Phenolic C-Glycosides and Aglycones from Marine-Derived

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Phenolic C‑Glycosides and Aglycones from Marine-Derived Aspergillus sp. and Their Anti-Inflammatory Activities Huiling Wen,†,‡,⊥ Chunmei Chen,†,⊥ Weiguang Sun,†,⊥ Yi Zang,† Qin Li,† Wenxuan Wang,§ Fanrong Zeng,† Junjun Liu,† Yuan Zhou,† Qun Zhou,† Jianping Wang,† Zengwei Luo,† Hucheng Zhu,*,† and Yonghui Zhang*,† †

J. Nat. Prod. Downloaded from pubs.acs.org by AUBURN UNIV on 04/23/19. For personal use only.

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation and Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ School of Pharmaceutical Sciences, Gannan Medical University, Ganzhou 341000, People’s Republic of China § School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan, Hubei 430074, People’s Republic of China S Supporting Information *

ABSTRACT: A chemical investigation of the secondary metabolites of a marine-derived Aspergillus sp. led to the isolation and characterization of 13 phenolic compounds, including 10 new compounds (1−10). Seven new compounds (1−7) are unusual phenolic C-glycosides, while the other new compounds (8−10) are structurally related aglycones. The chemical structures of these new compounds were elucidated by 1D and 2D NMR and HRESIMS spectroscopic analyses. The absolute configurations of these new C-glycosides were determined by comparison of experimental electronic circular dichroism spectra with those of calculated ones. In addition, the anti-inflammatory activities of these compounds were evaluated, and compound 9 significantly inhibited nitric oxide production with an IC50 value of 6.0 ± 0.5 μM in lipopolysaccharide-induced RAW264.7 cells. Moreover, compound 9 also showed anti-inflammatory activity by inhibiting the NF-κB-activated pathway.

compounds in the literature, anti-inflammatory activities were tested for all obtained compounds. The results showed that compound 9 significantly inhibited nitric oxide (NO) production with an IC50 value of 6.0 ± 0.5 μM, and it showed anti-inflammatory activity by inhibiting the NF-κB-activated pathway. Herein, we report the isolation and structure determination of the new isolates in addition to their biological evaluation.

C-Glycosides are characterized by a key C−C glycosidic bond, and the anomeric carbon of the sugar moiety is directly attached to a carbon atom of the aglycone. Normally, this C− C glycosidic bond is stable to both enzymatic and chemical hydrolysis, which confers C-glycosides a broad range of biological activities, such as antidiabetic properties via inhibition of the sodium-dependent glucose transporter 2 (SGLT2),1−3 antioxidant activity,4 and anti-inflammatory activity.5 In the past decade, C-glycosides have attracted considerable attention from scientists for their diverse structures and biosynthetic enzymes6−13 and as targets for synthesis.14,15 Fungi of the genus Aspergillus are a rich source of structurally intriguing and biologically active secondary metabolites, and in our previous research, a series of fungi belonging to the genus Aspergillus have been investigated, such as Aspergillus terreus and Aspergillus f lavipes.16−19 In this study, a marine-derived Aspergillus sp. was investigated as part of our ongoing search for biologically active compounds from the genus Aspergillus, leading to the isolation and characterization of 10 new phenolic compounds (1−10) and three known compounds (11−13). Literature investigation revealed that phenolic Cglycosides and alkylresorcinol derivatives have commonly had anti-inflammatory activities.5,20−22 Therefore, considering the structural relationship between compounds 1−10 and those © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Chemical investigation of the culture broth of a marine-derived Aspergillus sp. led to the isolation of 13 phenolic compounds, including 10 new compounds (1−10), and of these, compounds 1−7 are unusual phenolic C-glycosides and compounds 8−10 are structurally related aglycones. The known compounds carnemycin B (11),23 carnemycin A (12),23 and 2,4-dihydroxy-6-((3E,5E)-nona-3,5-dien-1-yl)benzoic acid (13)24,25 were identified by comparing their spectroscopic data with those reported in the literature, respectively. Compound 1 was obtained as an amorphous, reddish gum.26,27 Based on its HRESIMS data, it has a molecular Received: September 1, 2018

A

DOI: 10.1021/acs.jnatprod.8b00744 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

The relative configuration of the sugar moiety was established on the basis of the coupling constants of the observed protons and key NOESY correlations.28 The NOESY correlation between H-1′ and H-5′ indicated those protons were on the same face of the six-membered ring. The coupling constants (in pyridine-d5) of H-1′/H-2′ (J = 9.8 Hz), H-2′/H3′ (J = 8.8 Hz), and H-3′/H-4′ (J = 8.8 Hz) indicated transdiaxial relationships. These analyses, along with the anomeric proton resonating at δH 5.86 (H-1′), suggested the sugar moiety in compound 1 was a β-glucose unit. The Econfigurations of the two double bonds in the octadienyl unit were determined from the coupling constants of J10,11 (J = 14.5 Hz) and J12,13 (J = 14.7 Hz). Thus, compound 1 was found to have an unusual methyl 2,4-dihydroxy-3-C-(βglucosyl)benzoate, and some literature investigations revealed that only three compounds, 11, 12, and stromemycin, possess such a substructure.23,24 The absolute configurations of most previously reported Cglycosides were not determined or were indirectly assigned based on the biosynthesis pathway.29−31 In this study, the absolute configuration of the sugar moiety of 1 was determined by calculation of the electronic circular dichroism (ECD) spectrum. Considering that the chromophore in the octadienyl chain should have no significant effect on the ECD spectrum and that the flexible side chain will complicate the ECD calculation, the side chain was truncated to obtain a model structure to simplify the computations. As shown in Figure 2, the Cotton effects in the experimental ECD spectrum were consistent with the calculated ECD curve of model structure

formula of C22H30O9, which corresponds to eight degrees of unsaturation. The IR absorption bands at 3380 and 1641 cm−1 implied the presence of hydroxy and unsaturated carboxyl or ester carbonyl moieties. The 1H NMR (Table 1) and HSQC spectra of 1 indicated the presence of an aromatic proton (δH 6.28, s, H-5), four olefinic protons [δH 5.57 (m, H-10), 6.02 (m, H-11), 5.97 (m, H-12), and 5.62 (m, H-13)], a methyl group [δH 0.99 (t, J = 7.4 Hz, H-15)], a methoxy group [δH 3.91 (s, H-16)], and three methylenes [δH 2.89 (m, H-8), 2.26 (m, H-9), and 2.06 (m, H-14)]. The remaining oxygenated methine and methylene proton resonances at δH 3.38−4.91 suggested the presence of a hexose unit in the structure of 1. The 13C NMR and DEPT data (Table 2) revealed the presence of 22 carbons, which were assigned as an unsaturated carboxyl or ester carbonyl, 10 aromatic or olefinic carbons, three methylenes, a methyl group, a methoxy group, and six carbons belonging to the C-glycopyranosyl unit. Analyses of the COSY spectrum revealed two spin systems, including an octadienyl chain of H2-8/H2-9/H-10/H-11/H12/H-13/H2-14/H3-15 and a hexose unit of H-1′/H-2′/H-3′/ H-4′/H-5′/H2-6′. In addition, the HMBC correlations from H-8 to C-1, C-5, and C-6; from H-1′ to C-2, C-3, and C-4; and from H-5 to C-1, C-3, and C-4 defined a pentasubstituted aromatic ring and also revealed the location of the octadienyl chain at C-6 and the hexose at C-3. Finally, a methyl ester group, indicated by the HMBC interaction from Me-16 to C-7 (δC 173.4) (Figure 1), and two hydroxy groups were located at C-1, C-2, and C-4, respectively, according to the chemical shifts of C-1 (δC 105.8), C-2 (δC 164.3), and C-4 (δC 162.5). B



Article

Table 1. 1H NMR Spectroscopic Data for Compounds 1−10 (δ in ppm, J in Hz) no. 1 2 3 4 5 6 7 8

1a

2a

3b

4b

5b

6a

6.67, s

6.28, s

6.27, s

6.65, s

9b

6.09, t (2.2)

6.91, t (2.2)

6.15, d (2.2)

6.77, d (2.2)

5.06, m 2.97, m 6.24, d (2.3)

6.15, d (2.2) 2.51, m 2.42, m; 2.13, m 5.30, m

6.77, d (2.2) 2.71, m 2.47, m

6.20, d (2.3)

6.23, s

6.22, s

2.60, m 2.40, m

2.50, m 2.40, m

2.53, m 2.35, m

5.65, dt (14.8, 7.4) 6.10, dd (14.8, 10.6) 6.02, dd (14.5, 10.6) 5.55, dt (14.5, 7.0) 1.96, m

5.29, dt (11.0, 7.4) 5.93, t (11.0) 6.27, dd (15.0, 11.0) 5.64, dt (15.0, 7.2) 2.07, m

5.65, dt (15.2, 6.7) 6.34, dd (15.2, 11.0) 5.92, t (11.0) 5.30, dt (11.0, 7.4) 2.13, m

2.36, m

5.70, dt (14.7, 6.8) 6.12, dd (14.7, 10.4) 6.05, dd (14.7, 10.4) 5.56, dt (14.7, 7.0) 1.97, m

1.30, m 0.81, t (7.3)

1.42, m 0.92, t (7.4)

1.40, m 0.92, t (7.4)

2.07, m 1.41, m 0.91, t (7.4)

1.31, m 0.82, t (7.4)

5.83, d (9.8)

4.91, m

4.91, m

2.86, m

2.93, m

9

2.26, m

2.26, m

2.51, m

2.44, m

10

5.57, m

5.54, m

11

6.02, m

6.00, m

5.45, dt (10.9, 7.6) 6.12, t (10.9)

12

5.97, m

6.00, m

5.81, dt (14.6, 7.5) 6.55, dd (14.6, 10.9) 6.11, t (10.9)

13

5.62, m

5.59, m

14 15 16 17 1′

2.06, m 0.99, t (7.4) 3.91, s

1.71, d (6.1) 3.91, s

4.91, d (9.9)

4.91, d (9.9)

2′

4.05, m

4.03, m

3′

3.47, m

4′

6.50, dd (14.7, 10.9) 5.71, dt (14.7, 7.1)

5.38, dt (10.9, 7.6)

2.06, 1.35, 0.84, 3.81, 5.86,

2.14, 1.34, 0.85, 3.81, 5.85,

m m t (7.4) s d (9.8)

3.47, m

4.96, dd (9.8, 8.8) 4.41, t (8.8)

4.97, dd (9.8, 8.8) 4.41, t (8.8)

4.75, dd (9.8, 8.8) 4.38, t (8.8)

3.90, dd (9.8, 8.8) 3.49, m

3.91, dd (9.8, 8.8) 3.47, m

3.47, m

3.47, m

4.50, m

4.50, m

4.45, m

3.49, m

3.47, m

5′

3.38, m

3.40, m 3.85, dd (12.1, 2.2) 3.73, dd (12.1, 5.1)

4.04, dt (9.4, 3.5) 4.45, m

3.40, m

3.85, dd (12.1, 2.2) 3.73, dd (12.1, 5.1)

4.12, dt (9.4, 3.5) 4.50, m

3.39, m

6′a

4.13, dt (9.4, 3.5) 4.50, m

3.85, dd (12.3, 2.5) 3.76, dd (12.1, 4.7)

3.85, dd (12.1, 2.3) 3.76, dd (12.1, 4.7)

7′

10a

6.22, s

6.67, s

2.88, m

6′b

8a

6.64, s

2.89, m

m m t (7.4) s d (9.8)

6.23, s

7a

5.93, t (11.0) 6.27, m 5.64, m

5.71, dd (15.3, 6.9) 6.40, dd (15.3, 10.4) 6.09, dd (15.3, 10.4) 5.82, dt (15.3, 7.1) 2.09, m 1.44, m

0.92, t (7.4)

a

Recorded at 400 MHz in CD3OD. bRecorded at 400 MHz in pyridine-d5.

Compound 3 was obtained as an amorphous, reddish gum. Its molecular formula was assigned as C23H32O9 based on the [M + Na]+ ion at m/z 475.1938 in the HRESIMS spectrum. Compound 4 was obtained and had the same molecular formula. Their UV, IR, and NMR spectra closely resembled those of 1, except for an additional sp3 methylene signal. Further analyses of the COSY and HMBC spectra of 3 and 4 indicated that the octadienyl chain of 1 was replaced by nonadienyl residues in 3 and 4. Compounds 3 and 4 were considered to be a pair of isomers based on a comparison of their NMR data, which differed by the coupling constants of the double-bond protons. The configurations of Δ10 and Δ12 in 3 were assigned as Z and E, respectively, according to the coupling constants (J10,11 = 10.9 Hz and J12,13 = 14.7 Hz). However, the opposite configurations were observed in 4 according to the coupling constants (J10,11 = 14.6 Hz and J12,13 = 10.9 Hz). The nearly identical experimental ECD spectra of 3, 4, and 1 indicated that their sugar moieties share the same absolute configuration. On the basis of the above data, the structures of 3 and 4 were established as methyl 2,4-dihydroxy6-[(3Z,5E)-3,5-nonadienyl]-3-C-(β-D-glucosyl)benzoate and methyl 2,4-dihydroxy-6-[(3E,5Z)-3,5nonadienyl]-3-C-(β-Dglucosyl)benzoate. Because they had similar structures to

1A. Therefore, compound 1 was identified as methyl 2,4dihydroxy-6-[(3E,5E)-3,5-octadienyl)]-3-C-(β-D -glucosyl)benzoate and was given the trivial name carnemycin C. Compound 2 was also obtained as an amorphous, reddish gum. The HRESIMS spectrum of 2 supported a molecular formula of C21H28O9, 14 mass units less than that of 1. The UV, IR, and NMR data of 2 were very similar to those of 1, indicating that 2 and 1 share the same structural scaffold. A detailed comparison of the 13C NMR spectra of 2 and 1 revealed the absence of a methylene group in 2. Further analyses of the COSY and HMBC spectra of 2 indicated that the octadienyl chain of 1 was replaced by a heptadienyl residue. The E-configurations of the two double bonds in the heptadienyl unit were determined by the coupling constants (J10,11 = 14.4 Hz and J12,13 = 14.1 Hz) in the 1H NMR spectrum of 2 recorded in pyridine-d5. The absolute configuration of 2 was determined by comparing its experimental ECD spectrum with that of 1. Therefore, compound 2 was defined as methyl 2,4-dihydroxy-6[(3E,5E)-3,5-heptadienyl]-3-C-(β-D-glucosyl)benzoate and designated carnemycin D. C


Journal of Natural Products Table 2.

13

Article

C NMR Spectroscopic Data for Compounds 1−10 (δ in ppm)

no.

1a

2a

3b

4b

5b

6a

7a

8a

9b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1′ 2′ 3′ 4′ 5′ 6′ 7′

105.8, C 164.3, C 110.7, C 162.5, C 112.3, CH 147.7, C 173.4, C 37.6, CH2 35.8, CH2 132.1, CH 132.2, CH 130.7, CH 135.1, CH 26.6, CH2 14.1, CH3 52.4, CH3

105.7, C 164.1, C 110.7, C 162.4, C 112.4, CH 147.6, C 173.3, C 37.6, CH2 35.8, CH2 132.1, CH 131.8, CH 132.9, CH 127.9, CH 18.1, CH3 52.4, CH3

108.9, CH 158.0, C 110.1, C 158.0, C 108.9, CH 145.0, C 36.9, CH2 30.3, CH2 129.5, CH 130.3, CH 127.0, CH 135.6, CH 36.0, CH2 23.6, CH2 14.0, CH3

108.9, CH 158.0, C 110.1, C 158.0, C 108.9, CH 144.9, C 36.8, CH2 35.3, CH2 134.4, CH 127.4 CH 130.0 CH 130.8, CH 30.6, CH2 23.8, CH2 14.0, CH3

159.4, C 101.2, CH 159.4, C 108.0, CH 145.5, C 108.0, CH 37.1, CH2 30.5, CH2 129.5, CH 130.3, CH 127.0, CH 135.5, CH 36.0, CH2 23.6, CH2 14.0, CH3

160.6, C 102.1, CH 160.6, C 108.1, CH 145.2, C 108.1, CH 36.8, CH2 35.2, CH2 132.1, CH 131.5, CH 131.7, CH 132.9, CH 35.0, CH2 23.1, CH2 14.1, CH3

76.1, 73.0, 80.1, 71.6, 82.6, 62.7,

106.6, C 163.6, C 112.2, C 163.6, C 112.2, CH 145.9, C 172.4, C 36.9, CH2 35.6, CH2 134.6, CH 126.9, CH 129.8, CH 130.8, CH 30.3, CH2 23.5, CH2 14.2, CH3 52.2, CH3 76.9, CH 73.9, CH 80.7, CH 71.8, CH 83.2, CH 62.6, CH2

109.0, CH 158.4, C 111.5, C 158.4, C 109.0, CH 143.8, C 36.2, CH2 34.7, CH2 131.9, CH 131.5, CH 131.3, CH 132.8, CH 35.0, CH2 23.0, CH2 14.0, CH3

76.1, 73.0, 80.1, 71.6, 82.6, 62.6,

106.9, C 163.5, C 112.3, C 163.0, C 112.3, CH 146.0, C 172.5, C 37.0, CH2 30.6, CH2 129.8, CH 130.0, CH 126.9, CH 135.6, CH 35.6, CH2 23.3, CH2 14.3, CH3 52.4, CH3 77.0, CH 74.0, CH 80.8, CH 72.0, CH 83.3, CH 62.7, CH2

77.6, 74.6, 80.3, 71.3, 82.8, 62.0,

76.9, 73.8, 80.0, 71.4, 82.4, 62.4,

76.8, 73.8, 80.0, 71.4, 82.4, 62.3,

CH CH CH CH CH CH2

CH CH CH CH CH CH2

CH CH CH CH CH CH2

CH CH CH CH CH CH2

CH CH CH CH CH CH2

10a 171.4, C 80.8. CH 34.4, CH2 108.0, CH 165.6, C 102.2, CH 166.4, C 101.6, C 143.1, C

127.9, CH 135.4, CH 130.5, CH 138.4, CH 35.8, CH2 23.4, CH2 14.0, CH3

a

Recorded at 100 MHz in CD3OD. bRecorded at 100 MHz in pyridine-d5.

Figure 1. Key COSY and HMBC correlations of 1, 5, 9, and 10.

carnemycin B (11),23 they were given the trivial names (10Z,12E)-carnemycin B and (10E,12Z)-carnemycin B, respectively. Compound 5, an amorphous, reddish gum, was assigned a molecular formula of C21H30O7 based on its HRESIMS data. Comparison of the 1D NMR data of 5 with those of 3 and 4 revealed that the structures of these compounds were similar. The only significant differences between their 13C NMR spectra were the absence of the ester carbonyl and methoxy carbons. These differences indicated that the methyl ester group located at C-1 of 3 was missing in 5, which was supported by HMBC correlations from H-7 to C-1, C-5, and C-6. In addition, Δ9 and Δ11 both had trans configurations, according to the coupling constants (J9,10 = 14.8 Hz and J11,12 = 14.5 Hz) in pyridine-d5. The absolute configuration of the sugar was also determined by comparing the calculated and experimental ECD spectra (Figure 3). Finally, compound 5

was determined to be 2,4-dihydroxy-6-[(3E,5E)-3,5-nonadienyl]-3-C-(β-D-glucosyl)benzene and was named carnemycin E. The 1D and 2D NMR data of 6 and 7 were very similar to those of 5. The main differences were the configuration of the two double bonds. Δ9 and Δ11 in 6 were in cis and trans configurations, respectively, according to the coupling constants (J9,10 = 11.0 Hz and J11,12 = 15.0 Hz) in pyridined 5 , while the double bonds in 7 had the opposite configurations according to the coupling constants (J9,10 = 15.2 Hz and J11,12 = 11.0 Hz) in pyridine-d5. The close resemblance of the experimental ECD spectra of 5, 6, and 7 indicated their sugars had the same absolute configuration (Figure 3). Therefore, the structures of compounds 6 and 7 were determined, and they were named carnemycin F and carnemycin G, respectively. D



Article

Compound 9 was obtained as an amorphous, reddish gum. Its molecular formula was also determined to be C15H20O2 by HRESIMS analysis. The NMR data of 9 were almost identical to those of 8, except for the coupling constant between H-9 and H-10 (J9,10 = 14.7 Hz) in pyridine-d5. This difference suggested Δ9 in 9 was in the trans configuration. The structure of 9 was further confirmed by 2D NMR spectroscopy, including COSY and HMBC experiments (Figure 1). Therefore, compound 9 was named 5-[(3E,5E)-3,5-nonadienyl]-1,3benzenediol. Compound 10 was isolated as an amorphous, reddish gum with a molecular formula of C16H18O4 on the basis of its HRESIMS peak at m/z 273.1141, which is one carbon more than in 9. The overall 1H and 13C NMR data of 10 were similar to those of 9; however, some significant differences were observed. Signals of the methylene (C-8) in 9 were replaced by an oxygenated methine in 10 based on the HMBC correlations from H-4 to C-5, C-9, and C-1′ and the COSY correlation of H-3/H-4. In addition, an ester carbonyl was located at C-9, as indicated by the chemical shifts of C-10 (δC 143.1), C-9 (δC 101.6), and C-8 (δC 166.4). As 10 did not show a strong Cotton effect and the value of its optical rotation ([α]25D −0.91) was very small, it is hard to determine its absolute configuration. Thus, compound 10 was named 3-[(1′E,3′E)1′,3′-heptadienyl]-6,8-dihydroxy-1′,3′-dienylisocoumarin in a systematic name. All new phenolic compounds (1−10) were recorded as reddish gums, although they do not have chromophores that would suggest a red color. Minor oxidized impurities related to the compounds may be the source of the red color. It is known that many compounds with a conjugated double bond will isomerize when exposed to UV light or high temperature. Therefore, stability experiments of these isolates were conducted with 6 and 7 as representatives because both of them possess a cis-double bond, which were thought to be less stable. After a water bath at 60 °C or exposed to UV light for 3 days, there are only very few impurity signals of 6 as compared with the control group (Figure S101), and there is no change of these signals with prolonged exposure or a water bath time of 7 days. Therefore, these compounds were thought to be stable in these conditions, but whether they are stable in the much more complex conditions during extraction is still uncertain. Meanwhile, to confirm whether these methyl or ethyl esters were formed during the isolation process, the free acid 13 was dissolved in MeOH and EtOH and water bathed at 60 °C for 3 days, respectively. The results displayed that esterification did not occur in the experiment (Figure S102). However, due to the complex condition during extraction, there are many other substances that could potentially catalyze ester formation, particularly when MeOH and EtOH were used in the isolation process. Therefore, the presence of both esters might suggest that compounds 1−4, 11, and 12 are artifacts, whereas the corresponding precursor compounds were actually secondary metabolites of Aspergillus sp. In this report, the inhibitory effects of compounds 1−13 on NO production in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells were investigated. Compound 9 significantly inhibited NO production with an IC50 value of 6.0 ± 0.5 μM, whereas the IC50 values of the other tested samples were greater than 9 μM. Therefore, subsequent studies of 9 were done to elucidate its underlying molecular mechanism. As shown in Figure 4, compound 9 decreased the level of IL-1β

Figure 2. Calculated ECD spectra of the model structure and experimental ECD spectra of 1, 2, 3, and 4.

Figure 3. Calculated ECD spectrum of 5 and experimental ECD spectra of 5, 6, and 7.

Compound 8 was isolated as an amorphous, reddish gum. Its molecular formula, C15H20O2, with six degrees of unsaturation, was determined from its HRESIMS data. Detailed analysis of the 1H NMR and 13C NMR spectra of 8 revealed that the obvious signals of the sugar moiety in 6 were missing. The HMBC correlations from H-2 to C-1 and C-3 confirmed that compound 8 was the aglycone of 6. The configurations of Δ9 and Δ11 in 8 were the same as those in 6, and they were assigned as cis and trans, respectively, based on the coupling constants of H-9/H-10 (J9,10 = 11.0 Hz) and H-11/H-12 (J11,12 = 15.0 Hz) in CD3OD. Similarly to the known compound 5[(3Z,5Z)-3,5-nonadienyl]-1,3-benzenediol,32 compound 8 was named 5-[(3Z,5E)-3,5-nonadienyl]-1,3-benzenediol. E



Article

Figure 4. Effects of 9 on IL-1β (A) and IL-6 production (B) and protein expression levels of COX-2 (C, D) in RAW264.7 cells stimulated with LPS. The cells were pretreated for 2 h with the indicated concentrations of 9 and stimulated for 12 h with LPS (1 μg/mL). The measurement of the nitrite concentration and Western blot analyses were performed as described in the Experimental Section. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the group treated with LPS. spectrometer. Semipreparative HPLC separations were performed on an Agilent 1220 system equipped with an Ultimate XB-C18 column (5 μm, 10 × 250 mm). Gaussian 09 software was used for quantum chemical calculations. Fungus Material and Fermentation. The fungus Aspergillus sp. was isolated from superficial mycobiota of the brown alga Saccharina cichorioides f. sachalinensis collected from the South China Sea. rRNA gene sequence analysis results (DDBJ/EMBL/GenBank under accession No. 2167894) showed 99% similarities to Aspergillus ustus and Aspergillus calidoustus. Therefore, the strain was identified as an Aspergillus sp. Its static fermentation was performed at room temperature (rt) for 21 days in 240 × 1 L Erlenmeyer flasks containing 200 g of rice and 160 mL of distilled water. Extraction and Isolation. The fermented media were extracted with 95% EtOH and then partitioned with EtOAc and H2O to obtain an EtOAc extract (600 g). The extract was fractionated by silica gel column chromatography (CC) eluting with petroleum ether (PE)/ EtOAc and EtOAc/MeOH to generate four fractions (Fr. 1−Fr. 4). Fr. 3 was further purified by CC on RP-18, Sephadex LH-20 (MeOH), and semipreparative HPLC (CH3CN/H2O) to produce 1 (9.8 mg), 2 (2.0 mg), 3 (10.0 mg), 4 (6.6 mg), 5 (102.5 mg), 6 (1.8 mg), 7 (1.7 mg), 8 (1.3 mg), 9 (75.2 mg), 10 (2.3 mg), 11 (31.7 mg), 12 (2.3 mg), and 13 (24.7 mg). Carnemycin C (1): amorphous, reddish gum; [α]24D +25 (c 0.76, MeOH); UV (MeOH) λmax (log ε) 224 (4.74), 263 (4.27) nm; ECD (MeOH, 0.14 mM) λmax (Δε) 229 (−3.8), 266 (+4.7) nm; IR (KBr) νmax 3380, 2926, 1641, 1263 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 461.1801 [M + Na]+ (calcd for C22H30O9Na, 461.1788). Carnemycin D (2): amorphous, reddish gum; [α]25D +66 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 224 (4.94), 263 (4.35) nm; ECD (MeOH, 0.99 mM) λmax Δε 229 (−0.76), 266 (+0.71) nm; IR (KBr) νmax 3377, 2924, 1639, 1263 cm−1; 1H and 13C NMR spectroscopic data, Tables 1 and 2; (+)-HRESIMS m/z 447.1631 [M + Na]+ (calcd for C21H28O9Na, 447.1631). (10Z,12E)-Carnemycin B (3): amorphous, reddish gum; [α]25D +11 (c 2.17, MeOH); UV (MeOH) λmax (log ε) 224 (4.90), 264 (4.33) nm; ECD (MeOH, 1.85 mM) λmax Δε 236 (−0.66) nm; IR (KBr) νmax 3378, 2929, 1641, 1437, 1263 cm−1; 1H and 13C NMR data,

and IL-6 in a dose-dependent manner in LPS-induced RAW264.7 cells. Moreover, LPS-induced overexpression of cyclooxygenase-2 (COX-2) in RAW264.7 cells was significantly downregulated after treatment with compound 9. Moreover, the addition of 9 suppressed LPS-induced nuclear translocation of NF-κB p65 (Figure 5). Taken together, these results indicate that 9 can inhibit the expression of inflammatory mediators by inhibiting the NF-κB-activated pathway.

Figure 5. Effects of 9 on NF-κB activation detected by nuclear translocation of NF-κB p65; nuclear translocation was investigated by staining with an anti-p65 subunit antibody (green) and DAPI (blue).

■

EXPERIMENTAL SECTION

General Experimental Procedures. 1D and 2D NMR spectra were collected on a Bruker AM-400 spectrometer with tetramethylsilane as internal standard. Chemical shifts (δ) were acquired in ppm with reference to the solvent signals. Optical rotations were recorded on a PerkinElmer PE-341 instrument. UV absorption data were measured in MeOH on a PerkinElmer Lambda 35. ECD spectra were measured using a JASCO-810 spectrometer. IR spectra were measured with a Vertex 70 spectrophotometer using KBr pellets. HRESIMS data were acquired on a Bruker microTOF II F



Article

Tables 1 and 2; (+)-HRESIMS m/z 475.1938 [M + Na]+ (calcd for C23H32O9Na, 475.1944). (10E,12Z)-Carnemycin B (4): amorphous, reddish gum; [α]25D +21 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 225 (4.94), 264 (4.37) nm; ECD (MeOH, 0.18 mM) λmax Δε 231 (−2.86) nm; IR (KBr) νmax 3383, 2928, 1641, 1263 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 475.1930 [M + Na]+ (calcd for C23H32O9Na, 475.1944). Carnemycin E (5): amorphous, reddish gum; [α]25D +20 (c 0.67, MeOH); UV (MeOH) λmax (log ε) 232 (4.64), 283 (3.44) nm; ECD (MeOH, 0.46 mM) λmax Δε 233 (−3.12) nm; IR (KBr) νmax 3364, 2925, 1632, 1450, 1078 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 417.1874 [M + Na]+ (calcd for C21H30O7Na, 417.1889). Carnemycin F (6): amorphous, reddish gum; [α]25D +17 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 232 (4.74), 283 (3.52) nm; ECD (MeOH, 0.40 mM) λmax Δε 235 (−2.21) nm; IR (KBr) νmax 3382, 2927, 1633, 1450 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 417.1869 [M + Na]+ (calcd for C21H30O7Na, 417.1889). Carnemycin G (7): amorphous, reddish gum; [α]25D +15 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 233 (4.73), 283 (3.58) nm; ECD (MeOH, 0.32 mM) λmax Δε 231 (−2.25) nm; IR (KBr) νmax 3388, 2928, 1632, 1449 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 417.1875 [M + Na]+ (calcd for C21H30O7Na, 417.1889). 5-[(3Z,5E)-3,5-Nonadienyl]-1,3-benzenediol (8): amorphous, reddish gum; UV (MeOH) λmax (log ε) 232 (4.86), 282(3.67) nm; IR (KBr) νmax 3391, 2925, 1601, 1154 cm−1; 1H and 13C NMR data, Tables 1 and 2; (−)-HRESIMS m/z 231.1408 [M − H]− (calcd for C15H19O2, 231.1385). 5-[(3E,5E)-3,5-Nonadienyl]-1,3-benzenediol (9): amorphous, reddish gum; UV (MeOH) λmax (log ε) 232 (5.35), 275 (3.77) nm; IR (KBr) νmax 3324, 2927, 1604, 1479, 1156 cm−1; 1H and 13C NMR data, Tables 1 and 2; (+)-HRESIMS m/z 233.1551 [M + H]+ (calcd for C15H21O2, 233.1542). 3-[(1′E,3′E)-1′,3′-Heptadienyl]-6,8-dihydroxy-1′,3′-dienylisocoumarin (10): amorphous, reddish gum; UV (MeOH) λmax (log ε) 229 (5.00), 269 (4.49), 302 (4.19) nm; IR (KBr) νmax 3427, 2923, 1632, 1450, 1078 cm−1; 1H and 13C NMR spectroscopic data, Tables 1 and 2; (−)-HRESIMS m/z 273.1141 [M − H]− (calcd for C16H17O4, 273.1127). Computational Section. The conformations of the model structure of 1 generated by BALLOON33,34 were optimized based on the PM3 semiempirical level of theory using the Gaussian 09 program. If the root-mean-square (RMS) distance was less than 0.5 Å for any two geometry-optimized conformations, duplicate conformations were identified and removed. The conformations left were further optimized at the B3LYP/6-31G(d) level. The solvation effect in MeOH was assessed using the IEFPCM model. The stability of the final conformers was confirmed by calculating the harmonic vibrational frequencies. ECD spectra were calculated via timedependent density functional theory (TDDFT) at the B3LYP/6311++G(d,p)//B3LYP/6-31G(d) level. The calculated ECD spectrum of each conformer was generated with a bandwidth σ = 0.4 eV. Confab was used to generate the conformers.35 All the generated conformers of compound 5 were optimized with MOPAC2016 on the PM7 semiempirical level of theory.36 The conformers that had 4 kcal/ mol higher energy than the lowest conformer were subjected to further optimization and frequency calculations at the B3PW91-D3/ SVP level of theory, the same as the above solvent model and software package. TDDFT calculations of each conformer were done at the same level of theory as was used for the optimization. ECD spectra were generated by SpecDis 1.71 with a bandwidth σ = 0.3 eV. The contribution of each conformer to the final ECD spectrum was Boltzmann weighted according to their Gibbs free energy. Determination of Nitric Oxide, IL-1β, and IL-6. RAW264.7 cells were dispensed in 96-well culture plates (2 × 105 cells/well). After preincubation for 24 h, the seeded cells were treated with concentrations of the test compounds ranging from 40 to 0.5 μM for

2 h. Subsequently the cells were stimulated in the presence of LPS (1 μg/mL) for another 12 h. According to previous literature, the concentration of NO in each well was determined by Griess reagent. After collecting and centrifuging the culture medium at 16 000 rpm for 20 min, Griess reagent (100 μL) containing 0.1% naphthyl ethylenediamine dihydrochloride, 1% sulfanilamide, and 2% phosphoric acid was added to the cell culture supernatants (50 μL) in a 96-well plate for 10 min of incubation at rt. Then, the absorbance was measured at 540 nm with a microplate reader. Furthermore, in order to further evaluate the anti-inflammatory effect of compound 9 on cytokine release, two inflammatory mediators, IL-6 and IL-1β, were investigated by ELISA (BOSTER Biological Technology Co., Ltd.).

Table 3. Inhibitory Effects of Compounds Isolated from Aspergillus sp. on NO Production in LPS-Stimulated RAW 264.7 Macrophage Cells compound 1 2 3 4 5 6 7 8 9 10 11 12 13

IC50 (μM)a 19 >40 >40 14 >40 >40 30 28 6.0 17 >40 15 9.9

±2

±1

± ± ± ±

2 1 0.5 1

±1 ± 0.7

a

The IC50 value is defined as the concentration that results in a 50% decrease in the production of NO. The values represent the means of the results of three independent experiments with similar patterns. NF-κB/p65 Nuclear Translocation Immunofluorescence. Cell were fixed with 4% paraformaldehyde for 20 min at rt. Subsequently, they were permeabilized with 0.2% Triton X-100 for 30 min. Then, the slides were incubated with a primary antibody specific to the NF-κB p65 subunit overnight at 4 °C after blocking with 5% bovine serum albumin for 1 h and then incubated with the secondary antibody conjugated with Alexa Fluor 594 (1:500) for 1 h at rt. After staining with DAPI (5 μg/mL in PBS) for 30 min at 37 °C, the coverslips were washed and sealed. Images were obtained using an OLYMPUS IX73 fluorescence microscope (Olympus, Tokyo, Japan) with excitation/emission wavelengths of 590 nm/617 nm for Alexa Fluor-594 and 360 nm/450 nm for DAPI. Western Blot Analysis. The protein levels of COX-2 were measured using Western blot analysis. The procedures for the Western blot analysis were performed according to previous work.37 The cells were lysed with 20 mM Tris HCl buffer (pH 7.4), which contained the protease inhibitor mixture [phenylmethylsulfonyl fluoride (0.1 mM), aprotinin (5 mg/mL), pepstatin A (5 mg/mL), and chymostatin (1 mg/mL)]. The protein concentration was measured with a protein assay kit. Cell protein extracts were placed onto SDS-PAGE gels. Then, proteins was transferred electrophoretically to polyvinylidene fluoride membranes, blocked in a mixed solution that contains phosphate-buffered saline, 0.1% Tween 20, and 5% skimmed milk for 1 h at rt, and then sequentially incubated with the first antibodies specific to COX-2 and β-actin overnight at 4 °C. The following further incubation with proper secondary antibodies were performed. Blots were developed using chemiluminescence (Fusion FX6 XT), and the bands were quantified using ImageJ software. Statistical Analysis. Data were presented as means ± SD (n = 3). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests was used to assess the differences among means of G



Article

(9) Chen, D.; Sun, L.; Chen, R.; Xie, K.; Yang, L.; Dai, J. Chem. Eur. J. 2016, 22, 5873−5877. (10) Hoffmeister, D.; Drager, G.; Ichinose, K.; Rohr, J.; Bechthold, A. J. Am. Chem. Soc. 2003, 125, 4678−4679. (11) Andersen-Ranberg, J.; Kongstad, K. T.; Nafisi, M.; Staerk, D.; Okkels, F. T.; Mortensen, U. H.; Møller, B. L.; Frandsen, R. J. N.; Kannangara, R. ChemBioChem 2017, 18, 1893−1897. (12) Durr, C.; Hoffmeister, D.; Wohlert, S. E.; Ichinose, K.; Weber, M.; Mulert, U. V.; Thorson, J. S.; Bechthold, A. Angew. Chem., Int. Ed. 2004, 43, 2962−2965. (13) Chen, D.; Chen, R.; Xie, K.; Yue, T.; Zhang, X.; Ye, F.; Dai, J. Org. Lett. 2018, 20, 1634−1637. (14) Kitamura, K.; Ando, Y.; Matsumoto, T.; Suzuki, K. Chem. Rev. 2018, 118, 1495−1598. (15) Bokor, É .; Kun, S.; Goyard, D.; Tóth, M.; Praly, J. P.; Vidal, S.; Somsák, L. Chem. Rev. 2017, 117, 1687−1764. (16) Zhu, H.; Chen, C.; Tong, Q.; Yang, J.; Wei, G.; Xue, Y.; Wang, J.; Luo, Z.; Zhang, Y. Angew. Chem., Int. Ed. 2017, 56, 5242−5246. (17) Zhu, H.; Chen, C.; Tong, Q.; Li, X. N.; Yang, J.; Xue, Y.; Luo, Z.; Wang, J.; Yao, G.; Zhang, Y. Angew. Chem., Int. Ed. 2016, 55, 3486−3490. (18) Zhu, H.; Chen, C.; Xue, Y.; Tong, Q.; Li, X. N.; Chen, X.; Wang, J.; Yao, G.; Luo, Z.; Zhang, Y. Angew. Chem., Int. Ed. 2015, 54, 13374−13378. (19) Qi, C.; Bao, J.; Wang, J.; Zhu, H.; Xue, Y.; Wang, X.; Li, H.; Sun, W.; Gao, W.; Lai, Y.; Chen, J. G.; Zhang, Y. Chem. Sci. 2016, 7, 6563−6572. (20) Yue, S. J.; Qu, C.; Zhang, P. X.; Tang, Y. P.; Jin, Y.; Jiang, J. S.; Yang, Y. N.; Zhang, P. C.; Duan, J. A. J. Nat. Prod. 2016, 79, 2644− 2651. (21) Kim, M. K.; Yun, K. J.; Lim, D. H.; Kim, J.; Jang, Y. P. Biomol. Ther. 2016, 24, 630−637. (22) Knödler, M.; Conrad, J.; Wenzig, E. M.; Bauer, R.; Lacorn, M.; Beifuss, U.; Carle, R.; Schieber, A. Phytochemistry 2008, 69, 988−993. (23) Zhuravleva, O. I.; Afiyatullov, S. S.; Denisenko, V. A.; Ermakova, S. P.; Slinkina, N. N.; Dmitrenok, P. S.; Kim, N. Y. Phytochemistry 2012, 80, 123−131. (24) Bringmann, G.; Lang, G.; Steffens, S.; Günther, E.; Schaumann, K. Phytochemistry 2003, 63, 437−443. (25) Sun, W. L.; Zhuang, C. L.; Li, X.; Zhang, B. W.; Lu, X. H.; Zheng, Z. H.; Dong, Y. S. Bioorg. Med. Chem. Lett. 2017, 27, 3382− 3385. (26) Jin, W. T.; Zjawiony, J. K. J. Nat. Prod. 2006, 69, 704−706. (27) Note: although it does not have chromophores that would suggest a red color, compound 1 was also recorded as reddish gums. Minor oxidized impurities related to the compound may be the source of the red color. (28) Diaz, F.; Chai, H. B.; Mi, Q.; Su, B. N.; Vigo, J. S.; Graham, J. G.; Cabieses, F.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.; Swanson, S. M.; Kinghorn, A. D. J. Nat. Prod. 2004, 67, 352−356. (29) Hsieh, P. W.; Chang, F. R.; Lee, K. H.; Hwang, T. L.; Chang, S. M.; Wu, Y. C. J. Nat. Prod. 2004, 67, 1175−1177. (30) Miyase, T.; Noguchi, H.; Chen, X. M. J. Nat. Prod. 1999, 62, 993−996. (31) Stark, T. D.; Matsutomo, T.; Lösch, S.; Boakye, P. A.; Balemba, O. B.; Pasilis, S. P.; Hofmann, T. J. Agric. Food Chem. 2012, 60, 2053− 2062. (32) Yang, S. W.; Chan, T. M.; Terracciano, J.; Loebenberg, D.; Patel, M.; Gullo, V.; Chu, M. J. Antibiot. 2006, 59, 190−192. (33) Vainio, M. J.; Johnson, M. S. J. Chem. Inf. Model. 2007, 47, 2462−2474. (34) Puranen, J. S.; Vainio, M. J.; Johnson, M. S. J. Comput. Chem. 2009, 31, 1722−1732. (35) O’Boyle, N. M.; Vandermeersch, T.; Hutchison, G. R. J. Cheminf. 2011, 3, 32−32. (36) Stewart, J. P. MOPAC2016; Stewart Computational Chemistry: Colorado Springs, CO, USA, HTTP://OpenMOPAC.net, 2016.

three or more groups. All statistical analyses were conducted using GraphPad Prism 4.0 version software.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00744. HRESIMS, IR, UV, and 1D and 2D NMR data of 1−10, H and 13C NMR of 11−13, experimental ECD of 11 and 12, and ECD computational details of the model structure of 1 and 5 (PDF)

1

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Junjun Liu: 0000-0001-9953-8633 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions ⊥

H. Wen, C. Chen, and W. Sun contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of the Ministry of Education of the People’s Republic of China (No. T2016088); the National Natural Science Foundation for Distinguished Young Scholars (No. 81725021); the National Science and Technology Project of China (2018ZX09201001-001-003); Innovative Research Groups of the National Natural Science Foundation of China (81721005); the National Natural Science Foundation of China (No. 81703580); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in the acquisition of the ECD, UV, and IR spectra.

■

REFERENCES

(1) Jesus, A. R.; Vila-Viçosa, D.; Machuqueiro, M.; Marques, A. P.; Dore, T. M.; Rauter, A. P. J. Med. Chem. 2017, 60, 568−579. (2) Xu, G.; Lv, B.; Roberge, J. Y.; Xu, B.; Du, J.; Dong, J.; Chen, Y.; Peng, K.; Zhang, L.; Tang, X.; Feng, Y.; Xu, M.; Fu, W.; Zhang, W.; Zhu, L.; Deng, Z.; Sheng, Z.; Welihinda, A.; Sun, X. J. Med. Chem. 2014, 57, 1236−1251. (3) Guo, C.; Hu, M.; DeOrazio, R. J.; Usyatinsky, A.; Fitzpatrick, K.; Zhang, Z.; Maeng, J. H.; Kitchen, D. B.; Tom, S.; Luche, M.; Khmelnitsky, Y.; Mhyre, A. J.; Guzzo, P. R.; Liu, S. Bioorg. Med. Chem. 2014, 22, 3414−3422. (4) Stark, T. D.; Germann, D.; Balemba, O. B.; Wakamatsu, J.; Hofmann, T. J. Agric. Food Chem. 2013, 61, 12572−12581. (5) Talhi, O.; Silva, A. M. S. Curr. Org. Chem. 2012, 16, 859−896. (6) Shrestha, A.; Pandey, R. P.; Dhakal, D.; Parajuli, P.; Sohng, J. K. Appl. Microbiol. Biotechnol. 2018, 102, 1251−1267. (7) Nagatomo, Y.; Usui, S.; Ito, T.; Kato, A.; Shimosaka, M.; Taguchi, G. Plant J. 2014, 80, 437−448. (8) Chen, D.; Chen, R.; Wang, R.; Li, J.; Xie, K.; Bian, C.; Sun, L.; Zhang, X.; Liu, J.; Yang, L.; Ye, F.; Yu, X.; Dai, J. Angew. Chem., Int. Ed. 2015, 54, 12678−12682. H



Article

(37) Ko, W.; Sohn, J. H.; Jang, J. H.; Ahn, J. S.; Kang, D. G.; Lee, H. S.; Kim, J. S.; Kim, Y. C.; Oh, H. Chem.-Biol. Interact. 2016, 244, 16− 26.

I


Phenolic C-Glycosides and Aglycones from Marine-Derived

Recommend Documents