Hexaricins, Pradimicin-like Polyketides from a Marine Sediment

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

Hexaricins, Pradimicin-like Polyketides from a Marine SedimentDerived Streptosporangium sp. and Their Antioxidant Effects Chunzhi Gao,† Zhengyan Guo,‡ Xingzhong Lu,§ Haiyan Chen,⊥ Liwei Liu,‡ Zhiguo Yu,*,† and Yihua Chen*,‡ †

College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China Institute of Microbiology, University of Chinese Academy of Sciences, Beijing 100101, People’s Republic of China § Liaoning Baihao Biotech Company Ltd, Benxi 117000, People’s Republic of China ⊥ Key Laboratory of Applied Chemistry Technology and Resource Development, Medical College of Guangxi University, Guangxi Colleges and Universities, Nanning 530004, People’s Republic of China

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‡

S Supporting Information *

ABSTRACT: Seven pradimicin-like polyketides were isolated from the dichloromethane extract of the marine sediment-derived Streptosporangium sp. CGMCC 4.7309, including five new hexaricins, D−H (1−5), and known hexaricins A (6) and C (7). Their structures were determined by HRESIMS, 1D and 2D NMR, and other spectroscopic analyses. The absolute configurations of compounds 1−5 were determined on the basis of circular dichroism and specific rotation data. All isolated compounds 1−7 were tested for their antioxidant capacities by DPPH• scavenging, •OH scavenging, and •O2̅ scavenging assays. Compounds 3 and 4 displayed stronger antioxidant activities than the positive control (tert-butylhydroquinone). The relationship between structure and antioxidant activity is discussed. These compounds could be effective natural antioxidants with considerable pharmaceutical value.

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macrolides, polyenes, and polyethers. The diverse bioactivities of polyketides depend on their diverse structures. For example, the ansamycin polyketide, geldanamycin, and azithromycin exhibit antiplasmodial activity.8,9 Minocycline, a derivative of tetracycline, is a bacteriostatic antibiotic that is used for the treatment of acne vulgaris.10 Angucyclinones are another important group of polyketides characterized by their benz[a]anthracene backbone. To date, there are only a few benz[a]anthracene-containing natural products isolated from the less common actinomycete Streptosporangium. Angucyclinone R2, with antibacterial activity, was isolated from Streptosporangium sp. Sg3.11 Three compounds with the benz[a]anthracene backbone, WS79089 A−C, were isolated from Streptosporangium roseum. NO. 79089 in 1994.12 They showed highly selective endothelin converting enzyme inhibition activity. In our ongoing effort to discover novel bioactive natural products,13−15 Streptosporangium sp. CGMCC 4.7309 was isolated from marine sediments collected at Lijiao Bay, Huanghai Sea, China (N 39°01′24.19″; E 121°46′03.58″). In a previous study,16 hexaricins A−C were isolated from this strain by genome mining methods. Hexaricins A and B have the same planar structures as WS79089A and WS79089C, respectively.17 In the current study, five new pradimicin-like

n a biological context, reactive oxygen species (ROS) are formed as a natural byproduct of the normal metabolism of oxygen, including free radicals as well as non-free radical species. ROS have important roles in homeostasis and cell signaling. Under certain types of environmental stress (e.g., UV, X-ray, ionizing radiation, heat exposure, or air and water pollution) and life pressures, ROS levels can increase dramatically.1 The excessive production of ROS may result in severe damage to DNA strands or cell structures. Various antioxidants can be used to counteract the effects of ROS. Natural products are one of the most important sources for lead compounds in drug discovery. Effective antioxidants from natural products can be used to protect body health and prevent food oxidation. According to reports in the literature, the phylum actinobacteria provides almost 40% of the bioactive secondary metabolites that have potential applications in medicine, agriculture, and other areas.2−5 Owing to the special ecological environment, including physical properties and the competition for space and predation, marine habitats provide the largest reservoir of microorganisms and unknown microbial communities on earth. In recent years, marinederived microorganisms have given rise to more and more new secondary metabolites with various biological activities.6 Polyketides are a large family of natural products with significant diversity of structures, biological activities, and pharmacological properties.7 Frequently discovered polyketides include aromatic compounds such as anthracyclines, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 17, 2018

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DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX

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polyketides named hexaricins D−H (1−5), along with the known compounds hexaricins A (6) and C (7), were isolated from the fermentation broth of Streptosporangium sp. CGMCC 4.7309 grown in PGG liquid medium. Herein, we report the extraction, isolation, purification, and structure elucidation of the seven hexaricins. In addition, the antioxidant capacities of all isolated compounds were evaluated by DPPH• scavenging, • OH scavenging, and •O2̅ scavenging assays. The correlations between antioxidant activities and chemical structures of the hexaricins were subsequently analyzed.

Table 1. 1H NMR Data (800 MHz, DMSO-d6) for Compounds 1−5

Chart 1

6

δH, mult (J in Hz) position

1

2

3

4

5

11.90, brs

11.90, brs

12.00, brs

6.92, s 3.22, brd (16.8) 3.05, brd (16.8) 6.32, t (2.9)

6.91, s 3.22, brd (16.8) 3.07, brd (16.8) 6.36, t (3.0)

6.89, brs 3.29, overlap 2.41, m

11.80, brs 6.90, brs 3.29, overlap 2.39, m

2.90, m 2.58, m

2.91, m 2.59, m

17.80, brs 6.47, brs 2.91, brd (16.8) 2.73, brd (16.8) 5.21, m

3.82, s

3.82, s

3.82, s

3.82, s

5.00, brs 3.88, s

12.80, s 7.42, brd (8.0)

12.80, s 7.40, brd (8.2)

12.90, s 7.39, brd (8.0)

12.90, s 7.28, brd (8.2)

11

7.82, t (7.8)

7.80, t (7.7)

12

7.79, brd (8.0)

7.78, brd (7.8)

7.80, t (7.8) 7.77, brd (7.7)

7.73, t (8.0) 7.60, m

14-OH 16

14.00, brs 3.11, brd (16.4) 2.96, brdd (16.4, 12.1) 4.85, m

14.00, brs 3.12, brd (16.4) 2.96, brdd (16.4, 11.4) 4.89, m

12.90, s 7.39, dd (8.1, 1.3) 7.79, t (7.8) 7.76, dd (7.6, 1.3) 14.00, s 3.43, m

14.00, s 3.42, m

3.23, d (16.6)

3.23, d (16.6)

13.70, s 4.10, brd (16.6) 3.92, brd (16.6)

3.29, overlap 1.67, s

2.15, s

1-OH 4 5

6-OH 7OCH3 9-OH 10

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RESULTS AND DISCUSSION In order to find effective natural antioxidants, the CH2Cl2 extract from a fermentation culture (28.8 L) of Streptosporangium sp. CGMCC 4.7309 was chromatographed on Sephadex LH-20 columns, on a silica gel plate, and by reversed-phase HPLC to afford five new compounds (1−5) and two known compounds (6 and 7). Analysis of the HRESIMS data and the NMR spectra of compounds 6 and 7 indicated that they are hexaricins A and C.16 Compound 1 was obtained as a scarlet powder. Its molecular formula, C31H26O10, was established by HRESIMS data, indicating 19 degrees of unsaturation. The 1H NMR data (Table 1) of 1 revealed three hydroxy groups [δH 11.9 (1H, brs), 14.0 (1H, brs), 12.8 (1H, s)] and three methyl groups [δH 1.44 (d), 0.92 (d), 0.83 (d)]. In the 13C NMR (Table 2) data and HSQC spectrum (Figure S6), compound 1 possessed seven methine groups, two methylene groups, three methyl groups, one methoxy group, and 18 nonprotonated carbons. Among those nonprotonated carbons, four were carbonyl carbons (δC 169.7, 188.2, 186.8, 175.5) and 14 were olefinic carbons. The COSY correlations between H-10 (δH 7.42)/H11 (δH 7.82)/H-12 (δH 7.79), together with the HMBC correlations from 9-OH (δH 12.8) to C-8a (δC 116.9), C-9 (δC 161.5), and C-10 (δC 124.9); H-10 (δH 7.42) to C-9 (δC 161.5); and H-12 (δH 7.79) to C-12a (δC 132.5) indicated the presence of the ring A. The COSY correlation between H2-5 (δH 3.22, 3.05) and H-6 (δH 6.32), along with the HMBC correlations from H2-5 (δH 3.22, 3.05) to C-4a (δC 143.8) and C-14b (δC 116.8); H-6 (δH 6.32) to C-4a (δC 143.8), C-6a (δC

17 17-OH 17OCH3 18 2′

1.44, d (6.3) 2.32, m

3′ 4′

0.83, d (7.0) 0.92, d (6.9)

4.06, brs

1.44, d (6.3) 2.18, m 2.10, m 0.91, t (7.4)

1.67, s

139.0), C-7 (δC 150.9), and C-14a (δC 129.6); and 7-OMe (δH 3.82) to C-7 (δC 150.9) revealed the presence of rings C and D (Figure 1). Ring C was connected to ring A via two ketone carbonyl groups between C-7a (δC 122.6) and C-8a (δC 116.9), C-12a (δC 132.5), and C-13a (δC 115.9), which form ring B. The presence of rings E and F was deduced from the analysis of unsaturation degrees and six nonprotonated carbons including C-1 (δC 158.5), C-2 (δC 107.6), C-3 (δC 141.6), C4a (δC 143.8), C-14b (δC 116.8), and C-15 (δC 169.7). These results revealed that the pradimicin-like polyketide skeleton18 (rings A−F) of 1 was the same as that of 6. Notably, there were some striking differences in the data between 1 and 6. In the 1H and 13C NMR spectra, 6 possessed one shielded hydroxy proton (δH 5.26) that was not present in 1, whereas 1 had two more methyl groups [CH3-3′ (δC 18.8, δH 0.83) and CH3-4′ (δC 18.3, δH 0.92)], one more alkyl methine group [CH-2′ (δC 33.0, δH 2.32)], and one more carbonyl carbon C1′ (δC 175.5) than 6. These data suggested that 1 should possess an isobutyryl moiety. The presence of the isobutyryl moiety was further confirmed by the COSY correlations between H-2′ (δH 2.32)/H3-3′ (δH 0.83)/H3-4′ (δH 0.92) and the HMBC correlations from H-2′ (δH 2.32) to C-1′ (δC 175.5) and from H3-3′ (δH 0.83) to C-1′ (δC 175.5) (Figure 1). The HMBC correlation from H-6 (δH 6.32) to C-1′ (δC B

DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data (200 MHz, DMSO-d6) for Compounds 1−5

methylene group at H2-2′ (δH 2.18, 2.10) instead of an alkyl methine group at H-2′ (δH 2.32) in 1. In the 13C NMR spectrum, compound 1 possessed a methyl carbon at C-4′ (δC 18.3), which was not present in 2. The absence of a methyl group in 2 was also confirmed by its HRESIMS data (Figure S10). These results suggested that the isobutyric acid moiety in 1 was substituted by a propanoic acid moiety in 2. Thus, the structure of compound 2 was elucidated as hexaricin E. Compound 3 was obtained as a scarlet powder. Its molecular formula, C27H20O9, was established by HRESIMS data, indicating 18 degrees of unsaturation. The mass difference between compound 3 and 7 was 16 amu, which indicated that compound 3 has one more oxygen atom. A careful comparison of the NMR data of 3 and 7 revealed that the main difference was located in ring F. The 1H NMR data (Table 1) of 3 showed one shielded exchangeable hydrogen (δH 4.06) that was not present in 7, whereas the H-17 proton signal (δH 4.65) in 7 was missing in 3. In the 13C NMR data (Table 2), the chemical shift value of C-17 at δC 75.4 in 7 was deshielded to δC 106.4 in 3, suggesting that the extra hydroxy group was attached to C-17 in 3. This was also supported by the HMBC correlation from 17-OH (δH 4.06) to C-17 (δC 106.4). Thus, the structure of compound 3 was elucidated as hexaricin F. Compound 4 was obtained as a scarlet powder. Its molecular formula, C28H22O9, was established by HRESIMS data, indicating 18 degrees of unsaturation. A careful comparison of the NMR spectroscopic data (Tables 1 and 2) of 4 and 3 revealed that the main difference was located in ring F. The 1H and 13C NMR spectra indicated that the 17-OH (δH 4.06) in 3 was changed to 17-OMe (δC 50.0, δH 3.29) in 4, which was also supported by the HMBC correlation from 17-OMe (δH 3.29) to C-17 (δC 105.9). These results suggested the hydroxy group in 3 was replaced by a methoxy group in 4. This conclusion was further supported by its HRESIMS data (Figure S28), 14 amu more than that of 3. Thus, the structure of compound 4 was elucidated as hexaricin G. Compound 5 was obtained as a scarlet powder. Its molecular formula, C27H20O10, was established by HRESIMS data, indicating 18 degrees of unsaturation. Careful comparison of NMR data (Tables 1 and 2) of compounds 5 and 6 revealed that the main difference was located in ring F. In the 1H NMR spectrum, the doublet methyl signal [δH 1.45 (d, H3-18)] in 6 was replaced by a singlet methyl signal [δH 2.15 (s, H3-18)] in 5. In the 13C NMR spectrum, the chemical shift value of the C17 at δC 76.1 in 6 was deshielded to δC 205.2 in 5, indicating that one of the C-17 alkyl methine groups in 6 was substituted by a carbonyl carbon at C-17 in 5. These results provided unambiguous evidence that ring F was opened at C-17 in 5, which was further confirmed by the HMBC correlations (Figure 1) from H3-18 (δH 2.15) to C-16 (δC 50.1) and C-17 (δC 205.2), along with the chemical shift value of the 1-OH at δH 11.8 in 6, which was deshielded to δH 17.8 in 5. Thus, the structure of compound 5 was elucidated as hexaricin H. The absolute configurations of compounds 1 and 2 were determined by comparison of their electronic circular dichroism (ECD) spectra (Figure S46) with that of compound 6.16 Compounds 1 and 2 possessed two stereogenic carbons C6 and C-17, both of which were determined to be in Rconfiguration as for compound 6. This conclusion was further confirmed by the hydrolysis reactions of 1 and 2 that generated 6 (Figure S48). The sole stereogenic carbon C-6 of compound 5 is proposed to be in the R-configuration by the fact that it is biosynthesized by the same machinery as the other hexaricins,

δC, type position

1

2

3

4

5

1 2 3 4 4a 5 6 6a 7 7-OCH3 7a 8 8a 9 10 11 12 12a 13 13a 14 14a 14b 15 16 17 17-OCH3 18 1′ 2′ 3′ 4′

158.5, C 107.6, C 141.6, C 118.7, CH 143.8, C 34.5, CH2 61.9, CH 139.0, C 150.9, C 62.3, CH3 122.6, C 186.8, C 116.9, C 161.5, C 124.9, CH 136.6, CH 118.6, CH 132.5, C 188.2, C 115.9, C 157.0, C 129.6, C 116.8, C 169.7, C 33.4, CH2 76.2, CH

158.7, C 107.8, C 141.5, C 118.5, CH 143.8, C 34.5, CH2 61.8, CH 138.7, C 150.9, C 62.3, CH3 122.4, C 186.8, C 116.8, C 161.5, C 124.8, CH 136.6, CH 118.5, CH 132.5, C 188.1, C 115.8, C 157.1, C 130.8, C 116.8, C 169.6, C 33.5, CH2 76.0, CH

158.9, C 106.8, C 140.0, C 117.7, CH 146.2, C 22.3, CH2 28.9, CH2 124.6, C 150.8, C 61.0, CH3 121.8, C 187.2, C 116.7, C 161.5, C 124.6, CH 136.5, CH 118.4, CH 132.5, C 187.7, C 113.7, C 157.1, C 130.0, C 117.6, C 169.8, C 38.1, CH2 106.4, C

160.8, C 120.1, C 134.9, C 122.4, CH 141.0, C 37.9, CH2 58.6, CH 140.4, C 150.1, C 62.5, CH3 123.0, C 182.8, C 116.6, C 161.3, C 122.4, CH 136.3, CH 117.8, CH 134.3, C 188.1, C 115.3, C 156.2, C 129.7, C 120.5, C 170.0, C 50.1, CH2 205.2, C

20.2, CH3 175.5, C 33.0, CH 18.8, CH3 18.3, CH3

20.2, CH3 173.0, C 26.8, CH2 8.9, CH3

27.4, CH3

158.9, C 106.4, C 139.8, C 117.7, CH 146.3, C 22.2, CH2 29.1, CH2 124.7, C 150.7, C 61.1, CH3 122.0, C 187.2, C 116.7, C 161.5, C 124.6, CH 136.6, CH 118.4, CH 132.6, C 187.8, C 113.7, C 157.2, C 129.8, C 117.6, C 168.9, C 37.7, CH2 105.9, C 50.0, CH3 22.2, CH3

29.9, CH3

Figure 1. Key COSY (bold lines), HMBC (single arrows), and ROESY (double arrows) correlations of compounds 1 and 5.

175.5) revealed that the isobutyryl moiety was attached to C-6 (δC 61.9) via an oxygen atom. The presence of an isobutyryl moiety in 1 was also confirmed by its HRESIMS data (Figure S1), indicating 70 amu more than that of 6. Thus, the structure of compound 1 was elucidated as hexaricin D. Compound 2 was obtained as a scarlet powder. Its molecular formula, C30H24O10, was established by HRESIMS data, indicating 19 degrees of unsaturation. The NMR spectroscopic data of 2 were similar to those of 1 (Tables 1 and 2), indicating that they have the same rings A−F. The main difference was located at the isobutyric acid moiety that was attached to C-6 of 1. The 1H NMR spectrum of 1 showed one methyl group (H3-4′, δH 0.92) that was missing in 2, whereas 2 had one C

DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX

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which all have the R-configuration at C-6, and the specific rotation direction of the theoretical calculation for 6R-5 is the same as that measured by the experiment (Table S1). Analysis of the ECD spectra showed that both 3 and 4 were racemic mixtures, which indicated that the hemiketal moiety of 3 and the ketal moiety of 4 were formed by nonenzymatic reactions. Compound 4 may be an artifact generated during the isolation process, in which MeOH was used as a solvent. Antioxidant Activity Assays. The antioxidant capacities of compounds 1−7 were evaluated through DPPH free radical scavenging assay, hydroxyl radical scavenging assay, and superoxide anion radical scavenging assays. The results are summarized in Table 3 and Figure S47. In the three

reactive oxygen species in humans, protecting food against oxidative deterioration, and improving food storage stability.

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General Experimental Procedures. Optical rotations were measured with an AP-300 polarimeter (ATAGO) at the sodium D line (589 nm). UV spectra were obtained on a Lambda 950 UV spectrometer (PerkinElmer). ECD spectra were recorded on a Chirascan circular dichroism spectrophotometer (Applied Photophysics) using methanol as solvent. IR spectra were recorded on a Nicolet iS50 FT-IR spectrometer (Thermo Scientific). NMR spectra were recorded on an Ascend-800 NMR spectrometer (Bruker). Carbon signals and the residual proton signals of DMSO-d6 (δC 39.5 and δH 2.50) were used for calibration. High-resolution mass spectra (HRESIMS) were acquired on an Agilent 1260/6520 Q-TOF mass spectrometer. HPLC analyses were performed using an Agilent 1260 series system coupled with a ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm, Agilent). Semipreparative HPLC analyses were performed using an Agilent 1260 series system coupled with a ZORBAX Eclipse XDB-C18 column (250 × 9.4 mm, 5 μm, Agilent). Chromatographic separation was performed using a silica gel plate (carboxymethyl cellulose sodium 4 g, silica gel for thin-layer chromatography 80 g, pure water 400 mL) and Sephadex LH-20 (GE Healthcare). All chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd., and used without further purification. Actinomycetes Material. Streptosporangium sp. CGMCC 4.7309 was isolated from the sediment at Lijiao Bay, Huanghai Sea, China (N 39°01′24.19″; E 121°46′03.58″).16 The strain is stored in the Laboratory of Microbial Metabolites, College of Plant Protection, at Shenyang Agricultural University, China. Fermentation and Extraction. Streptosporangium sp. CGMCC 4.7309 was preserved as a spore suspension at −80 °C. This strain was cultured in PGG liquid medium in two-stage fermentation.12 In the first stage, a Streptosporangium sp. CGMCC 4.7309 spore suspension was added into 36 × 250 mL Erlenmeyer flasks (each containing 60 mL of the PGG liquid medium and 3 mL of the spore suspension) for preparing the seed culture. The flasks were cultivated at 28 °C on a shaker at 220 rpm for 2 days. In the second stage, the seed culture was transferred into 72 × 2 L Erlenmeyer flasks (each containing 400 mL of the PGG liquid medium, 20 g of Amberlite XAD-16 resin, and 30 mL of the seed culture). The flasks were incubated at 28 °C on a shaker at 220 rpm for 9 days. The XAD-16 resin was separated from the fermentation broth and then washed two times with tap H2O to remove remaining cells and supernatant. After the resin was dried at 28 °C, it was extracted three times with MeOH. The MeOH fractions were collected and concentrated to yield 8 g of a combined MeOH extract. The combined MeOH extract was evaporated and further dissolved in 1.2 L of MeOH−H2O (1:1). The solution was extracted three times with the same volume of CH2Cl2. The CH2Cl2 fraction was concentrated on a rotary evaporator under vacuum at 30 °C to yield 4.3 g of organic extract. Isolation and Purification. The CH2Cl2 fraction was subjected to Sephadex LH-20 column (1000 mm × 40 mm i.d.) chromatography and eluted with a mixture of CH2Cl2−MeOH (1:1) to remove culture medium components or impurities. Purified extract was then separated on a silica gel plate using a mixture of CH2Cl2−MeOH (97:3) as eluent, which resulted in four fractions, A−D. Fraction A was isolated by reversed-phase HPLC using a semipreparative C18 column and applying 70% MeOH with 0.1% HCOOH (v/v) solvent at a flow rate of 3.6 mL/min (UV absorption at 254 and 450 nm) to yield fractions A1 (30.1 mg, tR = 29.4 min), A2 (40.3 mg, tR = 41.7 min), and A3 (36.7 mg, tR = 47.2 min). Fractions C and D were subjected to reversed-phase HPLC applying 58% and 71% CH3CN with 0.1% HCOOH (v/v) as solvents, respectively, under the same instrument conditions to obtain fractions C1 (34.8 mg, tR = 19.5 min), C2 (18.2 mg, tR = 36.8 min), and D1 (26.4 mg, tR = 24.5 min). Then, these fractions and fraction B were refined by

Table 3. Antioxidative Activities of Compounds 1−7 EC50 (μM)a •

compd

DPPH scavenging

1 2 3 4 5 6 7 TBHQb

30 ± 3 30 ± 2 2.9 ± 0.8 2.7 ± 1.2 25 ± 2 21 ± 3 42 ± 2 10 ± 1

•

OH scavenging 804 ± 3 831 ± 2 619 ± 4 519 ± 2 840 ± 2 723 ± 4 1526 ± 1 1923 ± 3

•

EXPERIMENTAL SECTION

O2− scavenging 746 ± 3 754 ± 4 83 ± 2 139 ± 2 681 ± 3 589 ± 3 1104 ± 4 482 ± 1

a Data are presented as mean ± standard deviation. Statistical analysis was done by using SPSS version 22.0 at the 5% level. EC50 = concentration at which the DPPH, OH, and O2− radical scavenging activity was 50%. bTBHQ (tert-butylhydroquinone) as a positive control.

antioxidant activity assays, all isolated compounds displayed antioxidant activities. In the DPPH free radical scavenging assay, compounds 3 and 4 displayed stronger scavenging activities than the positive control, tert-butylhydroquinone (EC50 = 10 μM), with EC50 values of 2.9 and 2.7 μM, respectively. Compounds 1, 2, 5, and 6 showed moderate antioxidant capacities with EC50 values of 30, 30, 25, and 21 μM, respectively. However, compound 7 exhibited poor DPPH scavenging activity (EC50 = 42 μM). In the O2− and OH radical scavenging assays, compounds 3 and 4 displayed scavenging capacities, with EC50 values ranging from 83 to 619 μM, surpassing that of the positive control (EC50 = 482, 1923 μM). Compounds 1, 2, 5, and 6 showed moderate antioxidant capacities, and compound 7 exhibited weak antioxidant activity. Hexaricins A (6), B, and C (7) were previously examined for antibacterial activity and for cytotoxicity in our lab, but did not show any activity. From the above results, we propose that the antioxidant activities of the isolated compounds are influenced by the presence of the phenolic hydroxy groups in the pradimicin-like polyketide skeleton and by the lactone ring (ring F). By comparing the structures of compounds 1−7, we deduced that the substituents of compounds 3 and 4 attached to C-17 contributed to their stronger antioxidant activities. In addition, substitution at C-6 as in compounds 1, 2, 5, and 6 resulted in increased antioxidant activity. These results suggested that substituents at C-6 and C-17 positions may play important roles in the antioxidant activities of the tested compounds. In this context, Streptosporangium sp., rich in hexaricins, which are structurally related to pradimicins and benastatins, could be a potential resource of natural antioxidants for scavenging D

DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Sephadex LH-20 column (500 mm × 20 mm i.d.) chromatography, respectively, and eluted with a mixture of CH2Cl2−MeOH (1:1). Ultimately, pure compounds 1 (6.9 mg), 2 (13.4 mg), 3 (6.5 mg), 4 (8 mg), 5 (12 mg), 6 (10 mg), and 7 (13.7 mg) were obtained. Hexaricin D (1): scarlet powder; [α]24 D −400 (c 0.05 CHCl3); UV (CH2Cl2) λmax (log ε) 244 (4.24), 266 (4.17), 307 (3.77), 481 (3.87) nm; ECD (c 0.000 94 M, MeOH) λmax (Δε) 207 (+28.0), 228 (−31.2), 263 nm (−19.8); IR (ATR) νmax 2974, 2925, 2851, 1731, 1666, 1615 cm−1; 1H NMR (DMSO-d6, 800 MHz) and 13C NMR (DMSO-d6, 200 MHz) data, Tables 1 and 2; HRESIMS m/z 559.1610 [M + H]+ (calcd for C31H27O10, 559.1604). Hexaricin E (2): scarlet powder; [α]24 D −540 (c 0.08 CHCl3); UV (CH2Cl2) λmax (log ε) 245 (4.23), 266 (4.16), 306 (3.78), 481 (3.88) nm; ECD (c 0.000 94 M, MeOH) λmax (Δε) 207 (+22.5), 228 (−26.1), 263 nm (−16.3); IR (ATR) νmax 2979, 2924, 2851, 1732, 1662, 1615 cm−1; 1H NMR (DMSO-d6, 800 MHz) and 13C NMR (DMSO-d6, 200 MHz) data, Tables 1 and 2; HRESIMS m/z 545.1449 [M + H]+ (calcd for C30H25O10, 545.1447). Hexaricin F (3): scarlet powder; UV (CH2Cl2) λmax (log ε) 246 (4.51), 267 (4.49), 356 (3.65), 477 (4.20) nm; IR (ATR) νmax 2926, 2850, 1651, 1644, 1614 cm−1; 1H NMR (DMSO-d6, 800 MHz) and 13 C NMR (DMSO-d6, 200 MHz) data, Tables 1 and 2; HRESIMS m/ z 489.1194 [M + H]+ (calcd for C27H21O9, 489.1185). Hexaricin G (4): scarlet powder; UV (CH2Cl2) λmax (log ε) 247 (4.19), 267 (4.18), 356 (3.35), 478 (3.88) nm; IR (ATR) νmax 2923, 2850, 1657, 1614 cm−1; 1H NMR (DMSO-d6, 800 MHz) and 13C NMR (DMSO-d6, 200 MHz) data, Tables 1 and 2; HRESIMS m/z 503.1343 [M + H]+ (calcd for C28H23O9, 503.1342). Hexaricin H (5): scarlet powder; [α]24 D +330 (c 0.06 MeOH); UV (CH2Cl2) λmax (log ε) 245 (4.51), 269 (4.44), 315 (4.08), 486 (4.10) nm; ECD (c 0.000 94 M, MeOH) λmax (Δε) 208 (+29.9), 232 (−37.8), 260 nm (−28.4); IR (ATR) νmax 3334, 2923, 1699, 1609 cm−1; 1H NMR (DMSO-d6, 800 MHz) and 13C NMR (DMSO-d6, 200 MHz) data, Tables 1 and 2; HRESIMS m/z 505.1134 [M + H]+ (calcd for C27H21O10, 505.1134). Optical Rotation Calculation of Compound 5. Because the two enantiomers will have equal and opposite specific rotations, compound 5 (6R) was used in the absolute configuration determination. The OR (optical rotation) of 5 was calculated based on the following procedures. At first, a systematic conformational search of the compound was carried out by the Conflex 7.0 program with the MMFF94 force field (the other parameters were set to default values).19 Within a window of 5 kcal mol−1, nine conformers were found for 6R-5. All of the conformers were further optimized at the B3LYP/6-31G(d,p) level. The same level of harmonic vibrational frequencies was further calculated to confirm the stability of the optimized structures with all positive frequencies and to estimate their relative Gibbs free energies (GFE) at 297.15 K. Solvent effects were considered by the Integral Equation Formalism Polarizable Continuum Model (IEFPCM) with MeOH. The ORs of 6R-5 were calculated using the density functional theory (DFT) method at the PCM/B3LYP/6-311G(d,p) level. Finally, the optical rotation of the conformers was Boltzmann weighted according to the calculated GFE (6R: +32.4). All DFT calculations used Gaussian 09.20 All calculations were performed on the High-Performance Computing Platform of Guangxi University. Antioxidant Activity Assays. DPPH Free Radical Scavenging Assay. The DPPH• scavenging assay was conducted with 96-well plates using a revised method.21 All tested compounds were dissolved in MeOH at concentrations of 1.875−120 μg/mL. The absorbance of the mixture was measured at 517 nm using a microplate reader (Multiskan GO, Thermo Scientific). The DPPH• scavenging percentage of the samples was calculated as follows:

absorbance of sample solutions in MeOH. The experiment was independently performed at least three times. Hydroxyl Radical Scavenging Assay. The •OH (hydroxyl radical) scavenging assay was performed with a modified Smirnoff method.22 Sample solutions (2 mL) in MeOH were added to 2 mL of FeSO4 solution (6 mM) and 2 mL of salicylic acid solution in EtOH (6 mM) to obtain final concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg/mL. A 2 mL H2O2 solution (6 mM) was added to the mixture, which was then mixed thoroughly in 96-well plates and kept at room temperature for 10 min. The absorbance of the mixture was determined at 510 nm in a microplate reader (Multiskan GO, Thermo Scientific). The •OH scavenging percentage of the samples was calculated as follows: Scavenging rate (%) =

[A 0 − (A 2 − A1)] × 100 A0

A0 is the absorbance of negative control, A1 is the absorbance of the samples/standard without H2O2 solution, and A2 is the absorbance of the samples/standard with H2O2 solution. The experiment was independently performed at least three times. Superoxide Anion Radical Scavenging Assay. The •O2̅ (superoxide anion radical) scavenging assay was based on Li’s method with some modification.23 To 1 mL of various concentrations (0.075−4.8 mg/mL) of the sample solutions in MeOH and 0.4 mL of pyrogallol solution (25 mM) was added 4.5 mL of Tris-HCl solution (0.05 M), and the mixture was incubated in a water bath at 25 °C for 20 min. The mixture was then placed in the 25 °C water bath for 4 min. A 0.5 mL HCl (8 M) solution was immediately added to the mixture in order to stop the reaction. The absorbance of the mixture was determined at 320 nm in 96-well plates by a microplate reader (Multiskan GO, Thermo Scientific). The •O2̅ scavenging percentage of the samples was calculated as follows: Scavenging rate (%) =

[A 0 − (A 2 − A1)] × 100 A0

A0 is the absorbance of the negative control, A1 is the absorbance of the samples/standard without pyrogallol solution, and A2 is the absorbance of the samples/standard with pyrogallol solution. The experiment was independently performed at least three times.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00397.

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UV, IR, NMR, and HRESIMS spectra of compounds 1− 5; ECD spectra of compounds 1, 2, and 5; scavenging effects on DPPH, OH, and O2̅ radicals of compounds 1−7; hydrolysis reaction of compounds 1 and 2; computational and experimental specific rotations of 5 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86 24 88487148. Fax: +86 2488487038. E-mail: zyu@ syau.edu.cn. *Tel: +86 10 64806121. Fax: +86 10 64806121. E-mail: [email protected].

ÄÅ É ÅÅ ij A1 − A 2 yzÑÑÑÑ ÅÅ j z zÑÑ × 100 Scavenging rate (%) = ÅÅ1 − jj j A 0 zzÑÑÑ ÅÅ ÅÇ k {ÑÖ

ORCID

Zhiguo Yu: 0000-0002-0909-6788 Notes

A0 is the absorbance of the negative control (DPPH in MeOH, 0.1 mM), A1 is the absorbance of the samples/standard, and A2 is the

The authors declare no competing financial interest. E

DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Rev. D 01; Gaussian, Inc.: Wallingford, CT, 2013. (21) Zhou, L.; Wang, Y.; Wang, X. L.; Liang, Y.; Huang, Z.; Zeng, X. X. J. Agric. Food Chem. 2017, 65, 1229−1238. (22) Smirnoff, N.; Cumbes, Q. J. Phytochemistry 1989, 28, 1057− 1060. (23) Li, X. C. J. Agric. Food Chem. 2012, 60, 6418−6424.

ACKNOWLEDGMENTS This work was financially supported by National Key R&D Program of China (2017YFD0201104) and the National Natural Science Foundation of China (31600016). We thank Dr. J. Ren, Dr. G. Ai, and Dr. W. Wang, Institute of Microbiology, University of Chinese Academy of Sciences, for technical assistance with MS, ECD, and NMR spectra. We also thank the High-Performance Computing Platform of Guangxi University for generous support of our computational chemistry research.

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DOI: 10.1021/acs.jnatprod.8b00397 J. Nat. Prod. XXXX, XXX, XXX−XXX