tested by the Cell Titer Glo (CTG) assay and doxorubicin (Dox) was used as the positive control. The results indicated that 4-O-methylcandidusin A (1) was active against 21 tumor cell lines with IC50 values of 0.98-19.1 μmol/L in the 26 tested tumor cell lines (Table 2). Especially, compound 1 showed a stronger or comparable inhibitory activity to the positive control (doxorubicin) against the triple negative breast cancer (MDA-MB-468), the breast invasive ductal carcinoma (BT474), and the epidermoid carcinoma (A431) cell lines, with the IC50 values of 1.84, 6.05, and 0.98 μmol/L, respectively (Table 2).

    Cell linea 1 Dox
    N87 6.05±0.24 0.05±0.01
    A673 16.75±0.42 0.10±0.01
    MV4-11 1.66±0.05 0.16±0.01
    K562 23.12±1.19 0.02±0.01
    A549 5.42±0.08 0.11±0.01
    BT474 6.05±0.04 1.90±0.01
    H1299 5.56±0.34 0.50±0.03
    HUCCT1 9.55±0.86 0.05±0.01
    MDA-MB-468 1.84±0.02 > 100
    H1975 8.45±0.41 0.09±0.01
    HL-60 4.16±0.01 0.20±0.01
    Karpas299 > 100 0.38±0.01
    U87 > 100 0.11±0.01
    A431 0.98±0.02 0.16±0.01
    U251 19.10±0.53 0.17±0.01
    HCC1954 4.93±0.07 0.05±0.01
    MCF-7 7.75±0.10 0.10±0.01
    MKN-45 7.52±0.45 0.19±0.01
    DU145 1.79±0.11 0.05±0.01
    SPC-A1 9.25±0.67 0.18±0.01
    HCT116 9.31±0.16 0.10±0.01
    MDA-MB-231 1.89±0.10 0.18±0.01
    143B 5.86±0.13 0.10±0.01
    B16F10 > 100 0.02±0.01
    H2228 > 100 0.10±0.01
    Hep3B > 100 16.29±1.39
    aThe details of the 26 cell lines are listed in the supplementary material

    Table 2.  Cytotoxicity of compound 1 against 26 human cancer cell lines (IC50 μmol/L)

    The antimicrobial activities of p-terphenyl alcohols 1-10 against human pathogenic bacteria, Enterobacter aerogenes (ATCC 13408), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC1 1775) and Pseudomonas aeruginosa (ATCC10145) and fungus Candida albicans (ATCC 10231), were evaluated using the filter disk method. Ciprofloxacin and ketoconazole were used as the positive controls for bacteria and fungus, respectively. The results (Table 3) showed that new compound 1 and the known compounds 2, 3 and 8 displayed a weak antimicrobial activity against E. coli, E. aerogenes, C. albicans and P. aeruginosa with MIC values of 27.3, 33.2, 37.2 and 71.7 μmol/L, respectively.

    Compound E. aerogenes S. aureus P. aeruginosa E. coli C. albicans
    1 > 200 > 200 > 200 27.3 > 200
    2 33.2 > 200 > 200 > 200 > 200
    3 71.7 > 200 71.7 143.4 > 200
    4 > 200 > 200 > 200 55.8 55.8
    5 > 200 115.0 > 200 > 200 > 200
    6 > 200 > 200 > 200 > 200 > 200
    7 > 200 141.0 > 200 141.0 > 200
    8 > 200 > 200 > 200 148.8 37.2
    9 > 200 > 200 > 200 > 200 > 200
    10 > 200 > 200 123.0 > 200 > 200
    Ciprofloxacin 3.2 0.4 13.0 0.1
    Ketoconazole 9.4

    Table 3.  The MIC values (μmol/L) of p-terphenyl alcohols 110 against pathogenic microbes

Discussion
  • p-Terphenyl alcohols are a kind of natural products mainly derived from fungi. The structural core is a C-18 tricyclic skeleton formed by three benzene rings that the two phenyl groups (rings A and C) connect at the 1, 4-positions of the middle benzene ring (ring B) (Wang et al. 2019b). The structural diversity of the natural p-terphenyls mainly resulted from the variation of the ring B, the number and the position of hydroxy groups, and the changes of the phenol to quinone functions. Approximately 230 natural p-terphenyls have been identified from the year 1877 to 2018 (Li et al. 2018; Liu 2006), and this number is growing every year (Kalansuriya et al. 2019; Kalvo et al. 2018; Lin et al. 2019; Lu et al. 2019; Narmani et al. 2019; Ren et al. 2018; Sangsopha et al. 2019; Song et al. 2019; Wang et al. 2019b; Xu et al. 2018). Our present work isolated and identified a new p-terphenyl alcohol with a dibenzofuran moiety from the marine sponge-derived fungus A. candidus OUCMDZ-1051, as well as nine known p-terphenyl alcohols. The strong and selective inhibition of compound 1 on MDA-MB-468, BT474 and A431 tumor cell lines indicated the potential use of 4-O-methylcandidusin A (1) as a lead for inhibiting triple negative breast cancer, invasive ductal breast cancer and epidermoid cancer. The antimicrobial activity of metabolites 1, 2, 3 and 8 hints at chemical protection of symbiotic microbes for sponge against pathogenic microorganisms.

Materials and methods

    General experimental procedures

  • Analytical HPLC were carried out on a Shimadzu LC-6AD equipped with a Shimadzu SPD-M10A detector (250 mm×4.6 mm YMC-Pack ODS-A column). Preparative HPLC was performed on a Waters 1525 equipped with Waters 2487 Dual λ Absorbance Detector (250 mm×10 mm YMC-Pack ODS-A column); UV data were obtained on a NanoDrop One Microvolume UV-Vis Spectrophotometer. IR spectra were measured in KBr disks on a Nicolet Nexus 470 spectrophotometer. NMR spectra were recorded on a JNM-ECP600 spectrometer, and the residual DMSO signals at δH/C 2.50/39.52 were used to reference the chemical shifts. HRESIMS data were measured using a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. TLC (thin-layer chromatography) plates pre-coated with silica gel GF254 (10-40 μm) and silica gel H (Qingdao Marine Chemical Factory) were respectively used for TLC, column chromatography (CC), and vacuum liquid chromatography (VLC). Sephadex LH-20 (Amersham Biosciences) was soaked overnight in a CH2Cl2/MeOH (1:1) mixture before use.

  • Strain information

  • Strain OUCMDZ-1051 was isolated from an unidentified sponge sample (No. XS-3) collected from Xisha islands, South China Sea in 2009, by a serial dilution method using a PDA agar medium that was prepared by dissolving 200 g Potato infusion, 20 g glucose and 20 g agar in 1 l natural sea water supplemented with 0.02% chloramphenicol. In brief, the washed and ground sponge tissue (1 g) was added into 9 ml of sterile natural seawater. Then, 100 μl of suspension was deposited on a PDA age plate incubated at 28 ℃ for 3 days. The fungus was purified using the same media and characterized as A. candidus (GenBank accession no. MN582997) by the comparison of its 18S rRNA gene sequence with the type strains. The strain was preserved in our Laboratory at -80 ℃, Ocean University of China.

  • Production, extraction and purification

  • Spores of the strain, grown on PDA agar plates, were inoculated into 1-l Erlenmeyer flasks; each contained 300 ml liquid medium (20 g mannitol, 10 g glucose, 20 g maltose, 3 g yeast extract, 10 g glutamate, 1 g corn syrup, 20 g CaCO3, 0.5 g KH2PO4, 0.3 g MgSO4·7H2O and 1 l natural seawater). After 35 days of static culture at room temperature, the mycelium was separated from the liquid by gauze. The 69 l ferment liquor was extracted three times with equal volume of ethyl acetate (EtOAc). The EtOAc layers were combined and evaporated to dryness at 37 ℃ using a rotary evaporator. The mycelium was extracted three times by 80% acetone aqueous, each for 1 h in an ultrasonic cleaning instrument. The acetone was removed from extracting solution at 37 ℃ using the rotary evaporator and the residual solution was extracted three times using triple volume of EtOAc. A total of 42 g crude extract was obtained.

    The extract was separated by VLC using a CH2Cl2/MeOH mixture solvent gradient from 100% CH2Cl2 to 100% MeOH, resulting in 11 fractions (Fr.1-Fr.11). Fr.4 (4.3 g) was separated into 12 subfractions (Fr.4.1-Fr.4.12) via a silica gel flash column eluting with petroleum ether (PE) and acetone mixture gradient. Fr.4.10 was purified on a semi-preparation ODS column eluting with 80% MeOH to obtain compound 5 (12.0 mg, tR 9.8 min). Fr.4.9 was purified by semi-preparation HPLC on an ODS column using the solvent system of 75% MeOH to yield compounds 1 (7.9 mg, tR 5.0 min) and 10 (5.9 mg, tR 8.0 min). Fr.4.4 was separated into four subfractions (Fr.4.4.1-Fr.4.4.4) on Sephadex LH-20 eluting with a CH2Cl2/MeOH (1:1) mixture. Fr.4.4.3 was further purified by semi-preparation HPLC on an ODS Column using 82% MeOH to obtain compounds 4 (12.3 mg, tR 15.0 min) and 6 (6.2 mg, tR 6.9 min). Fr.3 (1.4 g) was separated into five subfractions (Fr.3.1-Fr.3.5) via a silica gel flash column eluting with a PE/acetone mixture gradient. Fr.3.4 was separated into three subfractions (Fr3.4.1-Fr3.4.3) on Sephadex LH-20 eluting with a CH2Cl2/MeOH (1:1) mixture. Fr3.4.2 and Fr3.4.3 were respectively purified by HPLC on an ODS column using 80% MeOH and 60% MeOH to yield compounds 2 (21 mg, tR 13.0 min) and 9 (4.5 mg, tR 17.0 min). Fr.5 was purified on an LH-20 column eluting with a CH2Cl2/MeOH (1:1) mixture to yield compound 8 (132.0 mg). Fr.7 was separated into 10 subfractions (Fr.7.1-Fr.7.10) via a silica gel flash column eluting with a PE/acetone mixture gradient. Fr.7.8 was performed on an LH-20 column eluting with a CH2Cl2/MeOH (1:1) mixture to give five subfractions (Fr.7.8.1-Fr.7.8.5). Fr.7.8.3 was separated into compound 3 (11.7 mg, tR 8.0 min) and a subfraction (Fr.7.8.5.1) on an ODS column using 65% MeOH; Fr.7.8.5.1 was further purified on the same column eluting with 65% MeOH to give compound 7 (35.0 mg, tR 10.0 min).

    4-O-Methylcandidusin A (1) White powder; UV (MeOH) λmax (log ε) 218 (4.40), 281 (4.15), 295 (4.12), 325 (4.20), 334 (4.24) nm; IR νmax (KBr) 3417, 2938, 2838, 2766, 1635, 1610, 1522, 1483, 1459, 1393, 1357, 1338, 1267, 1230, 1190, 1130, 1105, 1025, 913, 824, 586 cm-1; 1H and 13C NMR data (Table 1); HRESIMS m/z 367.1166 [M+H]+ (calcd for C21H19O6, 367.1176).

  • Cytotoxicity and antimicrobial assay

  • 4-O-Methylcandidusin A (1) was evaluated for cytotoxicity against 26 human cancer cell lines by the CTG assay (Shen et al. 2019; Wang et al. 2019b). The cell suspensions were prepared at 37 ℃ by growing cells in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution under a humidified atmosphere of 5% CO2 and 95% air. The cell suspension (100 μl) at a density of 20 cell/μl and 90 μl fresh medium was plated into a well of a 96-well microtiter plate and incubated overnight. Then, 10 μl of the compound solution in DMEM with varying concentrations from 100.0 to 0.032 μM by a consecutive twofold dilution was added and incubated for 72 h. To each well was added 100 μl of CTG solution and incubated in the dark for 10 min at room temperature. The absorbance of each well was recorded by a BioTek Synergy H1m multi-mode microplate reader.

    The antimicrobial activities against four human pathogenic bacteria (E. aerogenes, S. aureus, P. aeruginosa and E. coli) and one pathogenic fungus (C. albicans) were evaluated using the paper disk method as we previously described (Wang et al. 2017). In brief, the tested compounds and positive controls were respectively dissolved into methanol with the initial concentration of 0.1 mg/ml (> 200 μM). Only those samples with obvious inhibition zones to pathogenic microbes were further tested for MICs (presented as μM) by a double dilution method.

Acknowledgements
  • This work is supported by the National Natural Science Foundation of China (NSFC) (Nos. U1906213, 41876172, and U1606403).

Authors Contributions
  • D. Wang identifes the structures and write the draft of the manuscript. P. Qu isolates the fungus and compounds. J. Zhou and L. Wang test the bioactivities. Y. Wang identifes the fungus. W. Zhu designes the study and revises the manuscript and the proof.

Compliance with ethical standards

    Conflict of interest

  • The authors declare that they have no confict of interest.

  • Animal and human rights statement

  • This article does not contain any studies with human participants or animals performed by any of the authors.

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