Anti-inflammatory metabolites from a marine sponge-associated fungus Aspergillus sp. IMBC-FP2.05

Cite this paper: Vietnam J. Chem., 2021, 59(1), 52-56 Article DOI: 10.1002/vjch.202000099 52 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Anti-inflammatory metabolites from a marine sponge-associated fungus Aspergillus sp. IMBC-FP2.05 Tran Hong Quang1*, Le Thi Vien1, Le Ngoc Anh1, Nguyen Thi Thanh Ngan2, Tran Thi Hong Hanh1, Nguyen Xuan Cuong1, Nguyen Hoai Nam1, Chau Van Minh1 1Institue of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 2Institue of Genome Research, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Submitted June 17, 2020; Accepted July 15, 2020 Abstract Chemical investigation of the marine sponge-derived fungal strain Aspergillus sp. IMBC-FP2.05 resulted in isolation of five compounds, including JBIR-74 (1), homogentisic acid (2), methyl (2,5-dihydroxyphenyl)acetate (3), 3- chloro-2,5-dihydroxybenzyl alcohol (4), and p-hydroxybenzaldehyde (5). Their chemical structures were identified by comprehensive analyses of the 1D and 2D NMR and mass spectra in comparison with the previously reported data. Furthermore, nitric oxide (NO) inhibitory effects of the isolated compounds in LPS-stimulated RAW264.7 cells were also reported. Keywords. Aspergillus, marine-derived fungus, diketopiperazine, phenolic, nitric oxide. 1. INTRODUCTION The Aspergillus genus comprises of more than 300 species occurring in various habitats,[1] of which the marine-derived species have been shown to produce various types of secondary metabolites, including alkaloids, phenolics, terpenoids, and peptides with interesting biological effects such as cytotoxic, antimicrobial, and anti-inflammatory activities.[2] However, little is known about the chemical constituents and biological activities of Aspergillus aculeatinus so far, except that a strain isolated from Taxus chinensis var. mairei has been reported to produce the anticancer drug taxol.[3] In our continuing search for secondary metabolites from the Vietnamese marine-derived fungi,[4,5] we found that an EtOAc extract of the fungal strain Aspergillus sp. IMBC-FP2.05 displays NO inhibitory effects in LPS-stimulated RAW264.7 cells. In the present study, we report isolation and structural elucidation of five secondary metabolites from the EtOAc extract of the fermentation culture of the fungal strain Aspergillus sp. IMBC-FP2.05. In addition, NO inhibitory effects of the isolated metabolites in LPS-stimulated RAW264.7 cells were also evaluated. 2. MATERIAL AND METHODS 2.1. Fungal material The fungal strain IMBC-FP2.05 was isolated from an unidentified sponge collected from Quang Nam sea, Vietnam during May, 2019. After sterilizing the surface, the sponge sample was cut into small pieces and ground using a sterile mortar and pestle and mixed with sterile seawater. A portion of the sample was processed using the spread plate method in potato dextrose agar (PDA) medium containing seawater. The plate was incubated at 25 oC for 15 days. After sub-culturing the isolates several times, the final pure cultures were selected and preserved at -80 oC. The strain was identified by DNA amplification and analysis of the ITS region of the rDNA sequence. A GenBank search using the ITS rDNA gene of IMBC-FP2.05 revealed that Aspergillus aculeatinus CBS 121060 (NR 135417.1) is the closest match, with 99 % sequence identity. Thus, the fungal strain IMBC-FP2.05 was identified as Aspergillus sp. 2.2. General experimental procedures Optical rotations were determined using a Jasco P- 2000 digital polarimeter. The NMR spectra were recorded on Bruker AVANCE III HD 500 FT-NMR spectrometer. ESIMS data were obtained using an Vietnam Journal of Chemistry Tran Hong Quang et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 53 ESI Q-TOF MS/MS system (AB SCIEX Triple). TLC was performed on Kieselgel 60 F254 (Merck) or RP-18 F254s (Merck) plates. Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and YMC RP-18 resins. Preparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1200 Preparative HPLC System. 2.3. Fermentation and extraction The fungal strain Aspergillus sp. IMBC-FP2.05 was grown on 80 replicate 2L-Erlenmeyer flasks (each flask contained 100 mL of PDA mixed with 3 % NaCl). The seed cultures (2 mL) of the fungal strain was added to the growing media and incubated at 25 oC for 20 days. Subsequently, the fungal biomass was extracted with EtOAc (20 L x three times) to provide an organic phase which was further concentrated under reduced pressure to give a residue (8.25 g). 2.4. Isolation and identification The EtOAc extract of Aspergillus sp. IMBC-FP2.05 was introduced to reversed phase (RP) C18 flash column chromatography (CC), using a stepwise gradient elution of 20, 40, 60, 80, and 100 % MeOH in H2O to give six fractions (F1-F6), respectively. F1 was subjected to sephadex LH-20 CC, eluting with MeOH-H2O (1:2, v/v) to provide subfractions F1.1- F1.4). F1.4 was then purified by RP C18 prep. HPLC, using an isocratic elution of acetonitrile in H2O (7:93, v/v) over 60 min to release 2 (24 mg) and 3 (11 mg). Fraction F2 was separated by Sephadex LH-20 CC, using MeOH-H2O (1:2, v/v) as eluent to provide six subfractions (F2.1–F2.6). Compound 5 (14 mg) was isolated from the subfraction F2.5 by silica gel CC, using CH2Cl2-MeOH (10:1, v/v) as a mobile phase. F2.6 was purified through a RP C18 prep. HPLC, using acetonitrile in H2O (7:93, v/v) over 60 min to afford 4 (1.5 mg). F3 was fractionated by sephadex LH-20 CC, eluting with MeOH-H2O (2:1, v/v) to provide subfractions F3.1 and F3.2. Subfraction F3.1 was separated by silca gel CC, eluting with CH2Cl2- MeOH (15:1, v/v) to provide F3.1.1 and F3.1.2. From the subfraction F3.1.1, compound 1 (20 mg) was obtained by RP C18 CC, using acetone-H2O (1:2, v/v) as a mobile phase. JBIR-74 (1): White, amorphous powder; C12H16N4O2, M = 248; ESIMS: m/z 249 [M+H]+; 1H (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see table 1. Homogentisic acid (2): White, amorphous powder; C8H8O4, M = 168; ESIMS: m/z 169 [M+H]+; 1H (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see table 2. Methyl (2,5-dihydroxyphenyl)acetate (3): White, amorphous powder; C9H10O4, M = 182; ESIMS: m/z 183 [M+H]+; 1H (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see table 2. 3-Chloro-2,5-dihydroxybenzyl alcohol (4): White, amorphous powder; C7H7ClO3, M = 174; ESIMS: m/z 175 [M+H]+; 1H (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see table 2. p-Hydroxybenzaldehyde (5): Pale yellow, amorphous powder; C7H6O2, M = 122; ESIMS: m/z 123 [M+H]+; 1H (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see table 2. 2.5. Anti-inflammatory assay Effects of the isolated compounds toward NO overproduction in LPS-stimulated RAW264.7 cells were evaluated using the Griess assay as previously described.[4] 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white, amorphous powder. Its molecular formula was established as C12H16N4O2 by a protonated molecular ion [M+H]+ at m/z 249 in the ESIMS in combination with an analysis of the 1H and 13C NMR spectra. The 1H NMR spectrum exhibited signals of three olefinic protons at H 6.71 (s, H-3), 7.36 (s, H-5), and 7.79 (s, H-6) which showed HSQC correlations with C 107.0 (C-3), 119.8 (C-5), and 137.3 (C-6), respectively in the HSQC spectrum. In the HMBC spectrum, correlations observed from H-5 to C-4 and C-6 and from H-6 to C-4 and C-5 revealed the presence of an imidazole ring in the molecule (Figure 1). H-5 additionally exhibited an HMBC correlation with C-3, while H-3 was found to have HMBC correlations with an amide carbonyl carbon at C 162.6 (C-1), a non-protonated olefinic carbon at C 125.2 (C-2), and C-4, suggesting the presence of a 2-amino-3-(1H-imidazol-5-yl)acrylic acid unit.[6] In the 1H NMR spectrum, signals for one primary methyl at H 1.01 (t, J = 7.5 Hz, H3-5) and one secondary methyl at H 0.92 (d, J = 6.5 Hz, H3-6) were also observed. In addition, a down-field shifted proton signal at H 4.19 (H-2) showed an HSQC correlation with C 60.4 (C-2), suggesting the presence of a nitromethine group. HSQC interactions observed between a proton at H 2.07 (H-3) and an aliphatic carbon at C 41.9 (C-3) and between two protons at H 1.52/1.35 (each m, H2-4) and a carbon at C 26.5 (C-4) allowed to point out Vietnam Journal of Chemistry Anti-inflammatory metabolites from © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 54 Figure 1: Chemical structures of 1-5 and selected HMBC correlations of 1, 3, and 4 the appearance of one methine and one methylene group, respectively. In the HMBC spectrum, correlations observed from H3-5 to C-3 and C-4, from H3-6 to C-2, C-3, and C-4, and from H-3 to an amide carbonyl carbon at C 167.9 (C-1) and C-2 revealed that 1 possesses an isoleucine as a partial structure.[6] The conjunction of the 2-amino-3-(1H- imidazol-5-yl)acrylic acid and isoleucine units was deduced by HMBC correlations from H-2 to C-1 and from H-3 to C-1. The Z geometry of the 2,3- double bond was identified based on the W type long range correlation from H-3 to C-1.[6] Subsequently, the structure of 1 was confirmed by the good agreement when comparing its NMR data with those of the reported diketopiperazine alkaloid, JBIR-74.[6] On the basis of the spectroscopic analysis, compound 1 was identified as JBIR-74. Table 1: 1H and 13C NMR data for compound 1 Position C# 1 Ca,b Ha,c (mult., J in Hz) 1 166.8 167.9 - 2 59.2 60.4 4.19 (d, 2.0) 3 40.7 41.9 2.07 (m) 4 25.4 26.5 1.52 (m), 1.35 (m) 5 12.8 12.2 1.01 (t, 7.5) 6 11.0 14.0 0.92 (d, 6.5) 1 161.4 162.6 - 2 124.0 125.2 - 3 105.8 107.0 6.71 (s) 4 137.0 138.2 - 5 118.6 119.8 7.36 (s) 6 136.1 137.3 7.79 (s) aRecorded in CD3OD, b125 MHz, c500 MHz #δC of JBIR-74 in CD3OD.[6] Compound 2 was isolated as a white, amorphous powder, with the molecular C8H8O4 as deduced by a quasi-molecular ion m/z 169 [M+H]+ in the ESIMS. The 1H NMR spectrum showed signals characteristic of an ABX spin system [H 6.66 (d, J = 8.5 Hz, H-3), 6.57 (dd, J = 2.5, 8.5 Hz, H-4), and 6.64 (d, J = 2.5 Hz, H-6)] and a singlet of a methylene group at H 3.55 (s, H2-7). The 13C NMR spectrum contained eight signals, including one carbonyl carbon at C 176.2 (C-8), six sp2 carbons of a 1,2,5-trisubstituted benzene ring of which two were oxygenated at C 149.6 (C-2) and 151.0 (C-5), and one sp3 carbon at C 36.6 (C-7). Comparison of the 1H and 13C NMR data of 2 with those of the reported phenylacetic acid analog, homogentisic acid revealed the good match, suggesting the similarity of both structures.[7] Therefore, compound 2 was determined to be homogentisic acid. Compound 3 was obtained as a white, amorphous powder. Comparison of the 1H and 13C NMR data of 3 with those of 2 revealed the close similarity, except for the additional presence of a methoxy group in 3 at H 3.69 (s)/C 52.3 located at the carbonyl carbon position. This was supported by observation of an HMBC correlation from the proton signal of the methoxy group at H 3.69 to the carbonyl carbon signal at C 174.6 (C-8) in the HMBC spectrum. Thus, the structure of 3 was elucidated as methyl (2,5-dihydroxyphenyl)acetate.[8] Compound 4 was purified as a white, amorphous powder and its molecular formula was established as C7H7ClO3 based on the presence of a protonated molecular ion [M+H]+ at m/z 175, along with analysis of its 1H and 13C NMR spectra. In the 1H NMR spectrum, characteristic signals of two meta- coupling protons at H 6.69 and 6.75 (each d, J = 3.0 Hz, H-4 and H-6) were observed, suggesting the presence of a 1,2,3,5-tetrasubstituted aromatic ring. In addition, a singlet signal of an oxymethylene group at H 4.64 (s, H2-7) was also observed in the 1H NMR spectrum. The 13C NMR and HSQC spectra showed signals of six sp2 of which two non- Vietnam Journal of Chemistry Tran Hong Quang et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 55 protonated carbons at C 132.1 (C-1) and 121.9 (C-3) and two oxygenated carbons at C 144.4 (C-2) and 151.8 (C-5), along with one sp3 carbon at C 61.2 (C-7). The location of the oxymethylene group at C- 1 position was deduced by an HMBC correlation from H 4.64 (H2-7) to C-1, C-2, and C-6 in the HMBC spectrum (Figure 1). The remaining non- protonated sp2 carbon, with the more up-field chemical shift at C 121.9 compared with that of C-1 (C 132.1) was suggested to be a location of a halogen atom. Accordingly, comparison of the 1H and 13C NMR data of 4 with those of a previously reported chlorobenzyl derivative, 3-chloro-2,5- dihydroxybenzyl alcohol resulted in the good match, suggesting that the structures of both compounds are identical and the location of a chlorine atom at C-3 position.[9] Assignment of all the positions of 4 was further confirmed by detailed analysis of the HMBC spectrum as shown in figure 1. Thus, the overall structure of 4 was concluded to be 3-chloro-2,5- dihydroxybenzyl alcohol. Table 2: 1H- and 13C-NMR data for compounds 2-5 Pos. C#1 2 C#2 3 C#3 4 C#4 5 Ca,b Ha,c Ca,b Ha,c Ca,b Ha,c Ca,b Ha,c 1 124.3 123.6 - 124.3 123.2 - 132.0 132.1 - 129.9 130.3 - 2 150.4 149.6 - 148.2 149.6 - 144.3 144.4 - 132.5 133.4 7.80 (d, 9.0) 3 117.4 116.8 6.66 (d, 8.5) 115.8 116.7 6.65 (d, 8.5) 121.8 121.9 - 116.0 116.9 6.94 (d, 9.0) 4 116.0 115.6 6.57 (dd, 2.5, 8.5) 114.7 115.7 6.57 (dd, 3.0, 8.5) 115.4 115.5 6.69 (d, 3.0) 161.6 165.1 - 5 151.6 151.0 - 150.0 151.1 - 151.7 151.8 - 116.0 116.9 6.94 (d, 9.0) 6 119 118.6 6.64 (d, 2.5) 117.9 118.6 6.62 (d, 3.0) 114.5 114.5 6.75 (d, 3.0) 132.5 133.4 7.80 (d, 9.0) 7 37.7 36.6 3.55 (s) 35.5 36.5 3.57 (s) 61.1 61.2 4.64 (s) 191.2 192.8 9.78 (s) 8 177.0 176.2 - 172.2 174.6 - OCH3 51.8 52.3 3.69 (s) aRecorded in CD3OD, b125 MHz, c500MHz, multiplicity and coupling constants (Hertz) are given in parentheses; #1δC of homogentisic acid in CD3OD;[7] #2δC of methyl (2,5-dihydroxyphenyl)acetate in DMSO-d6;[8] #3δC of 3-chloro-2,5- dihydroxybenzyl alcohol in CD3OD;[9] #4C of p-hydroxybenzaldehyde in CDCl3.[10] Compound 5 was given as a pale yellow, amorphous powder. Its molecular formula was determined to be C7H6O2 by the ESIMS and the 1H and 13C NMR spectroscopic data. The 1H NMR spectrum contained signals of two pairs of ortho- coupling aromatic protons at H 7.80 (each d, J = 9.0 Hz, H-2 and H-6) and 6.94 (d, J = 9.0 Hz, H-3 and H-5), suggesting the presence of a 1,4-disubstituted benzene ring. The 1H NMR additionally showed a far down-field shifted signal of at H 9.78 (s, H-7), implying the presence of an aldehyde group. The 13C NMR spectrum displayed seven carbon signals, including one aldehyde carbon at C 192.8 (C-7) and six carbons of the para-substituted aromatic ring. This spectroscopic data suggested that 5 possesses the benzaldehyde skeleton type. Accordingly, the 1H and 13C NMR data of 5 were shown to be similar with those of the reported p-hydroxybenzaldehyde, indicating both compounds have the identical structures.[10] Thus, the structure of 5 was established as p-hydroxybenzaldehyde. The in vitro anti-inflammatory effects of the isolated compounds 1-5 were evaluated using LPS- induced NO overproduction in RAW264.7 cells, with the concentration range up to 80 M.[11] The result revealed that two 2,5-dihydroxyphenylacetic acid derivatives, including shomogentisic acid (2) and methyl (2,5-dihydroxyphenyl)acetate (3) among the tested compounds, showed the most inhibitory effects against NO overproduction, with IC50 values of 28.2 and 14.2 M, respectively (yable 3). The chloro-benzyl alcohol (4) exhibited an NO inhibitory effect (IC50 = 41.8 M) which was comparable with that of the positive control, NG-Monomethyl-L- arginine (L-NMMA) (IC50 = 44.5 M). Compounds 1 and 5 moderately inhibited NO overproduction in LPS-stimulated RAW264.7 cells, with IC50 values of 79.7 and 62.4 M, respectively. Table 3: Nitric oxide inhibitory effects of compounds 1-7 Compound IC50 (µM)a 1 79.72.4 2 28.23.0 3 14.21.5 4 41.83.8 5 62.42.9 L-NMMAb 44.56.2 aThe values are mean  SD (n = 3); bPositive control. Vietnam Journal of Chemistry Anti-inflammatory metabolites from © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 56 In conclusion, our chemical study of the marine- derived fungal strain Aspergillus sp. IMBC-FP2.05 led to the isolation of five metabolites: JBIR-74 (1), homogentisic acid (2), methyl (2,5- dihydroxyphenyl)acetate (3), 3-chloro-2,5- dihydroxybenzyl alcohol (4), and p- hydroxybenzaldehyde (5). It is noted that this is the first time to report the in vitro anti-inflammatory effects of compounds 1, 3, and 4. Acknowledgment. This work was financially supported by Vietnam Academy of Science and Technology (VAST04.05/19-20). REFERENCES 1. R. A. Samson, C. M. Visagie, J. Houbraken, S.-B. Hong, V. Hubka, C. H. Klaassen, G. Perrone, K. 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