Polyprenylated Tetraoxygenated Xanthones from ... - ACS Publications

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Polyprenylated Tetraoxygenated Xanthones from the Roots of Hypericum monogynum and Their Neuroprotective Activities Wen-Jun Xu, Rui-Jun Li, Olga Quasie, Ming-Hua Yang, Ling-Yi Kong,* and Jun Luo* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new polyprenylated tetraoxygenated xanthones, monogxanthones A−J (1−10), together with eight known analogues (4b, 11−17) were identified from the roots of Hypericum monogynum. The structures of these new polyprenylated xanthones (1−10), a class of compounds rarely found in plants of the genus Hypericum, were elucidated by the interpretation of their HRESIMS, 1D and 2D NMR, and electronic circular dichroism data. Compounds 1 and 2 exhibited neuroprotective effects against corticosterone (Cort)-induced lesions of PC12 cells at concentrations of 6.25, 12.50, and 25.00 μM, with cell viability greater than 75%, as well as inhibitory effects on nitric oxide production in lipopolysaccharide-induced BV2 microglia cells, with IC50 values of 7.47 ± 0.65 and 9.60 ± 0.12 μM, respectively. Collectively, these results shed new light on the potential of polyprenylated xanthones from the genus Hypericum in the development of antidepression therapies.

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(Cort)-induced PC12 cell injury was utilized to assess the neuroprotective effects of all compounds.7 Herein, the isolation and structure elucidation of these new compounds, along with an assessment of their neuroprotective effects, are reported.

olyprenylated xanthones, bearing a dibenzo-γ-pyrone scaffold and substituted with multiple C5 units, are a major class of xanthones. According to their oxygenation level, polyprenylated xanthones can be further divided into mono-, di-, tri-, tetra-, and higher oxygenated analogues.1 Owing to its diverse pharmacological properties,2 the xanthonoid structure has been described as a “privileged structure”3 and has attracted the attention of researchers in both chemistry and pharmacology. Polyprenylated xanthones have been reported to be mainly in plants of the family Guttiferae, particularly of the genus Garcinia.4 However, in plants of the genus Hypericum (Guttiferae), polyprenylated xanthones are rarely found.5 In previous studies on the bioactive metabolites of Hypericum plants, 36 polycyclic polyprenylated acylphloroglucinol derivatives (PPAPs) were identified from H. monogynum, H. attenuatum, and H. uralum.6 As part of this ongoing research, the roots of H. monogynum Linn have been investigated for polyprenylated xanthones, which are rare for this genus.5 As a result, 10 new polyprenylated tetraoxygenated xanthones named monogxanthones A−J (1−10), along with eight known analogues (4b, 11−17), were identified. Their structures were elucidated by a combination of HRESIMS, 1D and 2D NMR, and electronic circular dichroism (ECD) data. This study on the roots of H. monogynum, together with the previous study on the flowers of this plant, indicated that the secondary metabolites differed in different parts of this plant.6a In addition, an in vitro model of the corticosterone © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 95% ethanol extract of the fresh roots of H. monogynum was partitioned by column chromatography over D-101 macroporous resin and polyamide to obtain crude polyprenylated xanthone fractions. These fractions were further separated by repeated silica gel column chromatography as well as Sephadex LH-20, ODS RP-C18 column chromatography, and preparative HPLC, to afford 10 new polyprenylated xanthones, monogxanthones A−J (1−10), and eight known analogues, maculatoxanthone (11),8 biyouxanthones A−D (12, 4b, 13, and 14),5a calycinoxanthon D (15),9 garcinexanthone C (16),10 and toxyloxanthone B (17) (Figure S1, Supporting Information).11 Monogxanthone A (1) was isolated as a yellow, amorphous powder. Its molecular formula of C33H40O6 was assigned on the basis of its 13C NMR data and the observed [M + H]+ ion at m/z 533.2895 (calcd for C33H41O6, 533.2898). The FT-IR spectrum showed absorption bands of hydroxy (3416 cm−1), Received: March 19, 2016

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DOI: 10.1021/acs.jnatprod.6b00251 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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2, C-3, C-12, and C-16 and from H2-21 to C-3, C-4, C-4a, C22, and C-26 were consistent with the aforementioned assignment (Figure 1). Table 1. 1H NMR and 13C NMR Spectroscopic Data for Compounds 1a and 2b monogxanthone A (1) position

conjugated carbonyl (1642 cm−1), and double-bond (1612 and 1591 cm−1) functionalities. Its 1H NMR and 13C NMR spectra showed characteristic resonances of a 1,3,5,6-tetraoxygenated xanthone skeleton, including a hydrogen-bonded hydroxy group (δH 13.16, 1H, s, OH-1, ring A), a conjugated ketocarbonyl (δC 180.8, C-9, ring C), and two ortho-coupled aromatic protons [δH 7.74, 6.95 (each 1H, d, J = 8.3 Hz, H-8 and H-7), and δC 118.3, 112.5 (C-8 and C-7, ring B)]. The key HMBC correlations from OH-1 to C-1 (δC 159.6), C-2 (δC 110.0), C-9 (δC 180.8), and C-9a (δC 102.9); from H-8 to C-6 (δC 149.3), C-9, and C-10a (δC 145.1); and from H-7 to C-5 (δC 130.5), C-6, and C-8a (δC 114.4) confirmed this framework (Figure 1). This xanthone skeleton accounted for 13 carbon atoms; the remaining carbon and hydrogen atoms were assigned to two sets of monoterpene moieties at C-2 and C4, similar to the C-4 substituent of calycinoxanthon D (15),9 on the basis of its 1H NMR and 13C NMR resonances displayed in pairs (Table 1). The HMBC correlations from H2-11 to C-1, C-

δC

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11a 11b 12 13 14a 14b 15 16a 16b 17 18 19a 19b 20 21a 21b 22 23 24

159.6 110.0 159.8 105.5 152.7 130.5 149.3 112.5 118.3 114.4 180.8 102.9 145.1 27.8

25 26 27 28 29 30 OH-1 OH-3 OH-5 OH-6

20.4 32.5 122.8 134.4 26.0 18.2

47.0 149.8 110.9 20.4 32.2 122.9 133.0 25.9 18.1 27.9 48.1 150.6 111.2

δH, mult (J, Hz)

6.95, d (8.3) 7.74, d (8.3)

2.77, dd (14.2, 6.3) 2.71, dd (14.2, 8.2) 2.41, m 4.75, 4.69, 1.73, 2.16,

s s s m

5.12, t (6.8) 1.70, s 1.61, 2.93, 2.80, 2.34,

s dd (14.5, 5.5) dd (14.5, 7.2) m

4.77, 4.71, 1.72, 2.21, 5.23,

s s s s t (6.8)

1.77, s 1.64, s 13.16, s 5.95, s 5.65, s 6.17, s

monogxanthone B (2) δC 161.9 110.8 163.9 94.0 156.5 133.1 151.8 113.4 117.4 115.0 181.1 102.8 146.8 27.6 47.4 149.1 111.2 19.0 32.2

δH, mult (J, Hz)

6.48, s

6.93, d (8.7) 7.60, d (8.7)

2.74, dd (12.9, 7.8) 2.71, dd (12.9, 6.7) 2.61, m 4.56, 4.53, 1.70, 2.11,

s s s m

124.5 131.6 25.9

5.03, t (6.8) 1.52, s

17.9

1.57, s

13.44, s

a The 1H NMR spectra were measured at 500 MHz; the 13C NMR spectra were measured at 125 MHz. The NMR samples were dissolved in CDCl3. bSamples were dissolved in acetone-d6. The assignments were supported by the 2D NMR spectra.

The absolute configurations of C-12 and C-22 in 1 were established by comparison of its experimental ECD curve with the calculated ECD spectra using TDDFT performed with the Gaussian09 program.12 Four possibilities for the absolute conformations of (12R,22R)-1, (12R,22S)-1, and their enantiomers were considered. The (12R) and (22R) absolute

Figure 1. Selected HMBC correlations observed for compound 1. B



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Figure 2. (A) Experimental ECD spectrum of 1 and calculated ECD spectra of (12R,22R)-1, (12S,22S)-1, (12R,22S)-1, and (12S,22R)-1. (B) Experimental ECD spectrum of 2 and calculated ECD spectra of (12R)-2 and (12S)-2.

Figure 3. Selected HMBC and ROESY correlations observed for compounds 3, 5, and 6.

Figure 4. Chiral HPLC analyses of 3 and 10 on a Chiralpak AD-H column. (A) The chromatographic conditions for 3 used n-hexane/2-propanol (95:5) as mobile phase at a flow rate of 1 mL/min and a column temperature of 30 °C and with UV detection at 300 nm. (B) The chromatographic conditions for 10 used n-hexane/2-propanol (97:3) as mobile phase at a flow rate of 1 mL/min and a column temperature of 30 °C and with UV detection at 270 nm. C



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Figure 5. (A) Experimental ECD spectra of (+)-3 and (−)-3 and calculated ECD spectrum of (8R)-3. (B) Experimental ECD spectra of (+)-10 and (−)-10 and calculated ECD spectrum of (23S)-10.

HMBC correlations from Me-20 to C-17, C-18, and C-19 and from H2-19 to C-17, C-18, C-20, C-21, and C-22 (Figure 3). Isolates 3 (17Z) and 13 (17E) were separated using improved recycling HPLC techniques with a Shim-pack PRC-ODS (H) column (5 μm, 20 × 200 mm, i.d.). Although it has a single stereogenic center (C-8), 3 was found to be a racemic mixture. Chiral HPLC resolution of 3 afforded enantiomers (+)-3 and (−)-3 in a 50.4:49.6 ratio (Figure 4A). The enantiomers displayed typical antipodal ECD curves (Figure 5A) and specific rotations of opposite sign. The similarity between the experimental ECD curve of (+)-3 and the calculated ECD spectrum of (8R)-3 indicated the 8R configuration in (+)-3.14 On the basis of the above analyses, the structure of 3, (±)-monogxanthone C, was established as depicted. Compound 4a was obtained as a mixture with biyouxanthone B (4b)5a by preparative HPLC, and their extremely low polarity made them inseparable by many methods. The negative HRESIMS data of 4a/b displayed a deprotonated molecular ion [M − H]− at m/z 545.2908. These data in conjunction with the 13C NMR data indicated a molecular formula of C34H42O6. Interestingly, two sets of 1H and 13C NMR signals in a ratio of approximately 3:1, particularly the characteristic 13C NMR resonances of both 17Z (δC 32.1, 23.5) and 17E (δC 39.8, 16.4), were observed in the 1D NMR spectra. This observation suggested that 4a/b was a mixture of 4a (17Z) and 4b (17Eisomer, biyouxanthone B5a), which was confirmed by the diagnostic ROESY correlations of Me-20 and H-17 and of H219 and H2-16 for 4a. Chiral HPLC analysis suggested that 4a was also a racemate (Figure S6-7, Supporting Information). Thus, the structure of 4a, (±)-monogxanthone D, was found to be the Δ17,18 isomer of biyouxanthone B (4b) with a Z configuration. Compound 5 gave a molecular formula of C38H48O6 based on its HRESIMS and 13C NMR data, which indicated that it contained five more carbon atoms than biyouxanthone C (13).5a The 1D NMR spectra indicated the presence of one more isoprenyl group [(δH 5.28, 1H, t, J = 7.0 Hz; 3.46, 2H, d, J = 7.0 Hz; 1.85, 1.77 (each 3H, s) and δC 135.5, 121.8, 26.0, 22.0, 18.09)] than 13. The HMBC correlations from H2-11 to C-1, C-2, and C-3 suggested the C-2 linkage of the additional isoprenyl group. The diagnostic ROESY correlations between Me-25 and H2-21 and between H2-24 and H-22 (Figure 3) suggested the 22E configuration of the C-8 geranyl side chain. Similar to compound 3, the specific rotation of 5 was low ([α]25D −7), and no Cotton effect was observed in its ECD

configurations of the monoterpene moieties in 1 were assigned on the basis of the similarity between the experimental and the calculated spectra of (12R,22R)-1 at the B3LYP/6-31G** level in MeOH (Figure 2A). Thus, the structure of 1, monogxanthone A, which features a polyprenylated 1,3,5,6-tetraoxygenated xanthone skeleton, was defined as shown. Monogxanthone B (2) was obtained as a yellow, amorphous powder and showed a deprotonated molecule [M − H]− at m/ z 395.1497 (calcd for C23H23O6, 395.1500) in its HRESIMS spectrum. From these data and the 13C NMR data, the molecular formula was determined as C23H24O6. The 1H and 13 C NMR spectra of 2 were similar to those of calycinoxanthon D (15),9 with the exception of the resonances of H2-11 (ΔδH = −0.25, 0.22), C-2 (ΔδC = +2.6), and C-4 (ΔδC = −10.7). These 1D NMR data implied the C-2 substitution of the monoterpene moiety in 2 instead of C-4 in 15. This deduction was confirmed by the HMBC correlations from H2-11 to C-1, C-2, C-3, C-12, and C-16. The simulated ECD spectrum of 12R-2 agreed with the experimental spectrum (Figure 2B). The structure of compound 2, monogxanthone B, was thus assigned as shown. Compound 3 was obtained as a yellow, viscous oil with a molecular formula of C33H40O6 assigned on the basis of the HRESIMS (obsd [M + H]+ m/z 533.2896, calcd m/z 533.2898, Figure S5-6, Supporting Information) and 13C NMR data. Its 1 H NMR spectrum showed a hydrogen-bonded hydroxy group (δH 13.14, 1H, s), four olefinic protons of the isoprenyl groups (δH 5.30, 1H, t, J = 7.0 Hz; 5.04, 1H, t, J = 5.9 Hz; 4.64, 1H, t, J = 7.0 Hz; 4.63, 1H, t, J = 7.0 Hz), and seven methyl groups (δH 1.87−1.48, s) and indicated that compound 3 was also a polyprenylated xanthone. Comparison of the 1D and 2D NMR data of 3 with those of biyouxanthone C (13)5a from the roots of H. chinense revealed that 3 has the same 1,3,6,7tetraoxygenated xanthone skeleton as 13, with the difference being the C-8 geranyl moiety. A survey of the recent literature on the configurations of the geranyl moiety indicated that the Δ17,18 double bond in the C-8 geranyl moiety can occur in either a Z or E configuration.13 Thus, similar to the reported differences between garciniacowones B and A,13c the 17Z configuration in 3, instead of the 17E configuration in 13, was deduced from the 13C NMR spectrum, wherein the resonances of C-19 and C-20 near the Δ17,18 double bond were shielded (ΔδC‑19 = −7.6) and deshielded (ΔδC‑20 = +7.3), respectively. This deduction was confirmed by the ROESY cross-peaks of Me-20 and H-17 and those of H2-19 and H2-16, as well as the D



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Table 2. 1H NMR and 13C NMR Spectroscopic Data for Compounds 3−6a monogxanthone C (3)

a

position

δC

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16a 16b 17 18 19 20 21a 21b 22 23 24 25 26a 26b 27 28 29 30 31a 31b 32 33 34 35 OH-1 OH-3 OH-5 OH-6

161.0 99.9 160.6 104.6 153.9 108.8 159.2 201.4 56.0 116.6 179.8 105.4 151.9 21.9 121.2 135.51 25.79 18.08 37.6

1

118.4 139.2 32.1 23.5 26.7 124.3 131.8 25.9 17.8 38.2 117.9 135.48 25.83 18.12

δH, mult (J, Hz) 6.30, s

6.52, s

3.52, d (7.0) 5.30, t (7.0) 1.78, 1.87, 3.45, 2.83, 4.63,

s s dd (13.6, 7.0) dd (13.6, 7.0) t (7.0)

1.93, m 1.49, s 1.91, m 5.04, t (5.9) 1.67, 1.58, 3.42, 2.79, 4.64,

s s dd (13.4, 7.0) dd (13.4, 7.0) t (7.0)

1.49, s 1.48, s

monogxanthone D (4a) δC

δH, mult (J, Hz)

161.3 95.4 162.8 107.4 153.5 109.0 159.4 201.6 56.1 116.4 179.9 104.9 152.1 21.7 122.2 132.0 25.7 18.0 37.7 118.5 139.1 32.1 23.5 26.7 124.3 131.7 25.9 17.8 38.2 118.0 135.4 25.8 18.1 56.1

6.40, s

6.51, s

3.44, m 5.21, t (7.5) 1.70, 1.83, 3.46, 2.82, 4.64,

s s m m m

157.9 110.4 159.4 104.89 152.3 108.9 158.9 201.6 56.1 116.5 179.9 104.91 151.8 22.0 121.8 135.5 26.0 18.09 21.9 121.6 134.2 25.8 18.07 37.9

1.93, m 1.49, s 1.91, m 5.04, t (6.5) 1.67, 1.58, 3.43, 2.79, 4.65,

monogxanthone E (5) δC

s s m m m

118.0 139.1 39.8 16.4 26.8 124.1 131.5 25.9 17.7 38.1

1.49, s 1.47, s 3.90, s

118.1 135.3 25.7 18.11 13.14, s 6.01, brs

13.24, s

6.97, s

δH, mult (J, Hz)

6.50, s

3.46, d (7.0) 5.28, t (7.0) 1.77, s 1.85, s 3.48, d (7.0) 5.26, t (7.0) 1.86, 1.75, 3.44, 2.79, 4.67,

s s m m t (7.5)

1.78, 1.47, 1.82, 1.82, 4.88,

m s m m t (6.6)

1.60, 1.49, 2.81, 3.44, 4.64,

s s m m t (7.5)

1.49, s 1.49, s 13.46, s 6.30, s 6.93, s

monogxanthone F (6) δC 162.5 100.5 159.4 100.7 151.1 108.6 159.0 201.5 56.2 117.0 179.5 105.1 152.0 114.7 127.5 78.2 28.41 28.46 38.2 117.9 139.2 39.8 16.4 26.7 124.1 131.5 25.7 17.7 38.0 117.8 135.5 25.8 18.1


6.50, s

6.69, d (10.0) 5.59, d (10.0) 1.477, s 1.477, s 3.44, dd (14.0, 7.5) 2.79, dd (14.0, 7.5) 4.66, t (7.5) 1.78, m 1.477, s 1.84, m 4.87, t (6.2) 1.59, s 1.480, s 3.46, dd (14.4, 7.5) 2.81, dd (14.4, 7.5) 4.63, t (7.5) 1.49, s 1.49, s

13.22, s

6.99, s

13

The H NMR spectra were measured at 500 MHz; the C NMR spectra were measured at 125 MHz. The NMR samples were dissolved in CDCl3.

formula and the characteristic 1H NMR resonances of two ciscoupled olefinic protons [δH 6.69, 5.59 (each 1H, d, J = 10.0 Hz)] suggested that a pyran ring formed in 6 between the C-4 isoprenyl side chain and OH-3 of 13. The HMBC correlations from H-11 to C-3, C-4, C-4a, and C-13 and those from H-12 to C-4, C-13, C-14, and C-15 were consistent with this deduction (Figure 3). The 17E configuration of the C-8 geranyl side chain was confirmed by the diagnostic cross-peaks of Me-20 and H216 and of H2-19 and H-17 in the ROESY spectrum (Figure 3). No Cotton effects were observed in its ECD spectrum, which in conjunction with the chiral HPLC analysis (Figure S8-7,

spectrum (Figure S7-8, Supporting Information), suggesting that 5 was also a racemate. Therefore, the structure of compound 5, (±)-monogxanthone E, was established as shown. Compound 6 was isolated as a yellow, viscous oil. Its molecular formula was concluded to be C33H38O6 on the basis of its 13C NMR data and the deprotonated ion at m/z 529.2599 [M − H]− (calcd for C33H37O6, 529.2596) observed in its HRESIMS data. The 1D NMR spectroscopic data (Table 2) indicated that 6 was closely related to biyouxanthone C (13),5a which features a 1,3,6,7-tetraoxygenated xanthone skeleton with isoprenyl and geranyl side chains. Furthermore, the molecular E



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Figure 6. Selected HMBC and ROESY correlations observed for compounds 7, 8, and 9.

C5 units were assigned as one isoprenyl group at C-4 (ring A) and one geranyl side chain at C-5 (ring B) by comprehensive 1D and 2D NMR spectroscopic data analyses. Additionally, the 17E configuration of the C-5 geranyl chain was characterized by the chemical shifts of C-19 (δC 39.9) and C-20 (δC 16.5) and the ROESY correlations of Me-20/H2-16 and of H2-19/H-17 (Figure 6). Consequently, the structure of 8, monogxanthone H, was established as shown. Compound 9 was purified by recycling HPLC techniques as with 8 and was assigned the same molecular formula as 8 (C33H38O6) on the basis of its HRESIMS and 13C NMR data. Its 13C NMR data strongly resembled those of 8, except for the presence of the characteristic signals (δC‑19 32.3 and δC‑20 23.6) for the 17Z-configured double bond, suggesting that 9 is the 17Z-isomer of 8. This deduction was confirmed by the ROESY correlations between Me-20 and H-17 and between H2-19 and H2-16 (Figure 6). Thus, the structure of 9, monogxanthone I, was defined as depicted. Compound 10 was isolated as a yellow, viscous oil with the same molecular formula as 8 (C33H38O6), as determined from its HRESIMS and 13C NMR data. Analyses of its 1D and 2D NMR data suggested that 10 resembled 8. The 1H NMR spectrum of 10 also had the deshielded olefinic H-21 (δH 8.06, 1H, d, J = 10.2 Hz). The overlapped methyl groups in 8 were split into two distinct signals in 10, suggesting that the structural moiety around the pyran ring of 10 had changed. The key HMBC correlations from H2-24 to C-22, C-25, and C-26; from Me-25 to C-22, C-23, and C-24; and from H-21 to C-7, C-8, C-8a, and C-23 confirmed that a geranyl-like unit at C-8 in 10 was involved in the formation of the pyran ring instead of the prenyl-like group in 8. The remaining two C5 units were defined as isoprenyl groups at C-4 and C-5 from the HMBC correlations of H2-11 to C-3, C-4, and C-4a and of H2-16 to C5, C-6, and C-10a. Despite the presence of the C-23 stereogenic center in 10, the chiral HPLC and optical rotation data indicated that 10 was also racemic. Subsequent chiral resolution of 10 by HPLC using a Chiralpak AD-H column yielded the enantiomers (+)-10 and (−)-10 in a ratio of 48.9:51.1 (Figure 4B). The (23S) absolute configuration of (+)-10 was defined by comparison of the experimental ECD spectrum of (+)-10 and the calculated ECD spectrum of (23S)-10 (Figure 5B). Thus, the structure of 10, (±)-monogxanthone J, was elucidated as shown. In summary, 10 rare polyprenylated xanthones (1−10) from the genus Hypericum were characterized, together with four typical polyprenylated xanthones reported in 2010 (Figure S1, Supporting Information) from a congeneric plant of H.

Supporting Information) suggested that 6 was a racemic mixture. Therefore, the structure of 6, (±)-monogxanthone F, was determined as shown. Monogxanthone G (7) was obtained as a yellow, viscous oil. Its molecular formula was established as C28H32O6 on the basis of its HRESIMS (obsd [M + H]+ m/z 463.2123, calcd m/z 463.2126) and 13C NMR data. Its UV and IR spectra revealed the presence of a xanthone skeleton. The 1D NMR spectra of 7, with the aid of an HSQC experiment, indicated four hydroxy groups, two isolated aromatic protons, one isoprenyl group, and one geranyl side chain, similar to dulciol A.11 The differences between the 1H and 13C NMR of these two compounds were observed among the signals associated with ring A. The deshielded C-4 resonance (ΔδC = +10) and the presence of an aromatic proton singlet in 7, instead of two meta-coupled aromatic protons as in dulciol A, indicated that the isoprenyl group was located at C-4. The HMBC correlations from H2-11 to C-3, C-4, C-4a, C-12, and C-13 and from Me-14 and Me-15 to C-13 were consistent with this observation (Figure 6). The geranyl side chain was located at C-8, as revealed by the HMBC correlations between H2-16 (δH 4.39, 2H, d, J = 6.8 Hz) and C7, C-8, and C-8a (Figure 6). The substantial downfield chemical shift of H2-16 was attributed to the deshielding effect of the conjugated C-9 carbonyl. Finally, the 17E configuration was established on the basis of the chemical shifts of C-19 (δC 39.8) and C-20 (δC 16.5) and was further confirmed by ROESY correlations between Me-20 and H2-16 and between H2-19 and H-17 (Figure 6). Consequently, the structure of 7, monogxanthone G, was assigned as depicted. Monogxanthone H (8) had a molecular formula of C33H38O6, as established on the basis of its 13C NMR data and the observed pseudomolecular ion at m/z 529.2600 [M − H]− (calcd for C33H37O6, 529.2596) in its HRESIMS data, indicating four C5 units, along with one typical C13 xanthone skeleton. Its UV and IR spectra revealed the presence of a 1,3,6,7-tetraoxygenated xanthone skeleton similar to that of 7. A characteristic pyran ring was revealed by the 1H NMR spectroscopic data of two cis-coupled olefinic protons [δH 8.01, 5.78 (each 1H, d, J = 10.2 Hz, H-26, H-27)] and two overlapped methyls (δH 1.50, 6H, s), similar to the pyran ring in compound 6. Furthermore, the pyran ring was located in the “southern” region (C-7 and C-8 of ring B) of the xanthone skeleton, as suggested by the deshielded H-26 (ΔδH = +1.37 relative to H-11 in 6), the deshielding caused by the C-9 carbonyl group. The HMBC correlations from H-26 to C-7, C8, C-8a, and C-28 and those from Me-29 and Me-30 to C-28 and C-27 confirmed the regiochemistry. The remaining three F



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Table 3. 1H NMR and 13C NMR Spectroscopic Data for Compounds 7−10a monogxanthone G (7)

a

position

δC

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OH-1 OH-3 OH-6 OH-7

161.9 98.5 161.1 104.2 154.1 101.4 153.9 140.00 127.7 111.4 183.2 104.3 151.3 21.8 121.6 134.9 25.9 17.9 26.1 121.5 140.04 39.8 16.5 26.4 123.9 132.5 25.8 18.1

monogxanthone H (8)

δH, mult (J, Hz)

δC 161.8 98.6 161.3 104.29 154.2 115.7 148.9 136.8 117.4 108.4 183.4 104.33 151.3 22.0 121.7 135.5 25.7 17.8 22.8 121.19 136.7 39.9 16.5 26.8 124.4 131.5 25.9 18.1 121.23 131.6 77.2 27.6 27.6

6.23, s 6.87, s

3.53, d (7.2) 5.31, t (6.5) 1.76, 1.88, 4.39, 5.28,

s s d (6.8) t (6.8)

2.11, 1.87, 2.14, 5.04,

m s m t (5.4)

1.68, s 1.60, s

13.34, s 5.92, s 6.32, s 5.74, s

monogxanthone I (9)


3.57, d (6.9) 5.30, brs 1.76, 1.84, 3.63, 5.30,

s s d (6.7) brs

1.98, 1.81, 2.05, 5.04,

m s m t (6.8)

1.61, 1.55, 8.01, 5.78,

s s d (10.2) d (10.2)

1.50, s 1.50, s 13.34, s 5.97, s 6.32, s

δC

δH, mult (J, Hz)

161.8 98.6 161.3 104.34 154.2 115.6 148.8 136.8 117.4 108.4 183.4 104.32 151.3 22.1 121.8 135.4 25.8 17.8 22.6 121.9 137.0 32.3 23.6 26.7 124.5 131.8 25.9 18.1 121.2 131.6 77.2 27.6 27.6

6.25, s

3.57, d (7.0) 5.30, t (7.0) 1.77, 1.84, 3.63, 5.33,

s s d (6.8) t (7.0)

2.27, 1.70, 2.12, 5.17,

m s m t (7.0)

1.69, 1.63, 8.01, 5.78,

s s d (10.2) d (10.2)

1.49, s 1.49, s 13.34, s 5.96, s 6.32, s

monogxanthone J (10) δC

δH, mult (J, Hz)

161.8 98.5 161.2 104.4 154.2 115.6 148.7 136.9 117.3 108.3 183.4 104.3 151.2 22.0 121.8 135.3 25.91 18.08 22.94 121.5 133.0 25.91 18.05 121.6 130.8 79.5 40.7 25.86 22.88 123.9 132.2 25.75 17.8

6.25, s

3.57, brs 5.29, brs 1.76, 1.84, 3.62, 5.30,

s s d (6.5) brs

1.69, 1.81, 8.06, 5.75,

s s d (10.2) d (10.2)

1.78, 1.45, 2.13, 5.09,

m s brs t (6.1)

1.67, s 1.58, s 13.35, s 6.03, s 6.31, s

The 1H NMR spectra were measured at 500 MHz; the 13C NMR spectra were measured at 125 MHz. The NMR samples were dissolved in CDCl3.

Table 4. Neuroprotective Effects of Compounds 1, 2, 11, 14, and 15 and Their Inhibitory Effects on Nitric Oxide (NO) Productiona cell viability (% of control), concentration (μM) compound

25.00

control Cortb Fluc d L-NMMA 1 2 11 14 15

100 52.81 ± 2.52###

12.50

6.25

IC50 for NO inhibition (μM)

70.53 ± 4.08** 91.97 85.91 65.42 65.97 84.06

± ± ± ± ±

3.82*** 3.84*** 2.42* 3.77* 3.32***

83.77 ± 2.43*** 78.75 ± 2.06*** 71.18 ± 5.38* 60.17 ± 6.15 78.14 ± 4.02**

78.42 ± 5.73** 75.23 ± 3.27** 64.82 ± 1.79** 57.49 ± 6.86 60.68 ± 3.73

39.28 7.47 9.60 >50 5.66 8.35

± 1.81 ± 0.65 ± 0.12 ± 0.41 ± 0.72

Each value is expressed as the mean ± SEM from three independent experiments. Statistical significance was analyzed by ANOVA: ###P < 0.001 compared to the control, *P < 0.05, **P < 0.01, ***P < 0.001 compared to Cort. bModel group for neuroprotective effect. cPositive control for neuroprotective effect. dPositive control for NO inhibitory effect. a

monogynum.5a Moreover, some 13C NMR patterns were established on the basis of these diverse structures to

distinguish the Z or E configuration of the double bond of the geranyl side chain. The absolute configurations of the G



Article

Figure 7. Effects of compound 1 on the intracellular Ca2+ concentrations in PC12 cells. (A) PC12 cells were incubated with the indicated concentrations of fluoxetine (Flu) and compound 1 for 24 h and exposed to 200 μM Cort for 48 h. The intracellular Ca2+ concentrations were measured by flow cytometry analysis. (B) Alteration of the fluorescence intensities when the cells were treated with Cort, Flu + Cort, and compound 1 + Cort. The data are expressed as the mean ± SEM, n = 3. The statistical significance was analyzed by ANOVA: ###P < 0.001 compared to the control, ***P < 0.001 compared to Cort.

(CNS) inflammatory cascade, which in turn affects neural plasticity.19 Activated microglial cells in the CNS can produce inflammatory mediators such as nitric oxide (NO); the overproduction of NO in the CNS can result in uncontrolled neuroinflammation.20 Compounds 1, 2, 11, 14, and 15, which showed potential protective effects on the Cort-induced PC12 cell injuries, were assessed for their inhibitory effects on NO production stimulated by lipopolysaccharide (LPS) in BV2 microglia cells. N-Monomethyl-L-arginine (L-NMMA) was used as the positive control (IC50 value = 39.28 ± 1.81 μM). As a result, compounds 1, 2, 14, and 15 were observed to exhibit significant inhibitory effects on NO production, with IC50 values of 7.47 ± 0.65, 9.60 ± 0.12, 5.66 ± 0.41, and 8.35 ± 0.72 μM, respectively (Table 4). In addition, the cell viabilities were greater than 90% in all cases, as determined by an MTT assay. Collectively, these results shed new light on the potential of polyprenylated xanthones from the genus Hypericum in the development of antidepression therapies.

monoterpene moieties in 1 and 2 and C-8 in the xanthone skeleton of 3 were established on the basis of experimental and calculated ECD spectra. Structurally, ring A of compounds 1− 17 was a phloroglucinol-like structure, whereas ring B of the compounds was dioxygenated at C-5 and C-6 or C-6 and C-7 and had a 3,4-dihydroxybenzoic-like scaffold combined with the carbonyl group of the C-ring. Biosynthetically, the isolated xanthones were of mixed shikimate and acetate origin: the phloroglucinol-like ring A was derived from acetate, whereas the 3,4-dihydroxybenzoic-like ring B was developed from phenylalanine.15 Plants of the genus Hypericum have been used as traditional medicines in various parts of the world. Extracts of H. perforatum (St. John’s wort) are widely used in Europe as a drug for treating depression.16 Xanthones, characteristic secondary metabolites of the genus Hypericum, are recognized to contribute to the antidepressant properties of St. John’s wort.17 Hence, the protective effects of the isolates against Cort-induced PC12 cell injuries were assessed for their antidepressant potential in this study.7 Consequently, compounds 1 and 2 exhibited noticeable protective effects against injuries of PC12 cells at concentrations of 6.25, 12.50, and 25.00 μM with cell viabilities greater than 75% (Table 4). Additionally, the isolates did not cause cytotoxicity in PC12 cells at a concentration of 25.00 μM, as determined by an MTT assay. Because intracellular Ca2+ is a key regulator of many cellular processes and down-regulating intracellular Ca2+ is closely associated with antidepressant activity,18 efforts were made to probe whether the active compounds observed in the MTT assay could affect cellular Ca2+ homeostasis in PC12 cells. As a result, the overloaded intracellular Ca2+ concentration of PC12 cells induced by corticosterone can be largely reversed by incubation with compound 1 at concentrations of 6.25, 12.50, and 25.00 μM (Figure 7); this result is consistent with the neuroprotective effect of 1. Neuroinflammation has been found to be closely associated with depression. Stress can induce the activation of inflammatory responses and the central nervous system

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. The UV spectra were recorded on a UV-2450 UV/vis spectrophotometer. The ECD spectra were recorded on a JASCO J-810 spectrometer. The IR measurements were performed on a Bruker Tensor 27 spectrometer. The 1H, 13C, HSQC, HMBC, and ROESY NMR spectra were recorded on a Bruker Avance III NMR spectrometer using standard pulse sequences (1H: 500 MHz, 13C: 125 MHz) with tetramethylsilane as an internal standard. The HRESIMS spectra were acquired using an Agilent 6520B UPLC-Q-TOF mass spectrometer. Column chromatography was carried out using D-101 macroporous resin (pore size B 13−14 nm, 26−60 mesh, Qingdao Haiyang Chemical Co. Ltd., China), polyamide (80−100 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), silica gel (100−200 mesh and 200−300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH20 (40−70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden), and ODS RP-C18 (40−63 μm, Fuji, Japan). Fractions obtained from column chromatography were monitored by TLC on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). The spots were visualized under UV light and by spraying the H



Article

Monogxanthone A (1): yellow, amorphous powder; [α]25D +13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.48), 252 (4.53), 285 (3.95), 328 (4.26) nm; ECD (MeOH) λmax (Δε) 219 (+8.36), 250 (−1.58), 265 (+0.96) nm; IR (KBr) νmax 3416, 2967, 2924, 1642, 1612, 1591, 1558, 1460, 1338, 1165, 1118, 794 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 533.2895 [M + H]+ (calcd for C33H41O6, 533.2898). Monogxanthone B (2): yellow, amorphous powder; [α]25D −8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.37), 252 (4.56), 285 (3.95), 325 (4.26) nm; ECD (MeOH) λmax (Δε) 213 (+2.72), 252 (−0.58), 266 (−0.23) nm; IR (KBr) νmax 3421, 2970, 2923, 1647, 1614, 1454, 1292, 1241, 1207, 798 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 395.1497 [M − H]− (calcd for C23H23O6, 395.1500). (±)-Monogxanthone C (3): yellow, viscous oil; [α]25D +2 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 205 (4.11), 238 (3.77), 298 (3.77), 338 (3.43), 390 (3.29) nm; IR (KBr) νmax 3366, 2976, 2924, 1646, 1564, 1509, 1443, 1269, 1173, 1104, 835 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 533.2896 [M + H]+ (calcd for C33H41O6, 533.2898). (+)-(8R)-Monogxanthone C [(+)-3]: yellow, viscous oil; [α]25D +18 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 209 (+5.56), 245 (+1.58), 272 (−0.18) nm. (−)-(8S)-Monogxanthone C [(−)-3]: yellow, viscous oil; [α]25D −14 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 206 (−5.64), 245 (−1.33), 276 (+0.23) nm. (±)-Monogxanthone D (4a): yellow, viscous oil; [α]25D −3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 206 (4.30), 234 (3.97), 299 (3.97), 338 (3.67), 402 (3.51) nm; IR (KBr) νmax 3419, 2920, 2853, 1646, 1593, 1443, 1270, 1179, 827 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 545.2908 [M − H]− (calcd for C34H41O6, 545.2909). (±)-Monogxanthone E (5): yellow, viscous oil [α]25D −7 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 205 (4.45), 234 (4.12), 297 (4.13), 335 (3.86), 401 (3.57) nm; IR (KBr) νmax 3394, 2922, 2852, 1649, 1436, 1313, 1178, 853 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 599.3380 [M − H]− (calcd for C38H47O6, 599.3378). (±)-Monogxanthone F (6): yellow, viscous oil; [α]25D −5 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (4.22), 244 (4.18), 291 (3.92), 415 (2.62) nm; IR (KBr) νmax 3441, 2921, 2851, 1648, 1451, 1384, 1278, 1157, 1068, 836 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 529.2599 [M − H]− (calcd for C33H37O6, 529.2596). Monogxanthone G (7): yellow, viscous oil; [α]25D +21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.87), 241 (3.58), 261 (3.68), 315 (3.37), 369 (3.13) nm; IR (KBr) νmax 3398, 2921, 2850, 1728, 1618, 1467, 1355, 1121, 1069 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 463.2123 [M − H]− (calcd for C28H31O6, 463.2126). Monogxanthone H (8): yellow, viscous oil; [α]25D +12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.41), 247 (4.26), 267 (4.28), 337 (4.15), 384 (3.59) nm; IR (KBr) νmax 3450, 2921, 2851, 1648, 1567, 1428, 1256, 1126, 1075, 802 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 529.2600 [M − H]− (calcd for C33H37O6, 529.2596). Monogxanthone I (9): yellow, viscous oil; [α]25D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.29), 248 (4.24), 257 (4.23), 344 (4.05), 385 (3.56) nm; IR (KBr) νmax 3362, 2921, 2851, 1650, 1569, 1433, 1293, 1138, 1084, 826 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 529.2598 [M − H]− (calcd for C33H37O6, 529.2596). (±)-Monogxanthone J (10): yellow, viscous oil; [α]25D +4 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 205 (3.58), 248 (3.30), 261 (3.30), 334 (3.11), 381 (3.56) nm; IR (KBr) νmax 3421, 2924, 2854, 1647, 1514, 1438, 1277, 1123, 1058, 832 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 529.2601 [M − H]− (calcd for C33H37O6, 529.2596). (+)-(23S)-Monogxanthone J [(+)-10]: yellow, viscous oil; [α]25D +13 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 200 (+23.42), 233 (−1.30), 260 (+4.28), 290 (−3.60) nm.

plates with a 1% vanillin/H2SO4 solution, followed by heating. The instrument used for HPLC analysis was an Agilent 1100 series chromatograph equipped with a DAD detector and an Agilent ZORBAX Eclipse XDB-C18 (5 μm, 4.6 × 150 mm2, i.d.) column. Preparative HPLC was carried out using a Shimadzu LC-6AD series instrument equipped with a Shim-pack RP-C18 column (10 μm, 20 × 200 mm2, i.d.) and a binary-channel UV detector set to detect at 254 and 350 nm. Recycling preparative HPLC was run on a Shimadzu LC20A series instrument equipped with a Shim-pack PRC-ODS (H) column (5 μm, 20 × 200 mm2, i.d.). The equipment was controlled, the data were acquired and processed, and the chromatographic information was managed using the Shimadzu LC-solution software. Chiral HPLC was conducted on a JASCO LC-Net II/ADC instrument equipped with a Chiralpak AD-H column (5 μm, 4.6 × 250 mm2, i.d.; Daicel Chiral Technologies Co., Ltd., China); the eluents were detected by a CD-2095 Plus chiral detector. Plant Material. The fresh roots of H. monogynum were collected from China Pharmaceutical University (Nanjing, Jiangsu Province, People’s Republic of China) in November 2014. The plant material was authenticated by Professor Min-Jian Qin (China Pharmaceutical University). A voucher specimen (No. 2014-RHML) was deposited at the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The fresh roots of H. monogynum (10.0 kg) were cut into pieces and extracted with 95% aqueous EtOH (3 × 15.0 L) under ultrasonic agitation at 90 Hz and 40 °C. After removal of the EtOH in vacuo, a large quantity of a yellow solid was obtained. This solid product was filtered through a Büchner funnel to obtain the crude extract (140.7 g). This crude extract was partitioned by column chromatography over D-101 macroporous resin (1.7 kg; ⦶ 10.0 × 50.0 cm2) and eluted with EtOH/H2O (40:60 to 80:20 to 95:5, v/v) to produce three fractions (A−C). Fraction B (85.0 g) was applied to a 1.5 kg polyamide open column (⦶ 12.0 × 100.0 cm2) and eluted with EtOH/H2O (50:50 to 70:30 to 95:5, v/v) to give three subfractions (Fr. B1−B3). Fractions B2 and B3 were obtained as yellow powders, and their HPLC-DAD chromatograms contained numerous xanthonelike chromatographic peaks as observed from the analysis. Fraction B3 (22.0 g) was subsequently refined over a silica gel column (200−300 mesh; 300.0 g; ⦶ 8.0 × 30.0 cm2) using CH2Cl2/acetone (100:1 to 10:1, v/v); the elution yielded four subfractions (Fr. B3A−B3D). Subfraction B3B (1.4 g) was further separated using Sephadex LH-20 (MeOH), to afford three subfractions (Fr. B3B1−B3B3). The second subfraction, B3B2, was purified by separation over an ODS RP-C18 column (40−63 μm; 40.0 g; ⦶ 2.5 cm × 30.0 cm2) using MeOH/H2O (75:25, v/v) to afford compounds 1 (55.6 mg), 11 (30.2 mg), and 14 (120.7 mg). Compounds 7 (7.0 mg), 8 (5.2 mg), 9 (4.2 mg), and 10 (5.3 mg) were separated from the third subfraction, B3B3 (103.5 mg), on an ODS RP-C18 column, followed by preparative HPLC using 90% MeOH in H2O. Another crude xanthone fraction, B2 (11.4 g), was separated over a silica gel column (200−300 mesh; 180.0 g; ⦶ 4.5 × 30.0 cm2) and eluted with a gradient mixture of CH2Cl2/acetone (100:1 to 10:1, v/v) to afford seven major subfractions (Fr. B2A− B2G). Compounds 2 (80.3 mg) and 15 (20.2 mg) were purified from subfractions B2G and B2F using preparative HPLC with 70% and 75% MeOH in H2O, respectively. Subfraction B2C was refined over Sephadex LH-20 (MeOH) and further purified using preparative HPLC to yield one major fraction (70.2 mg), whose HPLC-DAD chromatogram showed two peaks. This fraction was further separated by recycling HPLC. The successful separation of compounds 3 (20.0 mg) and 13 (40.0 mg) was conducted using improved recycling HPLC techniques with a Shim-pack PRC-ODS (H) column (5 μm, 20 × 200 mm2, i.d.) after 7−10 consecutive cycles. Compound 12 (150.0 mg) was purified by preparative HPLC from subfraction B2D. Guided by HPLC-DAD analysis, the rapid isolation of compounds 4a/b (27.8 mg) and 5 (8.0 mg), which showed the same UV absorption bands as compounds 3 and 13, was done via repeated column chromatography over polyamide, silica gel, Sephadex LH-20, and ODS RP-C18 and finally preparative HPLC from the lower polarity fraction C. Compound 6 (6.5 mg) was also purified from fraction C, along with compounds 4a/b, using the same preparative HPLC procedure. I



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(−)-(23R)-Monogxanthone J [(−)-10]: yellow, viscous oil; [α]25D −8 (c 0.1, CHCl3); ECD (MeOH) λmax (Δε) 200 (−24.50), 233 (+2.05), 259 (−4.79), 287 (+3.61) nm. Maculatoxanthone (11): 8 yellow, amorphous powder; [α]25D +8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.96), 283 (4.31), 337 (4.17) nm; IR (KBr) νmax 3510, 2973, 2923, 1650, 1624, 1585, 1464, 1334, 1191, 1129, 878 cm−1; 1H NMR (CDCl3, 500 MHz) δ 13.20 (1H, s, OH-1), 7.72 (1H, d, J = 8.8 Hz, H-8), 6.95 (1H, d, J = 8.8 Hz, H-7), 6.73 (1H, d, J = 10.0 Hz, H-11), 6.14 (1H, s, OH-6), 5.65 (1H, s, OH-5), 5.58 (1H, d, J = 10.0 Hz, H-12), 5.21 (1H, t, J = 6.8 Hz, H22), 4.67 (1H, s, H-19a), 4.59 (1H, s, H-19b), 2.85 (1H, dd, J = 13.5, 6.0 Hz, H-16a), 2.79 (1H, dd, J = 13.5, 7.9 Hz, H-16b), 2.39 (1H, m, H-17), 2.22 (1H, m, H-21a), 2.14 (1H, m, H-21b), 1.75 (3H, s, H320), 1.73 (3H, s, H3-24), 1.61 (3H, s, H3-25), 1.489 (3H, s, H3-14), 1.485 (3H, s, H3-15); 13C NMR (CDCl3, 125 MHz) δ 180.7 (C, C-9), 158.4 (C, C-3), 156.3 (C, C-1), 154.3 (C, C-4a), 149.4 (C, C-6), 149.3 (C, C-18), 145.1 (C, C-10a), 133.4 (C, C-23), 130.6 (C, C-5), 127.2 (CH, C-12), 123.0 (CH, C-22), 118.3 (CH, C-8), 115.9 (CH, C-11), 114.4 (C, C-8a), 112.6 (CH, C-7), 110.9 (CH2, C-19), 107.0 (C, C-4), 104.6 (C, C-2), 102.8 (C, C-9a), 78.4 (C, C-13), 48.2 (CH, C-17), 32.2 (CH2, C-21), 28.7 (CH3, C-14), 28.6 (CH3, C-15), 26.9 (CH2, C16), 26.0 (CH3, C-24), 19.5 (CH3, C-20), 18.1 (CH3, C-25); HRESIMS m/z 463.2113 [M + H]+ (calcd for C28H31O6, 463.2115). Neuroprotective Assay. PC12 dopaminergic neuron cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 5% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Corticosterone and fluoxetine were purchased from Sigma (Darmstadt, Germany). The cells were plated in 96-well microplates at a density of 2 × 104 cells/well. After attachment, the cells were pretreated with different concentrations (6.25, 12.50, 25.00 μM) of the test compounds for 24 h (or without drug, in the case of the model (Cort) group) and exposed to 200 μM corticosterone. After 48 h the cell viability was measured using an MTT assay.21 The absorbance at 570 nm was recorded on a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, USA); the absorbance at 630 nm was used as reference. Fluoxetine (Flu, 12.50 μM) was used as the positive control. The viability of the treated cells was expressed as a percentage of the nontreated control. Measurement of the Free Intracellular Ca2+. The concentration of intracellular Ca2+ was measured using the fluorescence Ca2+ indicator Fluo-3 AM according to a published procedure.22 Briefly, PC12 cells were harvested at the end of each treatment [pretreated with Flu (12.50 μM) and different concentrations (6.25, 12.50, 25.00 μM) of compound 1 for 24 h and exposed to 200.00 μM Cort for 48 h]. Intracellular Ca2+ was detected by a BD Accuri C6 flow cytometer (Becton & Dickinson Co., Miami, FL, USA). The Ca2+ concentrations were expressed as the mean fluorescent intensities of each treated group compared with that of the vehicle control group. Inhibition of NO Production in LPS-Stimulated BV2Microglial Cells.23 The BV2 microglial cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The cells were seeded into 96well plates at a density of 2.5 × 105 cells/mL. After attachment, the cells were preincubated with the test compounds for 2 h, and 20 μL of an LPS solution (diluted with medium to a final concentration of 100 ng/mL) was subsequently added. After 18 h, the culture supernatants were mixed with an equal volume of Griess reagent at room temperature for 10 min. The absorbance was measured at 540 nm with a microplate reader. The concentration of NO was calculated using the stated linear equation of the NO assay kit. The percent inhibition of NO production was calculated using the formula (AL − A0 − AC)/(AL − A0) × 100, where AL is the absorbance of the LPS-treated group, A0 is the absorbance of the normal control, and AC is the absorbance of the test compound with the LPS-treated group. The IC50 values of the tested compounds were calculated using linear regression plots.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00251. 1D NMR, 2D NMR, HRESIMS, and ECD spectra of compounds 1−10 and maculatoxanthone (11) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-83271405. E-mail: [email protected] (L.-Y. Kong). *E-mail: [email protected] (J. Luo). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the National Natural Science Foundation of China (81430092), the Program for New Century Excellent Talents in University (NCET-20131035), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). This research was also supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63).

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