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Polyketide derivatives from the sponge associated fungus Aspergillus europaeus with antioxidant and NO inhibitory activities
Fitoterapia 130 (2018) 190–197
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Polyketide derivatives from the sponge associated fungus Aspergillus europaeus with antioxidant and NO inhibitory activities Xiaowen Dua, Dong Liua, Jian Huanga, Chanjuan Zhanga, Peter Prokschb, Wenhan Lina, a b
T ⁎
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, 100191, PR China Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine- Universität Düsseldorf, 40225 Düsseldorf, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspergillus europaeus Polyketides Structure elucidation Radical scavenging activity Inhibition of NO production Inhibition of NF-κB activation
DPPH assay of the in-house marine-derived fungi uncovered that the EtOAc extract of the cultured fungus Aspergillus europaeus WZXY-SX-4-1, which was isolated from the marine sponge Xestospongia testudinaria, possesses radical scavenging activity. Chromatographic separation of the bioactive extract resulted in the isolation of 20 polyketide derivatives, including six new compounds namely eurobenzophenones A-C (1–3), euroxanthones A-B (4–5), and (+)1-O-demethylvariecolorquinones A (6). The structures of new compounds were determined on the basis of the analyses of spectroscopic data, including the Snatzke method for the configurational assignment. Benzophenones 3, 9 and 10 exhibited potent radical scavenging activity against DPPH. All polyketides were evaluated for the inhibitory effects toward the LPS induced nitric oxide (NO) production in mouse microglia BV2 cells and the NF-κB activation in human colon carcinoma cell line SW480. Compound 9 with the significant DPPH radical scavenging activity is corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS. Compounds 2, 4, 16–18 exerted remarked down-regulation of NF-κB in LPSinduced SW480 cells with weak inhibitory effects against NO production and the DPPH radical scavenging activity.
1. Introduction Xanthone-related derivatives represent a large class of natural polyphenolic compounds, commonly occurring in plants and fungi, and exhibiting a broad spectrum of bioactivities [1–4]. The structure variation is highly owing to functionalization with diverse substituents at various positions. Basically, natural xanthone-based analogues are classified into simple xanthones, xanthone glycosides, prenylated xanthones, xanthonolignoids, bisxanthones, and miscellaneous xanthones. Initial interest in xanthone-related derivatives stemmed from their antimicrobial activities [5], while this aspect has expanded over the years to include antiproliferative properties as well as antidepressant, antiviral, antitubercular, cardiotonic, diuretic, choleretic, and cytotoxic activities. The pharmaceutical activities of xanthone derivatives are related to the structural patterns. Seven-ring polycyclic xanthone MDN0185 exerted potent antiplasmodial activity against Plasmodium falciparum parasites [6]. Prenylated xanthones such as mangostanin and owaxanthones are strongly inhibitory effects toward sensitive and methicillin-resistant strains of Staphylococcus aureus [7]. Xanthone dimers including phomoxanthones A and B exhibited significant in vitro antimalarial and antitubercular activities [8]. Sterigmatocystin and its
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analogues are the environmental toxins, but they are also the inhibitors of the growth of transplanted leukemias P-388 and L1210 in mice [9]. Marine-derived fungi are considered as the potential source to produce the polyketide metabolites, of which chlorination, heterocyclic substitution, and dimerization are commonly occurred in some of the marine derived xanthone-related compounds [10–12]. In the course of search for antioxidant compounds from marine-derived fungi, a DPPH assay of the in-house marine fungal strains was performed, finding the EtOAc extract of the marine sponge (Xestospongia testudinaria) associated fungus Aspergillus europaeus WZXY-SX-4-1 to have radical scavenging activity. The OSMAC method [13] was used to optimize the culture mediums, while the EtOAc extract from the fungus cultured in salty rice showed stronger radical scavenging activity in the DPPH assay (Fig. 1). Thus, the fermentation of the fungus in solid phase on large scale was performed. Subsequently, the EtOAc extract of the fungus was chromatographed using HPLC separation to obtain 20 compounds, including six new compounds (Fig. 2).
Corresponding author. E-mail address: [email protected] (W. Lin).
https://doi.org/10.1016/j.fitote.2018.08.030 Received 23 July 2018; Received in revised form 30 August 2018; Accepted 31 August 2018 Available online 05 September 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. DPPH radical scavenging activities for the EtOAc extracts of Aspergillus europaeus cultured in different mediums. 1–1: liquid germ cultured by PE medium; 1–2: mycelium cultured by PE medium; 2–1: liquid germ cultured by MnPY medium; 2–2: mycelium cultured by MnPY medium; 3–1: liquid germ cultured by ISP2 medium; 3–2: mycelium cultured by ISP2 medium; 5: rice cultured fungus.
Fig. 2. Structures of the isolated compounds.
Qingdao Marine Chemistry Co., Ltd., Sephadex LH-20 (18–110 μm, Pharmacia) and ODS (50 μm, YMC) were involved in the column chromatography. TLC analysis was carried out on precoated silica gel plates (0.20–0.25 mm, GF254, Qingdao Marine Chemistry Co. Ltd.). Semi-preparative HPLC was performed on semi-preparative column (YMC-pack ODS-A C18, 5 μm, 10 × 250 mm) with Alltech 426-HPLC pump and Alltech UVis-200 detector. DAD-HPLC was performed on Waters e2695 separations module with Waters 2998 photodiode array detector and Waters Symmetry C18 column (5 μm 4.6 × 250 mm). Mo2(OAc)4 (molybdenum reagent) for Snatzke method was purchased from Alfa Aesar Co. Ltd. Fetal bovine serum (FBS) was purchased from Gibco Co. Ltd. Dulbecco's modified Eagle's medium (DMEM) and phosphate buffer saline (PBS) was purchased from M and C Gene Tech Co. Ltd. Nitric oxide (NO) concentration was detected by a Thermofisher Multiskan FC microplate reader using Applygen E1030 NO assay kit. Bioluminescence intensity was detected by a Berthold LB960 microporous plate chemo-luminescence detector using Beyotime
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. ECD spectra were obtained on a Jasco J-810 spectropolarimeter. 1H and 13C NMR as well as 2D NMR spectra were recorded on a Bruker Avance III 400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13C, respectively). Chemical shifts were expressed in δ (ppm) and were referenced to the solvent peaks at δH 2.50 and δC 39.5 for DMSO‑d6, and coupling constants are in Hz. The temperature for NMR measurement is 298 K, the coupling constant for HMBC experiments is optimized as 8 Hz, and the mixing time used for ROESY experiments is 2 s. HRESIMS spectra were measured on a Thermo Scientific LTQ Orbitrap XL spectrometer. Silica gel (160–200 and 200–300 mesh) used for column chromatography was purchased from 191
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yield 9 (20.7 mg), 14 (12.0 mg), and SF2d1 (22 mg), while the latter was further purified by semi-preparative HPLC eluting with MeOH-H2O (83:17, 0.1‰ TFA, 2 mL/min) to obtain 15 (2.0 mg), 16 (2.1 mg). SF2e (104 mg) was subjected to semi-preparative HPLC eluting with MeOHH2O (40:60, 0.1‰ TFA, 2 mL/min) to obtain 10 (5.7 mg), 11 (17.7 mg), 12 (45.5 mg). Compounds 13 (3.0 mg), 19 (2.5 mg), and 18 (1.5 mg) were isolated from SF2f (89 mg) by the semi-preparative HPLC eluting with MeOH-H2O (70:30, 0.1‰ TFA, 2 mL/min). F3 (1.5 g) was separated by ODS column chromatography eluting with MeOH-H2O (from 3:7 to 100:0) to obtain six subfractions (SF3a-SF3f). SF3b (132 mg) was purified on semi-preparative HPLC eluting with MeOH-H2O (32:68, 0.1‰ TFA, 2 mL/min) to obtain 2 (8.2 mg) and 3 (5.5 mg). SF3d (72 mg) was subjected to semi-preparative HPLC eluting with MeOHH2O (43:57, 0.1‰ TFA, 2 mL/min) to obtain 6 (3.8 mg), 17 (4.0 mg), and 20 (3.1 mg). Compounds 1 (7.1 mg), 5 (5.8 mg), 7 (14.8 mg), and 8 (3.2 mg) were isolated from SF3e (68 mg) by semi-preparative HPLC eluting with MeOH-H2O (50:50, 0.1‰ TFA, 2 mL/min).
Biotechnology RG005 luciferase reporter gene assay kit. 2.2. Fungal strain and fermentation The fungal strain Aspergillus europaeus WZXY-SX-4-1 was isolated from the marine sponge Xestospongia testudinaria, collected at the Weizhou Island in Guangxi Province of China, in May 2016. The species was identified by the morphological characters and the ITS sequence (GenBank accession No. LT220221) with standard records, and was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China. Based on the OSMAC method, the culture mediums involving PE (starch 10 g,peptone 1 g,sea salt 3.3 g,H2O 100 mL), MnPY (peptone 2 g, yeast extract 2 g,mannitol 4 g, sea salt 3.3 g,H2O 100 mL), ISP2 (yeast extract 4 g, melt extract 10 g, glucose 4 g,sea salt 3.3 g,H2O 100 mL), and PDA (PDA medium (BD Co., USA) 36 g, sea salt 33 g, H2O 1000 mL) for liquid culture, and solid medium (rice 30 g,sea salt 1.65 g,H2O 50 mL). The DPPH assay revealed that the EtOAc extract of the fungus strain WZXY-SX-4-1 cultured in salty rice showed the most radical scavenging activity (Fig. 1). Thus, the fungus A. europaeus WZXY-SX-4-1 was cultivated at 25 °C for 21 days in forty 500 mL Erlenmeyer flasks, each of which contained 50 g rice and 50 mL seawater.
2.3.1. Eurobenzophenone A (1) White powder; [α]20 D + 15 (c 0.10, MeOH); UV (MeOH) λmax 203, 248, 324 nm; IR (KBr) νmax 3407, 1701, 1612 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 421.0779 [M - H]− (calcd for C19H17O11, 421.0771).
2.3. Extraction and isolation 2.3.2. Eurobenzophenone B (2) White powder; [α]24 D + 24 (c 0.10, MeOH); UV (MeOH) λmax 205, 248, 325 nm; IR (KBr) νmax 3427, 1658 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 435.0924 [M - H]− (calcd for C20H19O11, 435.0927).
The fermented material in 500 mL Erlenmeyer flask was extracted twice with 400 mL EtOAc and the solvent was removed under reduced pressure to obtain a crude extract (21.2 g). The EtOAc extract was subjected to silica gel column chromatography and eluted with PEEtOAc (3:1) to afford five fractions, while the amounts of the fractions were distributed to F1 (1.5 g), F2 (1.6 g), F3 (2.5 g), F4 (13.0 g), and F5 (2.0 g). The DPPH assay resulted in F2 to be the most active for radical scavenging with IC50 = 10 μg/mL (Fig. 3), while the other fractions showed weak activity with IC50 > 100 μg/mL. F2 (1.6 g) was fractionated by ODS column chromatography eluting with MeOH-H2O (from 30:70 to 100:0) to obtain seven subfractions (SF2a-SF2g). SF2c (25 mg) was purified on semi-preparative HPLC eluting with MeOH-H2O (42:58, 0.1‰ TFA, 2 mL/min) to obtain 4 (3.0 mg). SF2d (124 mg) was isolated by an ODS column eluting with MeOH-H2O (from 20:80 to 60:40) to
2.3.3. Eurobenzophenone C (3) Pale yellow powder; UV (MeOH) λmax 195, 284 nm; IR (KBr) νmax 3186, 2953, 1719, 1635, 1610, 1514 cm−1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 303.0507 [M - H]− (calcd for C15H11O7, 303.0505). 2.3.4. Euroanthone A (4) Pale yellow powder; [α]24 D + 48 (c 0.10, MeOH); UV (MeOH) λmax 203, 239, 312 nm; IR (KBr) νmax 3648, 3627, 3436, 1733, 1717, 1683, Fig. 3. DPPH radical scavenging activities of fractions F1-F5. Fractions F1 to F5 are yielded from the EtOAc extract by silica gel column chromatography. The fractions were dissolved in methanol and the concentration gradient was 200, 100, 50, 20, 10, 5 μg/mL. 100 μL methanol solution of the sample or 100 μL methanol together with 100 μL 100 μM DPPH were added to 96 hole plates and the plates were placed at room temperature in the dark. The absorbance was detected at 517 nm after 30 min. Each group was made in duplicate.
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Table 1 1 H NMR Data of 1–5 and 7 in DMSO-d6 (δ in ppm, J in Hz). No.
1
2
3
2 4 10 12 15 16 1′
6.75, d (2.3) 6.49, d (2.3)
6.74, d (2.0) 6.51, d (2.0)
6.16, s
6.17, s
6.76, 6.46, 6.06, 6.06, 2.14,
2.25, 4.12, 4.27, 3.75, 3.40,
2.27, 4.13, 4.28, 3.75, 3.14,
2′ 3′ OH-1 OH-3 OH-5 OH-9 OH-13 COOH OH-2′ OH-3′ OMe
s dd (6.3, 11.3) dd (4.2, 11.3) m m
9.71, s 9.72, s 13.28, s 11.37, s 12.67, br 4.98, d (4.2) 4.68, t (4.0)
d (2.3) d (2.3) s s s
s dd (6.5, 11.3) dd (3,6, 11.3) m m 9.63, s 9.56, s 11.48, s 11.48, s 12.57, br
9.88, br
4
5
7
6.35, d (2.0) 6.21, d (2.0) 7.39, s
6.39, d (2.0) 6.24, d (2.0) 7.27, s
6.86, 6.56, 6.07, 6.07, 2.14,
2.40, s 4.34, dd (6.2, 11.2) 4.48, dd (4.0, 11.2) 3.82, m 3.40, m 12.39, br 9.00, br
2.44, 4.33, 4.47, 3.82, 3.45, 12.4,
d (1.8) d (1.8) s s s
s dd (6.3, 11.1) dd (4.2, 11.1) m m br 9.76, br 11.50, br 11.50, br 12.80, br
4.97, d (4.0) 4.70, t (4.0) 3.63, s
5.05, br 4.74, br 3.88, s
5.05, br 4.77, br 3.75, s
C NMR data, see Table 3; HRESIMS m/z 403.0656 [M - H]− (calcd for C19H15O10, 403.0665). 13
Table 2 13 C NMR Data of 1–5 and 7 in DMSO‑d6 (δ in ppm). No.
1
2
3
4
5
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1′ 2′ 3′ OMe
130.7, C 107.6, CH 158.2, C 106.6, CH 155.1, C 124.5, C 201.5, C 110.1, C 163.0, C 109.8, C 146.1, C 109.6, CH 162.6, C 167.6, C 168.7, C 21.7, CH3 67.0, CH2 69.7, CH 63.2, CH2
129.7, C 106.9, CH 158.2, C 106.5, CH 155.4, C 123.7, C 200.2, C 110.4, C 162.5, C 109.5, C 145.8, C 109.2, CH 162.5, C 166.1, C 168.3, C 21.4, CH3 66.5, CH2 69.2, CH 62.7, CH2 52.0, CH3
129.8, C 107.0, CH 157.3, C 106.2, CH 154.4, C 124.8, C 200.9, C 107.4, C 161.7, C 109.4, CH 146.7, C 109.4, CH 161.7, C 167.0, C 21.5, CH3
157.1, C 95.1, CH 167.5, C 99.4, CH 163.0, C 102.3, C 178.0, C 115.3, C 134.0, C 124.5, CH 144.1, C 124.8, C 152.3, C 168.6, C 165.2, C 19.8, CH3 67.7, CH2 69.7, CH 63.0, CH2 53.2, CH3
156.5, C 94.4, CH 166.3, C 98.7, CH 162.6, C 101.9, C 178.1, C 114.0, C 134.5, C 123.8, CH 143.5, C 123.6, C 151.6, C 169.3, C 165.0, C 19.4, CH3 67.2, CH2 69.3, CH 62.5, CH2
130.0, C 104.7, CH 159.0, C 104.9, CH 154.4, C 126.3, C 200.4, C 109.5, C 161.6, C 107.4, CH 146.8, C 107.4, CH 161.6, C 168.0, C 21.5, CH3
2.3.6. (+)1-O-demethylvariecolorquinones A (6) Yellow powder; [α]22 D + 25 (c 0.05, MeOH),UV (MeOH) λMeOH max (log ε) nm 223 (2.4), 253 (1.5), 288 (1.7), 441 (0.9)。1H NMR (DMSO‑d6, 400 MHz): δH 2.40 (3H, s, H-11), 3.43 (2H, m, H-4′), 3.77 (1H, m, H-3′), 4.23 (1H, dd, J = 6.6, 11.1 Hz, H-2′), 4.39 (1H, dd, J = 3.9, 11.1 Hz, H-2′), 6.59 (1H, d, J = 2.2 Hz, H-7), 7.13 (1H, d, J = 2.2 Hz, H-5), 7.57 (1H, s, H-4)。13C NMR (DMSO‑d6, 100 MHz): δC 19.6 (CH3, C-11), 62.6 (CH2, C-4′), 67.0 (CH2, C-2′), 69.2 (CH, C-3′), 108.0 (CH, C-7), 108.7 (CH, C-5), 109.5 (C, C-8a), 114.1 (C, C-1a), 120.4 (CH, C-4), 133.0 (C, C-4a), 135.0 (C, C-5a), 144.1 (C, C-3), 157.9 (C, C-1), 164.6 (C,C-6), 165.6 (C, C-8),166.6 (C, C-1′), 181.1 (C, C-10), 188.9 (C, C-9); ESIMS m/z 387 [M – H]-. 1.1.1. 14-O-Demethylsulochrin (7) Pale yellow powder; UV (MeOH) λmax 204, 284 nm; IR (KBr) νmax 3200, 1719, 1634, 1593, 1514 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 317.0659 [M - H]− (calcd for C16H13O7, 317.0661).
55.2, CH3
1653, 1558 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 417.0828 [M - H]− (calcd for C20H17O10, 417.0822).
2.4. ICD measurement
2.3.5. Euroanthone B (5) Pale yellow powder; [α]24 D + 14 (c 0.10, MeOH); UV (MeOH) λmax 240, 311 nm; IR (KBr) νmax 3422, 1725, 1652, 1613, 1568 cm−1; 1H and
The test compounds were reacted with molybdenum reagent (final concentration 0.6 mg/mL) in DMSO at a molar ratio of 0.5 after deducting the influence of ICD from the test compound. ICD was measured at 250–550 nm every 10 min until the spectrum became stable. 2.5. Cell culture and viability
Table 3 Free radical scavenging activity of compounds against DPPH. Compound
IC50 (μg/mL)
2 3 7 8 9 10 11 12 14 trolox
18.5 ± 0.5 1.7 ± 0.2 11.6 ± 1.2 13.2 ± 0.7 2.3 ± 0.1 5.4 ± 0.1 14.3 ± 0.7 25.3 ± 1.4 17.5 ± 0.5 5.4 ± 0.2
BV2 and SW480 cells (supplied by the Chinese Academy of Medical Sciences) were cultured at 37 °C in 5% CO2 in DMEM with 10% FBS. Compounds 1–20 in DMSO were diluted to 100 μM by DMEM. BV2 and SW480 cells were plated in 96-well plates (1 × 105 cells/mL), respectively, and incubated with 100 μM test compounds for 24 h. MTT assays were used to determine the cell viability. The experiments were performed at least in triplicate. 2.6. DPPH assay DPPH (1,1-diphenyl-2-picryl-hydrazyl) was purchased from Sigma–Aldrich (USA). Stock solutions of DPPH were prepared in MeOH, 193
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and MeOH buffered with acetic acid buffer (0.1 M, pH 5.5), respectively. Buffered MeOH was prepared by mixing 40 mL of 0.1 M acetate buffer (pH 5.5) with 60 mL methanol. The solvents and other chemicals were of analytical grade. The reaction tubes, in triplicates, were wrapped in aluminum foil and kept at 30 °C for 30 min in dark. All measurements were done under dim light. Spectrophotometric measurements were done at 517 nm using Spectronic Genesys 5 spectrophotometer. The data are mean ± SD. 2.7. NO inhibition assay BV2 cells (1 × 105 cells/mL) were seeded in 96-well plates and treated with 1 μg/mL LPS. Test compounds (10 μM), and positive control curcumin were added to keep the incubation for 24 h, respectively. Griess reagent was added to the supernatants. The absorbance was detected at 540 nm on the microplate reader and the standard curve was established by NaNO2 dilution series. The experiments were performed at least in triplicate. 2.8. NF-κB inhibition assay Fig. 5. Experimental ECD curves of 1, 2, and 5.
SW480-NF-κB cells were plated in 24-well plates at a density of 5 × 104 cells per well and treated with 1 μg/mL LPS. Compounds (10 μM), positive control MG132, respectively, were added to incubate for 6 h. The cells were washed twice with PBS and lysed by 100 μL reporter gene cell lysate per well at 4 °C for 5 min, then collected the cells and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein concentration was measured in 96-well plates using 5 μL supernatant incubated with BCA at 37 °C for 30 min. The absorbance was detected at 570 nm on the microplate reader and the standard curve was established by BSA dilution series. The bioluminescence intensity was measured in 96-well white plates using 50 μL supernatant and the luciferase assay reagent by the microporous plate chemiluminescence detector through the steps of dispersion, shake, delay and measure. The experiments were performed at least in triplicate.
109.8), and the methyl carbon C-16 (δC 21.7), H3–16 (δH 2.25, s) to C10, C-11 (δC 146.1), and C-12 (δC 109.6), as well as OH-9 (δH 13.28, s) to C-8, C-9 (δC 163.0), and C-10, OH-13 (δH 11.37, s) to C-8, C-12, and C-13 (δC 162.6) confirmed the substitution of hydroxyl groups at C-9 and C-13, while the methyl group was located at C-11. The four-bond HMBC correlation between H-12 and a carbonyl carbon C-15 (δC 166.1) suggested the location of a carbonyl group at C-10. The connection of rings A and B across a ketone group C-8 (δC 200.2) was deduced by the weak HMBC correlation from H-4 and H-13 to C-13 (Fig. 4), thus the ketone bridge was linked to C-6 and C-8 accordingly. In addition, a glycerol group was recognized by the 1He1H COSY relationship of a methine proton H-2′ (δH 3.75, m) with the methylene protons H2–1′ (δH 4.12, 4.27) and H2–3′ (δH 3.40) in association with the correlation between H-2’/OH-2′ (δH 4.98) and H2–3’/OH-3′ (δH 4.68). The HMBC correlation between C-15 and H2–1′ demonstrated the formation of an ester bond of glycerol group at C-15. Compound 1 contains the sole stereogenic center at C-2′, thus the phase of specific rotation directly reflected its configuration. Referring to the literature, glycerol-bearing endocrotin showing negative specific rotation ([α]D22–30) was determined as 2S by a dibenzoate chirality method [14]. Variecolorquinone A with [α]D20 value (−18) was also assigned as S configuration [15], whereas (+)-variecolorquinone A, a co-isolated analogue with the positive optical rotation ([α]D20 + 16.8), was identified to R configuration for the glycerol moiety [16]. Thus, the positive specific rotation of 1 ([α]D20 + 15) was consistent with 2′R configuration. In addition, the induced ECD spectrum of the 1-Mo2(OAc)4 complex showed positive Cotton effect at 305 nm (band IV) (Fig. 5), inferring to positive torsion force between OH-2′ and OH-3′. This data further supported 2′R configuration based on the Snatzke method [17,18]. The molecular formula of eurobenzophenone B (2) was deduced as C19H18O11 based on the HRESIMS and NMR data, containing 11 degrees of unsaturation. Comparison of the NMR data reveled 2 to be
3. Results and discussion Eurobenzophenone A (1) has a molecular formula of C20H20O11 as determined by the HRESIMS and NMR data, requiring 11 degrees of unsaturation. The IR absorptions at 3427 and 1658 cm−1 suggested the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum exhibited a total of 20 carbon resonances, including 12 aromatic carbons for two phenyl rings, and three carbonyl carbons. The 1H NMR spectrum showed three aromatic protons, two methyl singlets, and five protons for oxymethylene and oxymethine. The observation of the metacoupling between the aromatic protons H-2 (δH 6.75, d, J = 2.0 Hz) and H-4 (δH 6.49, d, J = 2.0 Hz) suggested ring A to be tetra-substituted. The HMBC correlations from the phenol proton OH-3 (δH 9.72, s) to C-2 (δC 107.6), C-3 (δC 158.2), and C-4 (δC 106.6), and from OH-5 (δH 9.71, s) to C-4, C-5 (δC 155.1), and C-6 (δC 124.5) indicated C-3 and C-5 to be hydroxylated. The HMBC correlation between H-2 and a carbonyl carbon at δC 167.6 (C-14) located a carboxylic group at C-1 (δC 130.7). In regard to the second aromatic ring (ring B), the presence of only one aromatic proton at δH 6.61 (s, H-12) suggested ring B to be penta-substituted. The HMBC correlations of H-12 to C-8 (δC 110.1), C-10 (δC
Fig. 4. Key COSY and HMBC correlations of 1 and 5.
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data and literature. Thus it was found from nature for the first time, and this is the first report of its NMR data. In addition, the co-isolated compounds were identical to (+)-variecolorquinone A (8) [18], 3-de-O-methylsulochrin (9) [19], 14-de-Omethyl-5-methoxysulochrin (10) [19], sulochrin (11) [19], 5-methoxysulochrin (12) [20], calyxanthone (13) [21], yicathin C (14) [22], yicathin A (15) [22], yicathin B (16) [22], dermolutein (17) [23], methylemodin (18), 1-methoxy-14-dehydroxywentiquinone C (19) [24], wentiquinone C (20) [25]. In the biogenetic speculation, anthraquinones such as endocrocin and emodin were considered as the precursors. As shown in Scheme 1, the oxidative cleavage of anthraquinones by two pathways (a and b) to generate benzophenone nucleus was postulated [26]. Nucleophilic attack of the hydroxyl group in benzophenones in association with dehydration resulted in the formation of xanthone core [27,28]. Methylation of benzophenone and xanthone to yield the methylated derivatives is arbitrarily derived, while the formation of glyceride specifically occurs in the carboxylic acid of ring B (Scheme 1). DPPH assay guided fractionation of the EtOAc extract revealed that F2 fraction possessing the DPPH radical scavenging activity with IC50 = 10 μg/mL (Fig. 3), while the remaining fractions showed weak activity with IC50 > 100 μg/mL. Chromatographic separation of F2 yielded compounds 2, 7–12 as the main components. These compounds were subjected for DPPH assay. As shown in Table 3, benzophenones 3, 9 and 10 exhibited potent radical scavenging activity toward DPPH, and the data were comparable with that of the positive control (trolox). Nitric oxide (NO) is a major mediator produced by macrophages during inflammation responses, whereas an excess amount of NO can destroy and induce dysfunction in the macrophages as well as in surrounding normal cells [29]. Therefore, the level of NO produced by its homeostasis reflected the inflammation stage. Prior to the bioassay, the cytotoxic effects of compounds were detected by the MTT method, showing no cytotoxicity on mouse microglial BV2 cells and SW480 human colon carcinoma cells at a dose of 100 μM. Lipopolysaccharide (LPS) induced NO production in BV2 microglia cells was markedly increased with approximately 3.5-fold related to the control group (P < .001). Compounds 1–20 (10 μM) exerted inhibitory activities against the NO production in the LPS induced BV2 cells ranging from 17.4 to 42.2%, while compound 4 was the most active. The positive control curcumin showed the inhibitory rate of 60% in the same dose (Table 4). In order to insight the NO inhibition related to the cell signaling factors, all compounds were detected for the inhibition of NF-κB in the NF-κB luciferase reporter gene transfected SW480 cells which were activated by the LPS induction. The experimental results revealed that compounds 2, 5, and 8 (10 μM) significantly reduced the NF-κB expression with the inhibitory rates of 74.9%, 68.8%, and 81.2%, respectively, that were comparable with the NF-κB inhibitor MIG132 (90% inhibitory rate in a dose of 10 μM). The experimental data uncovered that the significant inhibition of compound 5 toward inflammatory mediator, nitric oxide (NO), was mediated by down-regulation of the transcription factor nuclear factor-κB (NF-κB), that is an essential factor in controlling inflammatory mediators. However, the remarkable inhibition of compounds 2, 4, 9 and 16–18 against NF-κB expression with weak activities toward NO production suggested these compounds to manage the inflammatory responses by other inflammatory mediator rather than NO.
structurally related to 1, while the distinction was attributed to the presence of a methoxy group in 2. The 2D NMR data further confirmed 2 as a methyl ester product of 1, as evident from the methoxy protons at δH 3.63 (s) correlated to the carbonyl carbon C-14 (δC 166.1) in the HMBC spectrum. The similar ICD data of both 1 and 2 (Fig. 4) and specific rotation assumed the absolute configuration of 2 to be 2′R. Eurobenzophenone C (3) has a molecular formula of C15H12O7, on the basis of the HRESIMS and NMR data, bearing 10 degrees of unsaturation. The 1H and 13C NMR data revealed two aromatic rings, two carbonyl carbons, and a methyl group. Aromatic ring A was identical to that of 1, based on the comparable NMR data and the analyses of 2D NMR data. Aromatic ring B contained four carbon resonances with the overlapped aromatic protons at δH 6.06 (2H, s) and phenol protons at δH 11.48 (2H, s), indicating a symmetric moiety. The HMBC correlations from the methyl protons H3–15 (δH 2.14, s) to C-10/C-12 (δC 109.4) and C-11 (δC 146.7), and the phenol protons (δH 11.48, OH-9/ OH-13) to C-8 (δC 107.4), C-9/C-13 (δC 161.7), and C-10/C-12 identified ring B to be 11-methyl-9,13-dihydroxy substitution. The linkage of rings A and B by a ketone bridge C-7 (δC 200.9) across C-6 and C-8 was established by the four-bond HMBC correlation of C-7 with H-4 and H10/H-12. The molecular formula of euroxanthone A (4) was determined to be C20H18O10 with 12 degrees of unsaturation, based on the HRESIMS and NMR data. The IR absorptions at 3648, 3627, 3436, 1733, 1717, 1683, and 1653 cm−1 suggested the presence of hydroxy, carbonyl, and phenyl groups. The NMR spectra presented 12 aromatic resonances for two phenyl rings and a ketone at δC 178.0 (C-7). These data were characteristic of a xanthone nucleus. The meta-coupling of the aromatic protons H-2 (δH 6.35, d, J = 2.0 Hz) and H-4 (δH 6.21, d, J = 2.0 Hz) in addition to the HMBC correlations of H-2 and H-4 to C-3 (δC 167.5) and C-6 (δC 102.3) along with the correlation between H-2/C-1 (δC 157.1) and H-4/C-5 (δC 163.0) declared a tetra-substituted ring A, in which C-1 and C-3 were hydroxylated. Thus, the singlet aromatic proton at δH 7.39 (s, H-10) and the methyl protons at δH 2.40 (s, H3–16) were assigned to aromatic ring B. The HMBC correlations from H3–16 to C-10 (δC 124.5), C-11 (δC 144.1), and C-12 (δC 124.8), H-10 to C-8 (δC 115.3), C-12, the methyl carbon (δC 19.8), and a carbonyl carbon C-14 (δC 168.6), as well as the correlation between the methoxyl protons (δH 3.88) and C-14, positioned a methyl group at C-11, while a methylformate was linked to C-9 (δC 134.0). Thus, the remaining carbonyl carbon (δC 165.2, C-15) was assumed to be positioned at C-12, and this was supported by the four-bond HMBC correlation between H-10 and C-15. In additional, a glycerol unit was identified by the COSY and HMBC correlations. The HMBC correlation between the methylene protons H2–2′ (δH 4,34, 4.48) and C-15 deduced the formation of a glyceride. Based on the Snatzke method, the positive Cotton effect at 350 nm in the ICD spectrum agreed 2′R configuration, while the positive specific rotation ([α]24 D + 48 in MeOH) further supported the configurational assignment. Euroxanthone B (5) was determined to be an analogue of 4 with a formic acid unit at C-9 to replace a methylformate unit, based on the similar NMR data with the absence of a methoxy group and the molecular formula (C19H16O10) showing CH2 unit less than that of 4. The same phase of the specific rotation of both 4 and 5 ([α]20 D + 14, MeOH) suggested 2′R configuration in 5. Analyses of 1D and 2D NMR data in association with the HRESIMS data revealed the planar structure of 6 to be identical to the demethylated analogue of variecolorquinone A [18], an anthraquinone derivative. The distinction was attributed to the specific rotation of 6 ([α]22 D + 25 in MeOH), whereas that of the known analogue with 2′S was [α]22 D − 23 (MeOH). Thus, the absolute configuration of 6 was assumed to be 2′R, namely (+)1-O-demethylvariecolorquinone A. The structure of compound 7 was determined to be a 3-methoxylated analogue of 3, based on the similar NMR data with the exception of a methoxy group whose protons (δH 3.75, s) correlated to C-3 (δC 159.0). The structure of 7 is hit in SciFinder with the absence of NMR
4. Conclusion Fungus Aspergillus europaeus is a new species closely related to A. wentii originally found from soil [30], while this is the first time to isolate it from marine environment. In addition, this is the first report to describe the secondary metabolites to be isolated from A. europaeus. In the selected culture medium, the PKS biogenetic pathway is activated to produce 20 xanthone-related metabolites including six new compounds, while the polyketides bearing glyceride unit are uncommonly 195
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Scheme 1. Postulation of the biogenetic pathway to derive the isolated compounds.
found from nature. The bioassay results revealed that the significant DPPH radical scavenging activity of compound 9 are corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS, suggesting the NF-κB being the signing mediator for the antioxidant agents. However, all compounds showed moderate to weak activity to inhibit the NO production in LPS induced BV2 cells. Moreover, compounds 2, 4, 16–18 exerted remarked down-regulation of NF-κB in LPSinduced SW480 cells with weak inhibitory effects against NO production and weak DPPH radical scavenging activity suggested that they are potential to act on the NF-κB induced immune response to infection excluding the mediation by the inflammatory mediators NO and ROS, while the detail mode of action is under progress.
Table 4 Inhibitory activities of 1–20 against NO production in BV2 microglia cells and NF-κB activation in SW480 cells induced by LPS.a No.
% inhibition NOb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Curcuminc MG132c
27.0 17.4 23.3 42.2 23.4 28.6 35.2 28.2 19.5 39.4 26.5 19.5 17.6 27.0 23.7 35.3 25.0 20.0 33.3 38.4 60.1
NF-κBb ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.5 2.6 1.3 2.3 3.3 7.2 7.8 3.8 5.0 2.9 3.2 1.6 5.1 3.2 4.8 3.9 6.3 1.2 5.1 5.0 1.6
31.6 74.9 31.6 68.8 52.3 23.6 31.0 28.2 71.0 31.3 61.7 45.3 63.7 56.8 13.0 81.2 73.1 75.9 58.4 24.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.9 3.8 4.9 7.0 10.6 6.5 4.2 6.7 10.0 12.0 0.9 7.5 5.6 5.7 9.8 8.3 12.7 8.3 6.8 7.9
Conflict of interest The authors declare no conflict of interest. Acknowledgments Financial supports from the MOST-973 Program (2015CB755906) and the National Natural Science Foundation of China (81630089, 41376127, U1606403) are highly appreciated. Appendix A. Supplementary data
88.9 ± 6.1
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2018.08.030.
Dose of compound is 10 μM. SW480 cells are stably transfected with NF-κB luciferase reporter gene. a n = 3. b Three independent experiments, data are shown as mean ± SD. c Positive control.
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Polyketide derivatives from the sponge associated fungus Aspergillus europaeus with antioxidant and NO inhibitory activities Xiaowen Dua, Dong Liua, Jian Huanga, Chanjuan Zhanga, Peter Prokschb, Wenhan Lina, a b
T ⁎
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, 100191, PR China Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine- Universität Düsseldorf, 40225 Düsseldorf, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspergillus europaeus Polyketides Structure elucidation Radical scavenging activity Inhibition of NO production Inhibition of NF-κB activation
DPPH assay of the in-house marine-derived fungi uncovered that the EtOAc extract of the cultured fungus Aspergillus europaeus WZXY-SX-4-1, which was isolated from the marine sponge Xestospongia testudinaria, possesses radical scavenging activity. Chromatographic separation of the bioactive extract resulted in the isolation of 20 polyketide derivatives, including six new compounds namely eurobenzophenones A-C (1–3), euroxanthones A-B (4–5), and (+)1-O-demethylvariecolorquinones A (6). The structures of new compounds were determined on the basis of the analyses of spectroscopic data, including the Snatzke method for the configurational assignment. Benzophenones 3, 9 and 10 exhibited potent radical scavenging activity against DPPH. All polyketides were evaluated for the inhibitory effects toward the LPS induced nitric oxide (NO) production in mouse microglia BV2 cells and the NF-κB activation in human colon carcinoma cell line SW480. Compound 9 with the significant DPPH radical scavenging activity is corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS. Compounds 2, 4, 16–18 exerted remarked down-regulation of NF-κB in LPSinduced SW480 cells with weak inhibitory effects against NO production and the DPPH radical scavenging activity.
1. Introduction Xanthone-related derivatives represent a large class of natural polyphenolic compounds, commonly occurring in plants and fungi, and exhibiting a broad spectrum of bioactivities [1–4]. The structure variation is highly owing to functionalization with diverse substituents at various positions. Basically, natural xanthone-based analogues are classified into simple xanthones, xanthone glycosides, prenylated xanthones, xanthonolignoids, bisxanthones, and miscellaneous xanthones. Initial interest in xanthone-related derivatives stemmed from their antimicrobial activities [5], while this aspect has expanded over the years to include antiproliferative properties as well as antidepressant, antiviral, antitubercular, cardiotonic, diuretic, choleretic, and cytotoxic activities. The pharmaceutical activities of xanthone derivatives are related to the structural patterns. Seven-ring polycyclic xanthone MDN0185 exerted potent antiplasmodial activity against Plasmodium falciparum parasites [6]. Prenylated xanthones such as mangostanin and owaxanthones are strongly inhibitory effects toward sensitive and methicillin-resistant strains of Staphylococcus aureus [7]. Xanthone dimers including phomoxanthones A and B exhibited significant in vitro antimalarial and antitubercular activities [8]. Sterigmatocystin and its
⁎
analogues are the environmental toxins, but they are also the inhibitors of the growth of transplanted leukemias P-388 and L1210 in mice [9]. Marine-derived fungi are considered as the potential source to produce the polyketide metabolites, of which chlorination, heterocyclic substitution, and dimerization are commonly occurred in some of the marine derived xanthone-related compounds [10–12]. In the course of search for antioxidant compounds from marine-derived fungi, a DPPH assay of the in-house marine fungal strains was performed, finding the EtOAc extract of the marine sponge (Xestospongia testudinaria) associated fungus Aspergillus europaeus WZXY-SX-4-1 to have radical scavenging activity. The OSMAC method [13] was used to optimize the culture mediums, while the EtOAc extract from the fungus cultured in salty rice showed stronger radical scavenging activity in the DPPH assay (Fig. 1). Thus, the fermentation of the fungus in solid phase on large scale was performed. Subsequently, the EtOAc extract of the fungus was chromatographed using HPLC separation to obtain 20 compounds, including six new compounds (Fig. 2).
Corresponding author. E-mail address: [email protected] (W. Lin).
https://doi.org/10.1016/j.fitote.2018.08.030 Received 23 July 2018; Received in revised form 30 August 2018; Accepted 31 August 2018 Available online 05 September 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. DPPH radical scavenging activities for the EtOAc extracts of Aspergillus europaeus cultured in different mediums. 1–1: liquid germ cultured by PE medium; 1–2: mycelium cultured by PE medium; 2–1: liquid germ cultured by MnPY medium; 2–2: mycelium cultured by MnPY medium; 3–1: liquid germ cultured by ISP2 medium; 3–2: mycelium cultured by ISP2 medium; 5: rice cultured fungus.
Fig. 2. Structures of the isolated compounds.
Qingdao Marine Chemistry Co., Ltd., Sephadex LH-20 (18–110 μm, Pharmacia) and ODS (50 μm, YMC) were involved in the column chromatography. TLC analysis was carried out on precoated silica gel plates (0.20–0.25 mm, GF254, Qingdao Marine Chemistry Co. Ltd.). Semi-preparative HPLC was performed on semi-preparative column (YMC-pack ODS-A C18, 5 μm, 10 × 250 mm) with Alltech 426-HPLC pump and Alltech UVis-200 detector. DAD-HPLC was performed on Waters e2695 separations module with Waters 2998 photodiode array detector and Waters Symmetry C18 column (5 μm 4.6 × 250 mm). Mo2(OAc)4 (molybdenum reagent) for Snatzke method was purchased from Alfa Aesar Co. Ltd. Fetal bovine serum (FBS) was purchased from Gibco Co. Ltd. Dulbecco's modified Eagle's medium (DMEM) and phosphate buffer saline (PBS) was purchased from M and C Gene Tech Co. Ltd. Nitric oxide (NO) concentration was detected by a Thermofisher Multiskan FC microplate reader using Applygen E1030 NO assay kit. Bioluminescence intensity was detected by a Berthold LB960 microporous plate chemo-luminescence detector using Beyotime
2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. ECD spectra were obtained on a Jasco J-810 spectropolarimeter. 1H and 13C NMR as well as 2D NMR spectra were recorded on a Bruker Avance III 400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13C, respectively). Chemical shifts were expressed in δ (ppm) and were referenced to the solvent peaks at δH 2.50 and δC 39.5 for DMSO‑d6, and coupling constants are in Hz. The temperature for NMR measurement is 298 K, the coupling constant for HMBC experiments is optimized as 8 Hz, and the mixing time used for ROESY experiments is 2 s. HRESIMS spectra were measured on a Thermo Scientific LTQ Orbitrap XL spectrometer. Silica gel (160–200 and 200–300 mesh) used for column chromatography was purchased from 191
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yield 9 (20.7 mg), 14 (12.0 mg), and SF2d1 (22 mg), while the latter was further purified by semi-preparative HPLC eluting with MeOH-H2O (83:17, 0.1‰ TFA, 2 mL/min) to obtain 15 (2.0 mg), 16 (2.1 mg). SF2e (104 mg) was subjected to semi-preparative HPLC eluting with MeOHH2O (40:60, 0.1‰ TFA, 2 mL/min) to obtain 10 (5.7 mg), 11 (17.7 mg), 12 (45.5 mg). Compounds 13 (3.0 mg), 19 (2.5 mg), and 18 (1.5 mg) were isolated from SF2f (89 mg) by the semi-preparative HPLC eluting with MeOH-H2O (70:30, 0.1‰ TFA, 2 mL/min). F3 (1.5 g) was separated by ODS column chromatography eluting with MeOH-H2O (from 3:7 to 100:0) to obtain six subfractions (SF3a-SF3f). SF3b (132 mg) was purified on semi-preparative HPLC eluting with MeOH-H2O (32:68, 0.1‰ TFA, 2 mL/min) to obtain 2 (8.2 mg) and 3 (5.5 mg). SF3d (72 mg) was subjected to semi-preparative HPLC eluting with MeOHH2O (43:57, 0.1‰ TFA, 2 mL/min) to obtain 6 (3.8 mg), 17 (4.0 mg), and 20 (3.1 mg). Compounds 1 (7.1 mg), 5 (5.8 mg), 7 (14.8 mg), and 8 (3.2 mg) were isolated from SF3e (68 mg) by semi-preparative HPLC eluting with MeOH-H2O (50:50, 0.1‰ TFA, 2 mL/min).
Biotechnology RG005 luciferase reporter gene assay kit. 2.2. Fungal strain and fermentation The fungal strain Aspergillus europaeus WZXY-SX-4-1 was isolated from the marine sponge Xestospongia testudinaria, collected at the Weizhou Island in Guangxi Province of China, in May 2016. The species was identified by the morphological characters and the ITS sequence (GenBank accession No. LT220221) with standard records, and was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China. Based on the OSMAC method, the culture mediums involving PE (starch 10 g,peptone 1 g,sea salt 3.3 g,H2O 100 mL), MnPY (peptone 2 g, yeast extract 2 g,mannitol 4 g, sea salt 3.3 g,H2O 100 mL), ISP2 (yeast extract 4 g, melt extract 10 g, glucose 4 g,sea salt 3.3 g,H2O 100 mL), and PDA (PDA medium (BD Co., USA) 36 g, sea salt 33 g, H2O 1000 mL) for liquid culture, and solid medium (rice 30 g,sea salt 1.65 g,H2O 50 mL). The DPPH assay revealed that the EtOAc extract of the fungus strain WZXY-SX-4-1 cultured in salty rice showed the most radical scavenging activity (Fig. 1). Thus, the fungus A. europaeus WZXY-SX-4-1 was cultivated at 25 °C for 21 days in forty 500 mL Erlenmeyer flasks, each of which contained 50 g rice and 50 mL seawater.
2.3.1. Eurobenzophenone A (1) White powder; [α]20 D + 15 (c 0.10, MeOH); UV (MeOH) λmax 203, 248, 324 nm; IR (KBr) νmax 3407, 1701, 1612 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 421.0779 [M - H]− (calcd for C19H17O11, 421.0771).
2.3. Extraction and isolation 2.3.2. Eurobenzophenone B (2) White powder; [α]24 D + 24 (c 0.10, MeOH); UV (MeOH) λmax 205, 248, 325 nm; IR (KBr) νmax 3427, 1658 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 435.0924 [M - H]− (calcd for C20H19O11, 435.0927).
The fermented material in 500 mL Erlenmeyer flask was extracted twice with 400 mL EtOAc and the solvent was removed under reduced pressure to obtain a crude extract (21.2 g). The EtOAc extract was subjected to silica gel column chromatography and eluted with PEEtOAc (3:1) to afford five fractions, while the amounts of the fractions were distributed to F1 (1.5 g), F2 (1.6 g), F3 (2.5 g), F4 (13.0 g), and F5 (2.0 g). The DPPH assay resulted in F2 to be the most active for radical scavenging with IC50 = 10 μg/mL (Fig. 3), while the other fractions showed weak activity with IC50 > 100 μg/mL. F2 (1.6 g) was fractionated by ODS column chromatography eluting with MeOH-H2O (from 30:70 to 100:0) to obtain seven subfractions (SF2a-SF2g). SF2c (25 mg) was purified on semi-preparative HPLC eluting with MeOH-H2O (42:58, 0.1‰ TFA, 2 mL/min) to obtain 4 (3.0 mg). SF2d (124 mg) was isolated by an ODS column eluting with MeOH-H2O (from 20:80 to 60:40) to
2.3.3. Eurobenzophenone C (3) Pale yellow powder; UV (MeOH) λmax 195, 284 nm; IR (KBr) νmax 3186, 2953, 1719, 1635, 1610, 1514 cm−1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 303.0507 [M - H]− (calcd for C15H11O7, 303.0505). 2.3.4. Euroanthone A (4) Pale yellow powder; [α]24 D + 48 (c 0.10, MeOH); UV (MeOH) λmax 203, 239, 312 nm; IR (KBr) νmax 3648, 3627, 3436, 1733, 1717, 1683, Fig. 3. DPPH radical scavenging activities of fractions F1-F5. Fractions F1 to F5 are yielded from the EtOAc extract by silica gel column chromatography. The fractions were dissolved in methanol and the concentration gradient was 200, 100, 50, 20, 10, 5 μg/mL. 100 μL methanol solution of the sample or 100 μL methanol together with 100 μL 100 μM DPPH were added to 96 hole plates and the plates were placed at room temperature in the dark. The absorbance was detected at 517 nm after 30 min. Each group was made in duplicate.
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Table 1 1 H NMR Data of 1–5 and 7 in DMSO-d6 (δ in ppm, J in Hz). No.
1
2
3
2 4 10 12 15 16 1′
6.75, d (2.3) 6.49, d (2.3)
6.74, d (2.0) 6.51, d (2.0)
6.16, s
6.17, s
6.76, 6.46, 6.06, 6.06, 2.14,
2.25, 4.12, 4.27, 3.75, 3.40,
2.27, 4.13, 4.28, 3.75, 3.14,
2′ 3′ OH-1 OH-3 OH-5 OH-9 OH-13 COOH OH-2′ OH-3′ OMe
s dd (6.3, 11.3) dd (4.2, 11.3) m m
9.71, s 9.72, s 13.28, s 11.37, s 12.67, br 4.98, d (4.2) 4.68, t (4.0)
d (2.3) d (2.3) s s s
s dd (6.5, 11.3) dd (3,6, 11.3) m m 9.63, s 9.56, s 11.48, s 11.48, s 12.57, br
9.88, br
4
5
7
6.35, d (2.0) 6.21, d (2.0) 7.39, s
6.39, d (2.0) 6.24, d (2.0) 7.27, s
6.86, 6.56, 6.07, 6.07, 2.14,
2.40, s 4.34, dd (6.2, 11.2) 4.48, dd (4.0, 11.2) 3.82, m 3.40, m 12.39, br 9.00, br
2.44, 4.33, 4.47, 3.82, 3.45, 12.4,
d (1.8) d (1.8) s s s
s dd (6.3, 11.1) dd (4.2, 11.1) m m br 9.76, br 11.50, br 11.50, br 12.80, br
4.97, d (4.0) 4.70, t (4.0) 3.63, s
5.05, br 4.74, br 3.88, s
5.05, br 4.77, br 3.75, s
C NMR data, see Table 3; HRESIMS m/z 403.0656 [M - H]− (calcd for C19H15O10, 403.0665). 13
Table 2 13 C NMR Data of 1–5 and 7 in DMSO‑d6 (δ in ppm). No.
1
2
3
4
5
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1′ 2′ 3′ OMe
130.7, C 107.6, CH 158.2, C 106.6, CH 155.1, C 124.5, C 201.5, C 110.1, C 163.0, C 109.8, C 146.1, C 109.6, CH 162.6, C 167.6, C 168.7, C 21.7, CH3 67.0, CH2 69.7, CH 63.2, CH2
129.7, C 106.9, CH 158.2, C 106.5, CH 155.4, C 123.7, C 200.2, C 110.4, C 162.5, C 109.5, C 145.8, C 109.2, CH 162.5, C 166.1, C 168.3, C 21.4, CH3 66.5, CH2 69.2, CH 62.7, CH2 52.0, CH3
129.8, C 107.0, CH 157.3, C 106.2, CH 154.4, C 124.8, C 200.9, C 107.4, C 161.7, C 109.4, CH 146.7, C 109.4, CH 161.7, C 167.0, C 21.5, CH3
157.1, C 95.1, CH 167.5, C 99.4, CH 163.0, C 102.3, C 178.0, C 115.3, C 134.0, C 124.5, CH 144.1, C 124.8, C 152.3, C 168.6, C 165.2, C 19.8, CH3 67.7, CH2 69.7, CH 63.0, CH2 53.2, CH3
156.5, C 94.4, CH 166.3, C 98.7, CH 162.6, C 101.9, C 178.1, C 114.0, C 134.5, C 123.8, CH 143.5, C 123.6, C 151.6, C 169.3, C 165.0, C 19.4, CH3 67.2, CH2 69.3, CH 62.5, CH2
130.0, C 104.7, CH 159.0, C 104.9, CH 154.4, C 126.3, C 200.4, C 109.5, C 161.6, C 107.4, CH 146.8, C 107.4, CH 161.6, C 168.0, C 21.5, CH3
2.3.6. (+)1-O-demethylvariecolorquinones A (6) Yellow powder; [α]22 D + 25 (c 0.05, MeOH),UV (MeOH) λMeOH max (log ε) nm 223 (2.4), 253 (1.5), 288 (1.7), 441 (0.9)。1H NMR (DMSO‑d6, 400 MHz): δH 2.40 (3H, s, H-11), 3.43 (2H, m, H-4′), 3.77 (1H, m, H-3′), 4.23 (1H, dd, J = 6.6, 11.1 Hz, H-2′), 4.39 (1H, dd, J = 3.9, 11.1 Hz, H-2′), 6.59 (1H, d, J = 2.2 Hz, H-7), 7.13 (1H, d, J = 2.2 Hz, H-5), 7.57 (1H, s, H-4)。13C NMR (DMSO‑d6, 100 MHz): δC 19.6 (CH3, C-11), 62.6 (CH2, C-4′), 67.0 (CH2, C-2′), 69.2 (CH, C-3′), 108.0 (CH, C-7), 108.7 (CH, C-5), 109.5 (C, C-8a), 114.1 (C, C-1a), 120.4 (CH, C-4), 133.0 (C, C-4a), 135.0 (C, C-5a), 144.1 (C, C-3), 157.9 (C, C-1), 164.6 (C,C-6), 165.6 (C, C-8),166.6 (C, C-1′), 181.1 (C, C-10), 188.9 (C, C-9); ESIMS m/z 387 [M – H]-. 1.1.1. 14-O-Demethylsulochrin (7) Pale yellow powder; UV (MeOH) λmax 204, 284 nm; IR (KBr) νmax 3200, 1719, 1634, 1593, 1514 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 317.0659 [M - H]− (calcd for C16H13O7, 317.0661).
55.2, CH3
1653, 1558 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 417.0828 [M - H]− (calcd for C20H17O10, 417.0822).
2.4. ICD measurement
2.3.5. Euroanthone B (5) Pale yellow powder; [α]24 D + 14 (c 0.10, MeOH); UV (MeOH) λmax 240, 311 nm; IR (KBr) νmax 3422, 1725, 1652, 1613, 1568 cm−1; 1H and
The test compounds were reacted with molybdenum reagent (final concentration 0.6 mg/mL) in DMSO at a molar ratio of 0.5 after deducting the influence of ICD from the test compound. ICD was measured at 250–550 nm every 10 min until the spectrum became stable. 2.5. Cell culture and viability
Table 3 Free radical scavenging activity of compounds against DPPH. Compound
IC50 (μg/mL)
2 3 7 8 9 10 11 12 14 trolox
18.5 ± 0.5 1.7 ± 0.2 11.6 ± 1.2 13.2 ± 0.7 2.3 ± 0.1 5.4 ± 0.1 14.3 ± 0.7 25.3 ± 1.4 17.5 ± 0.5 5.4 ± 0.2
BV2 and SW480 cells (supplied by the Chinese Academy of Medical Sciences) were cultured at 37 °C in 5% CO2 in DMEM with 10% FBS. Compounds 1–20 in DMSO were diluted to 100 μM by DMEM. BV2 and SW480 cells were plated in 96-well plates (1 × 105 cells/mL), respectively, and incubated with 100 μM test compounds for 24 h. MTT assays were used to determine the cell viability. The experiments were performed at least in triplicate. 2.6. DPPH assay DPPH (1,1-diphenyl-2-picryl-hydrazyl) was purchased from Sigma–Aldrich (USA). Stock solutions of DPPH were prepared in MeOH, 193
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and MeOH buffered with acetic acid buffer (0.1 M, pH 5.5), respectively. Buffered MeOH was prepared by mixing 40 mL of 0.1 M acetate buffer (pH 5.5) with 60 mL methanol. The solvents and other chemicals were of analytical grade. The reaction tubes, in triplicates, were wrapped in aluminum foil and kept at 30 °C for 30 min in dark. All measurements were done under dim light. Spectrophotometric measurements were done at 517 nm using Spectronic Genesys 5 spectrophotometer. The data are mean ± SD. 2.7. NO inhibition assay BV2 cells (1 × 105 cells/mL) were seeded in 96-well plates and treated with 1 μg/mL LPS. Test compounds (10 μM), and positive control curcumin were added to keep the incubation for 24 h, respectively. Griess reagent was added to the supernatants. The absorbance was detected at 540 nm on the microplate reader and the standard curve was established by NaNO2 dilution series. The experiments were performed at least in triplicate. 2.8. NF-κB inhibition assay Fig. 5. Experimental ECD curves of 1, 2, and 5.
SW480-NF-κB cells were plated in 24-well plates at a density of 5 × 104 cells per well and treated with 1 μg/mL LPS. Compounds (10 μM), positive control MG132, respectively, were added to incubate for 6 h. The cells were washed twice with PBS and lysed by 100 μL reporter gene cell lysate per well at 4 °C for 5 min, then collected the cells and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein concentration was measured in 96-well plates using 5 μL supernatant incubated with BCA at 37 °C for 30 min. The absorbance was detected at 570 nm on the microplate reader and the standard curve was established by BSA dilution series. The bioluminescence intensity was measured in 96-well white plates using 50 μL supernatant and the luciferase assay reagent by the microporous plate chemiluminescence detector through the steps of dispersion, shake, delay and measure. The experiments were performed at least in triplicate.
109.8), and the methyl carbon C-16 (δC 21.7), H3–16 (δH 2.25, s) to C10, C-11 (δC 146.1), and C-12 (δC 109.6), as well as OH-9 (δH 13.28, s) to C-8, C-9 (δC 163.0), and C-10, OH-13 (δH 11.37, s) to C-8, C-12, and C-13 (δC 162.6) confirmed the substitution of hydroxyl groups at C-9 and C-13, while the methyl group was located at C-11. The four-bond HMBC correlation between H-12 and a carbonyl carbon C-15 (δC 166.1) suggested the location of a carbonyl group at C-10. The connection of rings A and B across a ketone group C-8 (δC 200.2) was deduced by the weak HMBC correlation from H-4 and H-13 to C-13 (Fig. 4), thus the ketone bridge was linked to C-6 and C-8 accordingly. In addition, a glycerol group was recognized by the 1He1H COSY relationship of a methine proton H-2′ (δH 3.75, m) with the methylene protons H2–1′ (δH 4.12, 4.27) and H2–3′ (δH 3.40) in association with the correlation between H-2’/OH-2′ (δH 4.98) and H2–3’/OH-3′ (δH 4.68). The HMBC correlation between C-15 and H2–1′ demonstrated the formation of an ester bond of glycerol group at C-15. Compound 1 contains the sole stereogenic center at C-2′, thus the phase of specific rotation directly reflected its configuration. Referring to the literature, glycerol-bearing endocrotin showing negative specific rotation ([α]D22–30) was determined as 2S by a dibenzoate chirality method [14]. Variecolorquinone A with [α]D20 value (−18) was also assigned as S configuration [15], whereas (+)-variecolorquinone A, a co-isolated analogue with the positive optical rotation ([α]D20 + 16.8), was identified to R configuration for the glycerol moiety [16]. Thus, the positive specific rotation of 1 ([α]D20 + 15) was consistent with 2′R configuration. In addition, the induced ECD spectrum of the 1-Mo2(OAc)4 complex showed positive Cotton effect at 305 nm (band IV) (Fig. 5), inferring to positive torsion force between OH-2′ and OH-3′. This data further supported 2′R configuration based on the Snatzke method [17,18]. The molecular formula of eurobenzophenone B (2) was deduced as C19H18O11 based on the HRESIMS and NMR data, containing 11 degrees of unsaturation. Comparison of the NMR data reveled 2 to be
3. Results and discussion Eurobenzophenone A (1) has a molecular formula of C20H20O11 as determined by the HRESIMS and NMR data, requiring 11 degrees of unsaturation. The IR absorptions at 3427 and 1658 cm−1 suggested the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum exhibited a total of 20 carbon resonances, including 12 aromatic carbons for two phenyl rings, and three carbonyl carbons. The 1H NMR spectrum showed three aromatic protons, two methyl singlets, and five protons for oxymethylene and oxymethine. The observation of the metacoupling between the aromatic protons H-2 (δH 6.75, d, J = 2.0 Hz) and H-4 (δH 6.49, d, J = 2.0 Hz) suggested ring A to be tetra-substituted. The HMBC correlations from the phenol proton OH-3 (δH 9.72, s) to C-2 (δC 107.6), C-3 (δC 158.2), and C-4 (δC 106.6), and from OH-5 (δH 9.71, s) to C-4, C-5 (δC 155.1), and C-6 (δC 124.5) indicated C-3 and C-5 to be hydroxylated. The HMBC correlation between H-2 and a carbonyl carbon at δC 167.6 (C-14) located a carboxylic group at C-1 (δC 130.7). In regard to the second aromatic ring (ring B), the presence of only one aromatic proton at δH 6.61 (s, H-12) suggested ring B to be penta-substituted. The HMBC correlations of H-12 to C-8 (δC 110.1), C-10 (δC
Fig. 4. Key COSY and HMBC correlations of 1 and 5.
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data and literature. Thus it was found from nature for the first time, and this is the first report of its NMR data. In addition, the co-isolated compounds were identical to (+)-variecolorquinone A (8) [18], 3-de-O-methylsulochrin (9) [19], 14-de-Omethyl-5-methoxysulochrin (10) [19], sulochrin (11) [19], 5-methoxysulochrin (12) [20], calyxanthone (13) [21], yicathin C (14) [22], yicathin A (15) [22], yicathin B (16) [22], dermolutein (17) [23], methylemodin (18), 1-methoxy-14-dehydroxywentiquinone C (19) [24], wentiquinone C (20) [25]. In the biogenetic speculation, anthraquinones such as endocrocin and emodin were considered as the precursors. As shown in Scheme 1, the oxidative cleavage of anthraquinones by two pathways (a and b) to generate benzophenone nucleus was postulated [26]. Nucleophilic attack of the hydroxyl group in benzophenones in association with dehydration resulted in the formation of xanthone core [27,28]. Methylation of benzophenone and xanthone to yield the methylated derivatives is arbitrarily derived, while the formation of glyceride specifically occurs in the carboxylic acid of ring B (Scheme 1). DPPH assay guided fractionation of the EtOAc extract revealed that F2 fraction possessing the DPPH radical scavenging activity with IC50 = 10 μg/mL (Fig. 3), while the remaining fractions showed weak activity with IC50 > 100 μg/mL. Chromatographic separation of F2 yielded compounds 2, 7–12 as the main components. These compounds were subjected for DPPH assay. As shown in Table 3, benzophenones 3, 9 and 10 exhibited potent radical scavenging activity toward DPPH, and the data were comparable with that of the positive control (trolox). Nitric oxide (NO) is a major mediator produced by macrophages during inflammation responses, whereas an excess amount of NO can destroy and induce dysfunction in the macrophages as well as in surrounding normal cells [29]. Therefore, the level of NO produced by its homeostasis reflected the inflammation stage. Prior to the bioassay, the cytotoxic effects of compounds were detected by the MTT method, showing no cytotoxicity on mouse microglial BV2 cells and SW480 human colon carcinoma cells at a dose of 100 μM. Lipopolysaccharide (LPS) induced NO production in BV2 microglia cells was markedly increased with approximately 3.5-fold related to the control group (P < .001). Compounds 1–20 (10 μM) exerted inhibitory activities against the NO production in the LPS induced BV2 cells ranging from 17.4 to 42.2%, while compound 4 was the most active. The positive control curcumin showed the inhibitory rate of 60% in the same dose (Table 4). In order to insight the NO inhibition related to the cell signaling factors, all compounds were detected for the inhibition of NF-κB in the NF-κB luciferase reporter gene transfected SW480 cells which were activated by the LPS induction. The experimental results revealed that compounds 2, 5, and 8 (10 μM) significantly reduced the NF-κB expression with the inhibitory rates of 74.9%, 68.8%, and 81.2%, respectively, that were comparable with the NF-κB inhibitor MIG132 (90% inhibitory rate in a dose of 10 μM). The experimental data uncovered that the significant inhibition of compound 5 toward inflammatory mediator, nitric oxide (NO), was mediated by down-regulation of the transcription factor nuclear factor-κB (NF-κB), that is an essential factor in controlling inflammatory mediators. However, the remarkable inhibition of compounds 2, 4, 9 and 16–18 against NF-κB expression with weak activities toward NO production suggested these compounds to manage the inflammatory responses by other inflammatory mediator rather than NO.
structurally related to 1, while the distinction was attributed to the presence of a methoxy group in 2. The 2D NMR data further confirmed 2 as a methyl ester product of 1, as evident from the methoxy protons at δH 3.63 (s) correlated to the carbonyl carbon C-14 (δC 166.1) in the HMBC spectrum. The similar ICD data of both 1 and 2 (Fig. 4) and specific rotation assumed the absolute configuration of 2 to be 2′R. Eurobenzophenone C (3) has a molecular formula of C15H12O7, on the basis of the HRESIMS and NMR data, bearing 10 degrees of unsaturation. The 1H and 13C NMR data revealed two aromatic rings, two carbonyl carbons, and a methyl group. Aromatic ring A was identical to that of 1, based on the comparable NMR data and the analyses of 2D NMR data. Aromatic ring B contained four carbon resonances with the overlapped aromatic protons at δH 6.06 (2H, s) and phenol protons at δH 11.48 (2H, s), indicating a symmetric moiety. The HMBC correlations from the methyl protons H3–15 (δH 2.14, s) to C-10/C-12 (δC 109.4) and C-11 (δC 146.7), and the phenol protons (δH 11.48, OH-9/ OH-13) to C-8 (δC 107.4), C-9/C-13 (δC 161.7), and C-10/C-12 identified ring B to be 11-methyl-9,13-dihydroxy substitution. The linkage of rings A and B by a ketone bridge C-7 (δC 200.9) across C-6 and C-8 was established by the four-bond HMBC correlation of C-7 with H-4 and H10/H-12. The molecular formula of euroxanthone A (4) was determined to be C20H18O10 with 12 degrees of unsaturation, based on the HRESIMS and NMR data. The IR absorptions at 3648, 3627, 3436, 1733, 1717, 1683, and 1653 cm−1 suggested the presence of hydroxy, carbonyl, and phenyl groups. The NMR spectra presented 12 aromatic resonances for two phenyl rings and a ketone at δC 178.0 (C-7). These data were characteristic of a xanthone nucleus. The meta-coupling of the aromatic protons H-2 (δH 6.35, d, J = 2.0 Hz) and H-4 (δH 6.21, d, J = 2.0 Hz) in addition to the HMBC correlations of H-2 and H-4 to C-3 (δC 167.5) and C-6 (δC 102.3) along with the correlation between H-2/C-1 (δC 157.1) and H-4/C-5 (δC 163.0) declared a tetra-substituted ring A, in which C-1 and C-3 were hydroxylated. Thus, the singlet aromatic proton at δH 7.39 (s, H-10) and the methyl protons at δH 2.40 (s, H3–16) were assigned to aromatic ring B. The HMBC correlations from H3–16 to C-10 (δC 124.5), C-11 (δC 144.1), and C-12 (δC 124.8), H-10 to C-8 (δC 115.3), C-12, the methyl carbon (δC 19.8), and a carbonyl carbon C-14 (δC 168.6), as well as the correlation between the methoxyl protons (δH 3.88) and C-14, positioned a methyl group at C-11, while a methylformate was linked to C-9 (δC 134.0). Thus, the remaining carbonyl carbon (δC 165.2, C-15) was assumed to be positioned at C-12, and this was supported by the four-bond HMBC correlation between H-10 and C-15. In additional, a glycerol unit was identified by the COSY and HMBC correlations. The HMBC correlation between the methylene protons H2–2′ (δH 4,34, 4.48) and C-15 deduced the formation of a glyceride. Based on the Snatzke method, the positive Cotton effect at 350 nm in the ICD spectrum agreed 2′R configuration, while the positive specific rotation ([α]24 D + 48 in MeOH) further supported the configurational assignment. Euroxanthone B (5) was determined to be an analogue of 4 with a formic acid unit at C-9 to replace a methylformate unit, based on the similar NMR data with the absence of a methoxy group and the molecular formula (C19H16O10) showing CH2 unit less than that of 4. The same phase of the specific rotation of both 4 and 5 ([α]20 D + 14, MeOH) suggested 2′R configuration in 5. Analyses of 1D and 2D NMR data in association with the HRESIMS data revealed the planar structure of 6 to be identical to the demethylated analogue of variecolorquinone A [18], an anthraquinone derivative. The distinction was attributed to the specific rotation of 6 ([α]22 D + 25 in MeOH), whereas that of the known analogue with 2′S was [α]22 D − 23 (MeOH). Thus, the absolute configuration of 6 was assumed to be 2′R, namely (+)1-O-demethylvariecolorquinone A. The structure of compound 7 was determined to be a 3-methoxylated analogue of 3, based on the similar NMR data with the exception of a methoxy group whose protons (δH 3.75, s) correlated to C-3 (δC 159.0). The structure of 7 is hit in SciFinder with the absence of NMR
4. Conclusion Fungus Aspergillus europaeus is a new species closely related to A. wentii originally found from soil [30], while this is the first time to isolate it from marine environment. In addition, this is the first report to describe the secondary metabolites to be isolated from A. europaeus. In the selected culture medium, the PKS biogenetic pathway is activated to produce 20 xanthone-related metabolites including six new compounds, while the polyketides bearing glyceride unit are uncommonly 195
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Scheme 1. Postulation of the biogenetic pathway to derive the isolated compounds.
found from nature. The bioassay results revealed that the significant DPPH radical scavenging activity of compound 9 are corresponded to the potent inhibition against NF-κB in SW480 cells induced by LPS, suggesting the NF-κB being the signing mediator for the antioxidant agents. However, all compounds showed moderate to weak activity to inhibit the NO production in LPS induced BV2 cells. Moreover, compounds 2, 4, 16–18 exerted remarked down-regulation of NF-κB in LPSinduced SW480 cells with weak inhibitory effects against NO production and weak DPPH radical scavenging activity suggested that they are potential to act on the NF-κB induced immune response to infection excluding the mediation by the inflammatory mediators NO and ROS, while the detail mode of action is under progress.
Table 4 Inhibitory activities of 1–20 against NO production in BV2 microglia cells and NF-κB activation in SW480 cells induced by LPS.a No.
% inhibition NOb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Curcuminc MG132c
27.0 17.4 23.3 42.2 23.4 28.6 35.2 28.2 19.5 39.4 26.5 19.5 17.6 27.0 23.7 35.3 25.0 20.0 33.3 38.4 60.1
NF-κBb ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.5 2.6 1.3 2.3 3.3 7.2 7.8 3.8 5.0 2.9 3.2 1.6 5.1 3.2 4.8 3.9 6.3 1.2 5.1 5.0 1.6
31.6 74.9 31.6 68.8 52.3 23.6 31.0 28.2 71.0 31.3 61.7 45.3 63.7 56.8 13.0 81.2 73.1 75.9 58.4 24.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.9 3.8 4.9 7.0 10.6 6.5 4.2 6.7 10.0 12.0 0.9 7.5 5.6 5.7 9.8 8.3 12.7 8.3 6.8 7.9
Conflict of interest The authors declare no conflict of interest. Acknowledgments Financial supports from the MOST-973 Program (2015CB755906) and the National Natural Science Foundation of China (81630089, 41376127, U1606403) are highly appreciated. Appendix A. Supplementary data
88.9 ± 6.1
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2018.08.030.
Dose of compound is 10 μM. SW480 cells are stably transfected with NF-κB luciferase reporter gene. a n = 3. b Three independent experiments, data are shown as mean ± SD. c Positive control.
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