Polycyclic Polyprenylated Derivatives from Hypericum uralum

Article pubs.acs.org/jnp

Polycyclic Polyprenylated Derivatives from Hypericum uralum: Neuroprotective Effects and Antidepressant-like Activity of Uralodin A Zhong-Bo Zhou, Zhong-Rui Li, Xiao-Bing Wang, Jian-Guang Luo,* and Ling-Yi Kong* 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: The isolation of the new polycyclic polyprenylated acylphloroglucinols uraliones A−K (1−11) together with five known analogues (12−16) from a whole Hypericum uralum plant was reported. The structures of these compounds were established through spectroscopic methods, and a single-crystal X-ray diffraction analysis was used to confirm the absolute configuration of 1. The protective effects of the isolates against corticosterone-induced PC12 cell injury were assessed. Except for compound 9, all tested compounds exhibited significant protective effects against induced injury in PC12 cells. Uralodin A (14), orally administered in doses of 13 and 26 mg/kg, exhibited antidepressant-like activity in the tail suspension and forced-swimming tests in mice. administration.10 Recent findings have suggested that activation of nitrosative and oxidative stress and inflammatory pathways is a crucial pathophysiological determinant in depression.11 In vivo studies have also revealed that various classes of antidepressants can reduce the levels of oxidative stress markers and increase the release of some endogenous antioxidants.12 Hypericum uralum Buch.-Ham. ex D. Don is distributed in Tibet and in the Northwest Yunnan Province of China.13 The entire plant is used in the Yunnan Province for antiinflammation applications, for detoxification, and to relieve itching. To date, only two PPAPs have been identified from this plant.14 As a part of the phytochemical investigation of Hypericum spp.,15 the whole H. uralum plant was investigated, resulting in the isolation of 11 new (1−11) and five known PPAPs. Herein, the isolation, structure elucidation, and bioactivities of all the isolates are discussed.

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family of natural compounds named polycyclic polyprenylated acylphloroglucinols (PPAPs), which possess a phloroglucinol core decorated with prenyl, geranyl, and even more highly substituted moieties, are the main secondary metabolites of Hypericum.1 These metabolites exhibit extensive biological activities, such as antidepressant, antineurodegenerative, anti-inflammatory, anticancer, antioxidant, antibacterial, antiulcer, antimalarial, and anti-HIV activities.2 Hyperforin, a PPAP compound from Hypericum perforatum, is well known for its various bioactivities, particularly its clinical use for the treatment of depression.3 After the isolation of hyperforin in 1975,4 interest in the biological activities and structures of PPAPs has increased considerably.5 Investigations of hyperforin analogues from the genus Hypericum have revealed a number of biologically and structurally interesting PPAPs.6 The PC12 cell line, derived from the pheochromocytoma of the adrenal medulla in rats, is widely used in studies because of its typical neuron characteristics and large number of glucocorticoid receptors.7 PC12 neuronal damage can be induced by suitable concentrations of glucocorticoids under depressive disorder, leading to a useful in vitro experimental model for depression studies.8 Different types of classical antidepressants were found to be effective in previous studies in protecting against the cytotoxicity in PC12 cells induced by glucocorticoids.8b It has been reported that oxidative stress may contribute to corticosterone-induced neuronal injury.9 Depressive-like behaviors may be induced by lipopolysaccharide (LPS) through stimulation of cell signaling networks and elicitation of inflammatory responses; therefore, a commonly used model of depressive-like behaviors can be established by acute LPS © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Uralione A (1) was obtained as colorless crystals. The sodium adduct ion at m/z 575.3706 ([M + Na]+, calcd for C35H52NaO5, 575.3707) in the HRESIMS spectrum and the 13 C NMR data indicated that the molecular formula of 1 was C35H52O5. The IR spectrum showed absorption bands due to hydroxy (3396 cm−1) and carbonyl (1728 cm−1) groups. Analysis of the 13C (Table 1) and HSQC NMR data indicated the presence of an acylphloroglucinol group with an enolized Received: August 7, 2015

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DOI: 10.1021/acs.jnatprod.5b00667 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

(δH 5.02, m) to C-19 and C-20 confirmed the attachment of Me-14 and a 4-methyl-3-pentenyl side chain at C-3. Two prenyl side chains were connected to C-4 and C-6, which were established by the HMBC cross-peaks from H-31 (δH 2.45, m; 1.26, m) to C-1, C-6, and C-32 and from H-32 (δH 5.05, m) to C-34 and C-35 and the connections between H-21 (δH 2.12, dd, 14.0, 5.5; 1.70, m) and C-4. In addition, the 2,2-dimethyl2H-dihydropyran ring was linked to C-7 and C-8, as elucidated from the HMBC correlations of H-27 (δH 3.83, t, 4.5) with C-8, C-28, and C-29. The relative configuration of 1 was determined by comparison of its NMR data with those of related compounds and from ROESY data. H-4 was in the αorientation, based on the C-4 chemical shift (δC 45.1) and the chemical shift difference between H-5β and H-5α (Δδ ca. 0.36).17 The ROESY (Figure 1) correlations of H-5β (δH 1.42) and Me-14 (δH 1.08) indicated that Me-14 was β-orientated. The absolute configurations at the five stereogenic centers were elucidated as (2S, 3R, 4R, 6S, 27R) by single-crystal X-ray diffraction data analysis [Flack parameter, −0.09(7)]. Hence, the structure of compound 1, uralione A, was assigned as depicted. Uraliones B (2) and C (3) were both obtained as colorless oils. Their molecular formulas were assigned as C38H50O5 based

1,3-diketo functionality (δC 195.1, 113.4, and 165.8), two unconjugated carbonyl groups (δC 210.0 and 206.4), three quaternary carbons at δC 47.6, 64.5, and 77.1, a methine carbon (δC 45.1), and a methylene (δC 39.9) carbon. Three olefinic protons (δH 5.05, 1H, m; 5.02, 1H, m; and 4.92, 1H, t, 7.0), nine methyl singlets (δH 1.08−1.67), and an isopropyl group (δH 1.12, 3H, d, 6.5; 1.15, 3H, d, 6.5; 2.48, 1H, m) (Table 2) were also evident. Additionally, the 1H NMR signals at δH 3.83 (1H, t, 4.5), 2.71 (1H, dd, 17.5, 4.5), 2.51 (1H, dd, 17.5, 4.5), 1.44 (3H, s), and 1.30 (3H, s) together with the 13C NMR resonances at δC 82.5, 68.1, 26.1, 25.0, and 22.8 indicated the presence of an oxidized 2,2-dimethyl-2H-dihydropyran ring. These observations demonstrated that 1 was a polycyclic polyprenylated acylphloroglucinol. Comparing the NMR data of compound 1 with those of furohyperforin isomer 216 revealed their structural similarity, except for the signals of the C-26−C-30 segment. Further analyses of the NMR data indicated the presence of the oxidized 2,2-dimethyl-2Hdihydropyran ring in 1 rather than the 2-(2-hydroxypropyl)2H dihydrofuran ring in furohyperforin isomer 2. HMBC cross-peaks from Me-14 (δH 1.08, s) to C-3, C-4, and C-15, from H-15 (δH 1.99, d, 11.5; 1.24, m) to C-3 and C-16, from H-16 (δH 1.99, d, 11.5;2.21, m) to C-15, and from H-17 B



Article

Table 1. 13C NMR (125 MHz, CDCl3) Spectroscopic Data for Compounds 1−11 position

1

2

3

4

5

6

7

8

9

10

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

206.4 77.1 47.6 45.1 39.9 64.5 165.8 113.4 195.1 210.0 40.5 21.8 21.6 13.0 38.7 25.7 124.8 131.6 26.3 18.0 28.1 122.7 133.5 26.0 18.0 26.1 68.1 82.5 22.8 25.0 29.4 119.9 134.2 25.8 18.2

207.2 73.5 49.7 44.2 41.8 65.4 165.5 112.6 194.9 194.4 138.2 128.3 128.2 132.2 128.2 128.3 13.6 36.6 26.2 124.7 131.7 25.8 18.3 28.3 122.5 133.7 26.0 18.0 25.4 68.9 82.8 20.5 25.5 29.6 119.8 134.7 25.6 18.1

207.0 80.2 50.8 43.4 40.1 57.7 168.8 112.4 192.4 193.7 136.9 128.6 127.9 132.0 127.9 128.6 14.3 36.8 25.1 124.8 131.4 26.2 18.1 27.4 122.8 133.5 25.9 17.9 25.8 69.3 81.3 20.4 25.4 29.1 119.6 134.2 25.9 18.4

210.2 73.6 50.2 44.3 42.5 63.8 166.3 111.8 194.8 194.0 137.9 128.3 128.2 132.2 128.2 128.3 13.6 38.4 26.2 124.8 131.5 25.8 18.1 28.1 122.5 133.7 25.9 18.0 25.4 68.0 82.6 25.3 24.3 32.8 74.9 73.1 23.9 22.3

204.7 79.9 49.4 44.6 41.1 59.8 172.5 117.4 193.5 194.4 137.4 128.3 128.0 132.0 128.0 128.3 13.9 36.6 25.7 125.0 131.2 25.6 18.0 27.3 122.5 133.5 25.9 17.9 22.8 120.8 132.7 25.9 17.8 28.8 91.5 71.1 27.2 25.8

206.3 71.1 49.1 44.3 40.8 64.7 190.9 118.8 172.8 26.8 93.8 71.6 26.3 24.8 13.0 39.6 25.8 124.4 132.4 25.6 18.0 28.2 122.4 133.7 26.0 18.0 193.6 137.9 128.2 128.0 132.4 128.0 128.2 29.4 119.7 134.5 26.1 18.3

207.0 70.8 49.5 41.7 41.1 65.2 190.6 119.0 171.7 26.7 93.7 70.7 26.6 24.0 15.0 39.6 25.8 123.3 133.8 26.0 17.9 28.3 124.5 131.7 25.8 18.2 193.4 137.9 128.3 128.7 132.8 128.7 128.3 29.5 119.7 134.8 26.0 18.3

205.6 72.1 46.4 36.9 40.2 64.9 194.4 127.5 167.2 196.6 137.3 128.7 128.1 132.7 128.1 128.7 17.4 33.8 24.3 87.2 72.2 25.7 25.0 27.3 122.2 133.2 26.0 18.0 22.8 120.9 133.7 25.8 18.1 29.4 119.8 134.8 26.1 18.2

208.9 72.1 47.0 37.0 41.8 63.8 194.6 127.7 167.7 195.9 137.0 128.6 128.4 133.0 128.4 128.6 17.5 33.9 24.3 87.6 72.2 25.9 25.0 27.2 121.9 133.6 26.0 18.1 22.8 120.7 134.0 25.7 18.1 33.3 74.4 73.2 25.9 24.3

206.6 74.9 45.4 32.7 40.8 64.1 194.5 129.9 166.2 211.4 41.3 21.7 20.9 17.0 33.4 24.0 88.4 72.8 26.1 25.7 30.4 75.5 73.2 26.9 23.5 23.0 122.3 133.7 25.6 18.2 29.7 119.8 134.4 26.2 18.2

74.8 171.9 120.3 191.1 63.0 41.0 44.4 48.0 209.2 26.9 93.7 71.3 26.6 25.1 208.3 41.0 21.0 20.7 32.9 74.6 73.1 25.3 24.6 28.0 122.1 134.0 26.0 18.0 12.7 39.3 24.5 124.4 132.5 25.8 18.1

on the HRESIMS and 13C NMR data. Their NMR data (Tables 1 and 2) were similar to those of compound 1. Analysis of the NMR data revealed that compounds 2 and 3 possessed identical 2D structures. The 2-methylpropanoyl group at C-2 in compound 1 was replaced by an unsubstituted benzoyl moiety in compounds 2 and 3 (2: δC 138.2, 128.3 × 2, 128.2 × 2, 132.2; 3: δC 136.9, 128.6 × 2, 127.9 × 2, 132.0), as indicated by the comparison of their 1D and 2D NMR data with those of compound 1. The configurations of the stereogenic centers of compounds 2 and 3 were consistent with compound 1 based on the C-4 chemical shift, the chemical shift difference between H-5α and H-5β, and analysis of the ROESY data indicating the correlation of H-5β/Me-17. The H-30β orientation in 2 was confirmed via the ROESY correlations between H-14 and H-30. Compound 3 was considered to be the C-30 epimer of 2 based on the slight difference observed in the H-30 chemical shift [δH 3.63 in 2; 3.81 in 3] and the comparison between the chemical shifts of H-30 in 3 and H-27 [δH 3.83] in 1. Therefore, the structures of compounds 2 and 3, uraliones B and C, were established as shown.

Uralione D (4) was obtained as a colorless oil. The HRESIMS ion at m/z 621.3791 [M + H]+ (calcd. for C38H53O7, 621.3786) and 13C NMR data indicated a molecular formula of C38H52O7. Compound 4 was structurally similar to compound 2 except for the C-34−C-38 side chain, as deduced by analysis of the NMR data. This deduction was supported by the presence of a secondary hydroxy group [δH 3.54 (m), δC 74.9], the absence of one set of olefinic carbon signals, and the HMBC correlations from H-34 (δH 2.45, m) to C-6, C-9, and C-35 and from H-35 (δH 3.54, m) to C-36. The ROESY correlations from H-30 (δ 3.57) to H-12 (δ 7.67) and H-34 (δ 2.09) indicated a β-orientation for H-30. The in situ [Rh2(OCOCF3)4] complex-induced electronic circular dichroism (ECD) spectrum of 4 exhibited the typical Cotton effects (Figure 2) for a 35S configuration.18,19 Thus, the structure of compound 4, uralione D, was assigned as shown. Compound 5 was isolated as a colorless oil. The HRESIMS ion at m/z 587.3727 [M + H]+ (calcd for C38H51O5, 587.3731) and 13C NMR data indicated that its molecular formula was C38H50O5. The 13C NMR data (Table 1) together with the HSQC spectra indicated the presence of three carbonyl groups, C



Article

Table 2. 1H NMR (500 MHz, CDCl3) Spectroscopic Data for Compounds 1−6 position

1

2

3

4 5

1.56 m 1.78 dd (13.5, 4.5) 1.42 t (13.5)

1.68 m 1.93 dd (13.0, 4.5) 1.47 t (13.0)

1.85 dd (13.0, 4.5) 2.00 m 1.45 t (13.0)

1.83 m 1.99 dd (13.5, 4.0) 1.44 t (13.5)

1.71 m 2.43 dd (14.0, 4.5) 1.58 t (14.0)

7.53 7.28 7.42 7.28

7.47 7.24 7.37 7.24

7.67 7.29 7.41 7.29

7.45 7.22 7.37 7.22

4

5

6 1.68 1.94 1.43 3.02 2.84 4.12

10 11 12 13 14 15 16 17

2.48 1.15 1.12 1.08 1.99 1.24 2.21 1.99 5.02

m d (6.5) d (6.5) s m td (13.5, 2.5) m m m

18 19

1.66 s

20 21 22

1.53 2.12 1.70 4.92

23 24

1.66 s

25 26 27 28 29

1.61 2.71 2.51 3.83

s dd (14.5, 5.5) m t (7.0)

s dd (17.5, 4.5) dd (17.5, 4.5) t (4.0)

1.30 s

30 31 32 33 34

1.44 s 2.45 m 5.05 m

35 37 38

1.66 s

1.67 s

d (7.5) t (7.5) t (7.5) t (7.5)

d (8.0) t (8.0) t (8.0) t (8.0)

d (7.5) t (7.5) t (7.5) t (7.5)

d (7.5) t (7.5) t (7.5) t (7.5)

7.53 d (7.5)

7.47 d (8.0)

7.67 d (7.5)

7.45 d (7.5)

1.22 s

1.13 s

1.22 s

1.18 s

2.03 1.47 2.27 2.03 5.05

2.03 1.66 2.17 1.94 5.08

2.10 m 1.40 t (13.0) 2.26 m 2.01 m 5.03 t (6.5)

2.21 1.39 2.29 2.00 5.10

dd (13.0, 4.5) t (13.0) m dd (13.0, 4.5) m

dd (13.5, 5.5) m m m t (7.0)

1.65 s

1.66 s

1.65 s

1.64 s 2.14 dd (14.0, 4.0)

1.61 s 2.14 m 1.79 dd (13.0, 4.5)

1.63 s 2.18 dd (13.0, 3.5)

1.68 s 2.20 m

5.00 m

1.74 m 4.95 t (7.0)

1.81 m 4.99 t (6.5)

1.70 1.58 2.68 2.39

1.68 1.54 2.59 2.26

1.65 1.57 3.12 3.06

1.77 m 4.94 t (7.0)

1.68 s 1.55 s 2.74 dd (17.0, 5.0) 2.39 dd (17.0, 5.0) 3.63 m 0.66 s 1.20 s 2.53 m 5.05 m 1.68 s 1.68 s

s s dd (17.0, 5.0) dd (17.0, 5.0)

s s t (4.0) m

3.57 m

5.12 m

1.33 1.42 2.54 2.45 5.00 1.66 1.66

1.19 0.52 2.09 1.67 3.54 1.24 1.21

1.65 1.63 3.04 1.84 4.43 1.46 1.25

s s m m m s s

2.06 1.49 2.33 2.07 5.03

m dd (13.0, 3.5) m m t (6.5)

1.66 s 1.63 s 2.13 dd (13.5, 5.0) 1.75 m 4.95 t (6.0)

1.66 s 1.55 s

s s dd (14.0, 7.5) dd (14.0, 7.5)

3.81 t (6.0) s s dd (14.5, 6.0) dd (14.5, 6.0) m s s

1.13 s 1.10 s 1.22 s

m m m m m

1.68 s

m dd (13.0, 4.0) t (13.0) dd (15.0, 10.0) dd (15.0, 10.0) t (10.0)

7.56 d (7.5)

s s dd (13.5, 10.0) dd (13.5, 5.5) dd (10.0, 5.0) s s

7.30 7.43 7.30 7.56 2.55 2.46 5.03 1.66 1.66

t (7.5) t (7.5) t (7.5) d (7.5) dd (14.5, 6.5) dd (14.5, 6.5) t (6.5) s s

derivatives bearing a benzoyl group, structurally similar to the known hyperibone I.6b Comparison of the 13C NMR data of 6 and 7 with those of hyperibone I revealed that they differed from hyperibone I at the C-16−C-21 substituent. This was corroborated by the HMBC correlations from H-15 (δ 1.26, s) to C-3; from H-16 (δ 2.06, m; 1.49, dd, 13.0, 3.5) to C-3, C-15, C-17, and C-18; and from H-18 (δ 5.00, t, 8.0) to C-20 and C-21 in 6, as well as those from H-15 (δ 1.22, s) to C-3; from H-16 (δ 1.83, m; 1.68 m) to C-3, C-15, C-17, and C-18; and from H-18 (δ 5.03, t, 6.5) to C-20 and C-21 in 7. The α-orientation of H-11 in 6 was deduced from the ROESY correlation (Figure 3) between H-11 and H-16. In contrast, 7 was considered to be a C-11 epimer of 6 based on the difference in the chemical shifts of H-11 (δ 4.12

an unsubstituted benzoyl moiety, nine methyls, and a vicinal dioxygenated moiety. Analyses of 1D and 2D NMR data revealed that 5 was a C-35 epimer of uralodin A,20 which was supported by the ROESY correlation between Me-37 (δ 1.25) and H-5α (δ 2.43). Therefore, the structure of 5 (uralione E) was determined as 35-epiuralodin A. Compounds 6 and 7 possessed the same molecular formula of C38H50O5 based on their HRESIMS data. The 1D NMR spectra were similar, and each exhibited the presence of a benzoyl, a 4-methyl-3-pentenyl, two isoprenyl, and two unconjugated carbonyl groups, as well as an enolized 1,3dicarbonyl ether system (Tables 1−3). From the above data, 6 and 7 are assumed to be polyprenylated acylphloroglucinol D



Article

Figure 1. ORTEP drawing and selected key HMBC and ROESY correlations of compound 1.

Figure 2. ECD spectra of the [Rh2(OCOCF3)4] complexes of compounds 4 (a) and 10 (b) with the intrinsic ECD spectrum subtracted.

the resonances of the C-34−C-38 side chain. The isoprenyl residue at C-6 of 8 was replaced by a 3-methyl-2,3-pentanediol moiety in 9, as revealed by comparison of the NMR data between 8 and 9. The β-orientation of H-20 in 9 was based on the ROESY correlations between H-20 and H-18β, H-12, and H-16. The in situ [Rh2(OCOCF3)4] complex-induced ECD spectrum of 9 exhibited a positive E band, reminiscent of a 35S configuration.18,19 The aforementioned analyses led to assignment of the structure of 9 (uralione I) as 35-hydroxybenzoyloxepahyperforin. Compound 10 possessed a molecular formula of C35H54O7 based on the ion at m/z 587.3947 [M + H]+ in the HRESIMS (calcd for C35H55O7, 587.3942) and 13C NMR data. The NMR data of 10 (Tables 1 and 3) suggested that 10 and oxepahyperforin22 shared the same core structure and differed only at the C-4 substituent. H-17 was assigned a β-orientation based on the ROESY correlation between H-17 (δ 3.81) and Me-12 (δ 1.10). The in situ Rh2(OCOCF3)4 complex-induced ECD spectrum (Figure 2) was used to establish the 22S absolute configuration.18 Thus, the structure of 10 (uralione J) was determined as 22-hydroxybenzoyloxepahyperforin. Compound 11 was obtained as a colorless oil, and its molecular formula was C35H54O7 according to the m/z 587.3945 [M + H]+ ion in the HRESIMS data (calcd for C35H55O7, 587.3942) and 13C NMR data. The spectroscopic data suggested that 11 was structurally analogous to attenuatumione F,15a the only differences invoving the C-8 substituent. H-11 was assigned a β-orientation based on the ROESY correlation between H-30 (δ 1.84) and Me-13 (δ 1.84). The absolute configuration of 11 was deduced from the in situ Rh2(OCOCF3)4 complex-induced ECD spectrum.18

in 6; 4.60 in 7). The ECD spectra of 6 and 7 were similar to that of guttiferone M,21 indicative of their similar absolute configurations. Hence, the structures of compounds 6 and 7, uraliones F and G (11-epiuralione E), respectively, were elucidated. Compound 8 was obtained as a colorless oil. The HRESIMS ion at m/z 587.3734 [M + H]+ (calcd for C38H51O5, 587.3731) and 13C NMR data indicated a molecular formula of C38H50O5. The 1H NMR signals at δ 7.59 (2H, d, 7.5), 7.44 (1H, t, 7.5), and 7.28 (2H, t, 7.5) and the 13C NMR resonances at δ 137.3, 132.7, 128.7 (×2), and 128.1 (×2) suggested the presence of an unsubstituted benzoyl moiety. The 13C NMR spectrum of compound 8 also exhibited signals of two unconjugated carbonyls (δ 205.6 and 196.6) and one enolized 1,3-dicarbonyl ether moiety (δ 194.4, 127.5, and 167.2). Notably, the characteristic 13C NMR signals of an oxepane ring (δ 167.2, 87.4, 46.4, 33.8, and 24.3), which has only been reported in oxepahyperforin,22 were observed. Comparison of the NMR data of 8 with those of oxepahyperforin revealed the presence of an oxepane ring with a hydroxyisopropyl unit between C-3 and C-9 in 8. The ROESY correlation (Figure 3) of the oxymethine proton H-20 and H-18β and of H-20 with H-12 and H-16 indicated that the hydroxyisopropyl unit was αoriented. Its ECD spectrum closely resembled those of 2 and 3, suggesting the same absolute configurations for the core structure. On the basis of the above analyses, the structure of 8 (uralione H) was established as benzoyloxepahyperforin. A sodium adduct ion at m/z 643.3607 [M + Na]+ was observed in the HRESIMS data of compound 9 and, together with 13C NMR data, indicated a molecular formula of C38H52O7. The NMR data of 8 and 9 were similar except for E



Article

Table 3. 1H NMR (500 MHz, CDCl3) Spectroscopic Data for Compounds 7−11 position

7

8

4 5

1.80 m 1.99 dd (13.5, 4.0) 1.45 t (13.5)

2.09 m 1.90 dd (13.5, 5.0) 1.64 m

9 2.14 m 2.11 m 1.61 m

10

6 7 10 11 12 13 14 15 16

2.95 dd (10.5, 5.5) 4.60 t (10.0)

18

0.88 0.92 1.26 1.85 1.68 2.10 1.95 5.00

20 21

1.70 s 1.57 s

22

2.10 dd (13.0, 5.5) 1.78 dd (13.0, 5.5) 5.00 t (8.0)

17

23 24 25 26

s s s dd (15.0, 4.0) m dd (13.0, 5.5) dd (13.0, 4.0) t (8.0)

1.66 s 1.58 s

27 28 29

7.56 d (8.0)

30

7.35 t (8.0)

31

7.49 t (8.0)

32 33 34

7.35 7.56 2.58 2.49 5.05 1.68 1.70

35 37 38

t (8.0) d (8.0) dd (14.0, 6.5) dd (14.0, 7.5) t (7.5) s s

7.59 7.28 7.44 7.28 7.59

d (7.5) t (7.5) t (7.5) t (7.5) d (7.5)

7.68 7.31 7.46 7.31 7.68

d (7.5) t (7.5) t (7.5) t (7.5) d (7.5)

1.26 s

1.27 s

2.38 td (15.5, 2.0) 2.00 dd (15.5, 4.0) 3.72 d (9.0)

2.40 td (16.0, 2.0) 2.01 m 3.73 d (9.0)

0.95 s

0.95 s

0.93 2.09 1.67 4.96

s m m t (7.0)

0.93 2.09 1.67 4.97

s m m t (6.5)

1.68 1.56 3.18 3.04 4.87

s s dd (14.5, 7.0) dd (14.5, 7.0) t (7.0)

1.71 1.57 3.17 3.08 4.86

s s dd (15.0, 6.5) dd (15.0, 6.5) t (6.5)

2.51 1.16 1.10 0.97 2.47 1.76

m d (6.5) d (6.5) s m 1.88 dd (15.5, 5.5) dd (15.5, 5.5)

3.82 d (9.0)

5.15 t (7.0) 1.68 s 1.67 s

1.65 1.69 2.12 2.00 3.54 1.24 1.22

s s m m m s s

According to the bulkiness rule, the positive E band identified from the Rh2(OCOCF3)4 complex of 11 indicated the 20S configuration. Thus, the structure of compound 11 (uralione K) was defined as 20-hydroxyattenuatumione F. Five known compounds, namely, uralodin B,17 uralodin C,17 uralodin A,20 furohyperforin,23 and attenuatumione E,15a were identified by comparison of their spectroscopic data with reported data. Considering that PPAPs have been reported to possess antidepressant-like activities,1 the protective effects of all isolates on corticosterone-induced PC12 cell injuries were examined via the MTT method.24 As shown in Table 4, except for 9, all tested compounds exhibited significant protective

2.00 1.33 1.60 3.09 2.92 4.67

dd (13.5, 4.0) t (13.5) m dd (15.0, 10.0) dd (15.0, 10.0) t (10.0)

1.34 s 1.25 s 2.43 m 1.16 d (6.5) 1.07 d (6.5)

1.21 1.44 0.90 3.25

s t (12.0) m d (10.0)

1.26 s 1.12 3.26 3.17 5.03

s dd (15.0, 6.0) dd (15.0, 6.0) m

1.68 s 1.66 s

3.41 m

1.21 s 1.22 2.10 1.45 4.92

s m td (12.0, 4.0) t (6.5)

1.67 s 1.54 s 1.09 s

5.05 m

1.84 1.45 2.20 2.00 5.00

1.68 s

1.66 s

1.70 s

1.59 s

2.48 m 1.63 s 1.64 s 2.56 d (7.0)

11

2.37 m 1.95 dd (13.5, 5.0) 1.50 t (13.5)

m td (12.0, 4.0) m dd (13.5, 4.0) t (6.5)

effects against induced injury in PC12 cells within the concentration range tested (0.1−10.0 μM). Uralodin A (14), the most abundant compound among the isolates, exhibited the highest cell viability (>85%) under induced injury in PC12 cells and more potent protective activity than the positive control fluoxetine. Therefore, the antidepressant-like activity of uralodin A was further evaluated in a mouse tail suspension test (TST) and a forced swimming test (FST) as shown in Figure 4. In the FST test, the depressive mice induced by LPS exhibited a significant increase in immobility duration compared with the control animals (p < 0.001), with a percentage of 92.2%. Fluoxetine (25 mg/kg, p < 0.001) and uralodin A (14) at the test doses (13 and 26 mg/ kg) reduced the immobility time compared with the vehicle F



Article

fluoxetine (25 mg/kg) and uralodin A (14) at the test doses (13 and 26 mg/kg) shortened the immobility time by 23.1%, 6.6%, and 12.6%, respectively. These observations indicated that H. uralum is a source of PPAPs with potential antidepressant-like activity, which is related to the protective effects on neural cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. A UV−visible spectrophotometer (UV-2450, Shimadzu, Tokyo, Japan) was used to obtain UV spectra. Infrared spectra were acquired using a Tensor 27 infrared spectrometer (Bruker, Karlsruhe, Germany, KBr pellets). Optical rotations were determined using a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan). A JASCO 810 spectropolarimeter (Jasco, Tokyo, Japan) was used to record ECD spectra. 1D and 2D NMR spectra were recorded on a Bruker AV-500 spectrometer using tetramethylsilane as an internal standard. ESIMS data were obtained using an Agilent 1100 series LC/MSD Trap mass, and HRESIMS data were acquired using an Agilent 6529B Q-TOF spectrometer. Silica gel (200−400 mesh, Qingdao Marine Chemical Co. Ltd., Qindao, China), Fuji RP-C18 silica (40−63 μm, Tokyo, Japan), and Pharmacia Sephadex LH-20 (Uppsala, Sweden) columns were employed for column chromatography. An Agilent 1260 Series coupled with an RP-C18 column (20 × 200 mm i.d., Shim-pack, Shimadzu, Tokyo, Japan) and an Agilent 1100 Series (Agilent Technologies, Santa Clara, CA, USA) multiple wavelength detector were used to perform preparative HPLC. Plant Material. Whole plants of Hypericum uralum Buch.-Ham. ex D. Don were collected in Yunnan Province, People’s Republic of China, in June 2013 and were authenticated by Prof. Mian Zhang, School of Traditional Chinese Pharmacy, China Pharmaceutical University. Voucher specimens numbered 2013-SEJST were deposited at the State Key Laboratory of Natural Medicines, China Pharmaceutical University. Extraction and Isolation. Air-dried plant material powder (12 kg) was extracted using 95% aqueous EtOH (3 × 30 L) under reflux. A crude extract (1604 g) was obtained after removing the solvent under reduced pressure. The crude extract was suspended in distilled H2O (3 L) and successively extracted using petroleum ether (PE, 3 × 3 L) and

Figure 3. Selected key HMBC and ROESY correlations of compounds 6 (a) and 8 (b).

group, with percentages of 33.7%, 21.4%, and 28.1%, respectively. In the TST test, LPS-induced depressive mice exhibited a significantly prolonged immobility time compared to the control by 68.1%. Compared with the vehicle group,

Table 4. Protection Rates of the Target Compounds on Corticosterone-Injured PC12 Cellsa viability (%) control model fluoxetine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

10.0 μM

3.0 μM

1.0 μM

100 55.6 ± 0.87### 69.6 ± 0.75* 75.1 ± 0.43** 65.9 ± 0.68 68.9 ± 0.15 80.9 ± 0.32*** 86.6 ± 0.36*** 74.1 ± 0.72*** 68.1 ± 0.44 84.2 ± 0.24*** 53.2 ± 1.43 72.9 ± 0.43** 71.5 ± 0.75* 75.9 ± 0.68** 57.0 ± 0.77 85.3 ± 0.58*** 85.2 ± 0.28*** 61.0 ± 0.97

59.1 ± 0.12 91.5 ± 0.39*** 69.0 ± 1.03 82.2 ± 1.23*** 72.5 ± 0.43** 78.0 ± 0.28*** 81.1 ± 0.38*** 77.7 ± 0.49*** 81.6 ± 0.20*** 59.7 ± 0.54 90.7 ± 0.19*** 76.5 ± 0.23*** 72.9 ± 0.79* 74.4 ± 0.35** 90.2 ± 0.23*** 77.7 ± 0.06*** 63.8 ± 0.23

70.1 ± 0.52* 81.8 ± 0.30*** 77.6 ± 0.97*** 86.3 ± 0.38*** 78.5 ± 0.05*** 68.1 ± 0.39 76.6 ± 0.33*** 78.5 ± 0.49*** 83.5 ± 0.01*** 65.0 ± 0.25 93.5 ± 0.14*** 81.1 ± 0.12*** 74.7 ± 0.19** 72.2 ± 0.43* 85.9 ± 0.49*** 79.7 ± 0.47*** 77.0 ± 0.35***

0.3 μM

66.2 61.4 80.8 86.8 77.7 72.8 81.8 72.9 82.7 57.5 84.4 80.0 56.6 77.7 87.1 79.9 68.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.20 1.07 0.17*** 0.20*** 0.29*** 0.50* 0.40*** 0.26** 0.18*** 0.50 0.66*** 0.05*** 0.11 0.27** 0.08*** 0.35*** 0.17

0.1 μM

77.7 ± 0.31** 80.3 ± 0.54*** 70.0 ± 0.28 78.6 ± 0.18*** 77.9 ± 0.13*** 82.9 ± 0.30*** 76.7 ± 0.24*** 78.6 ± 0.29*** 83.4 ± 0.28*** 55.9 ± 0.70 91.7 ± 0.17*** 72.3 ± 0.31* 55.6 ± 0.68 69.9 ± 0.36 85.7 ± 0.48*** 77.2 ± 0.29*** 80.9 ± 0.11***

The cells were stabilized in the presence of corticosterone (200 μM) for 48 h at 37 °C. The cells were cultured for an additional 48 h while being incubated with the corresponding drugs in serum-free medium. *p < 0.05, **p < 0.01, ***p < 0.001 vs model; ###p < 0.001 vs control.

a

G



Article

fractions (CIa−CIe). Fraction CIe (1.0 g) was purified by passage over a Sephadex LH-20 column (CHCl3−MeOH, 1:1) and further purified using preparative HPLC (CH3CN−H2O, 70:30) to afford 9 (4.4 mg) and 16 (6.2 mg). A silica gel column (200−300 mesh; 300.0 g; ⦶ 4.5 × 40.0 cm2; PE−acetone, 3:1 to 0:1) was employed to purify fraction D (52 g), which provided three fractions (DI−DIII). Fraction DI (14 g) was fractionated by MPLC (ODS RP-C18 column; 40−63 μm; 40.0 g; ⦶ 2.0 cm × 40.0 cm2; 70%, 90%, and 100% MeOH) to afford three fractions (DIa−DIc). Fraction DIb (4.9 g) was reseparated using a silica gel column (200−300 mesh; 80.0 g; ⦶ 2.0 × 30.0 cm2; PE− acetone, 5:1 to 0:1) to produce seven fractions (DIb1−DIb7). Fraction DIb5 (2.4 g) was subjected to MPLC (75% MeOH; ODS RP-C18 column; 40−63 μm; 40.0 g; ⦶ 2.0 cm × 40.0 cm2) and then to preparative HPLC (CH3CN−H2O, 70:30) to afford 4 (10.4 mg), 10 (1.7 mg), and 11 (10.0 mg). Uralione A (1): colorless crystals (MeOH−H2O, 80:20); mp 117− 118 °C; [α]25 D +49 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 205 (4.31), 272 (3.82) nm; ECD (MeOH) λmax nm (Δε) 330 (+3.4), 299 (−41.4), 271 (+96.9); IR (KBr) νmax cm−1 3396, 2964, 2926, 1728, 1640, 1597, 1446, 1390, 1267, 1221, 1130, 1087, 931, 916, 853, 770, 585; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/z 575.3706 [M + Na]+ (calcd for C35H52NaO5, 575.3707). Uralione B (2): colorless oil; [α]25 D −28 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 204 (4.25), 248 (3.94), 272 (3.87) nm; ECD (MeOH) λmax nm (Δε) 383 (+16.1), 313 (−6.8), 288 (+1.1), 268 (−20.0), 248 (+82.7), 217 (−68.3), 204 (−36.6); IR (KBr) νmax cm−1 3442, 2968, 2923, 2854, 1722, 1699, 1653, 1612, 1446, 1384, 1269, 1221, 1125, 951, 829, 770, 728, 689; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/z 587.3729 [M + H]+ (calcd for C38H51O5, 587.3731). Uralione C (3): colorless oil; [α]25 D −41 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 204 (4.26), 247 (3.94), 277 (3.89) nm; ECD (MeOH) λmax nm (Δε) 380 (+13.3), 315 (−7.3), 302 (−3.5), 273 (−52.8), 250 (+39.3), 225 (−35.3); IR (KBr) νmax cm−1 3442, 2965, 2923, 2854, 1723, 1699, 1608, 1448, 1385, 1222, 1125, 1075, 868, 840, 799, 769, 688; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/ z 587.3736 [M + H]+ (calcd for C38H51O5, 587.3731). Uralione D (4): colorless oil; [α]25 D −28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.19), 248 (3.96), 274 (3.87) nm; ECD (MeOH) λmax nm (Δε) 371 (+8.4), 348 (+1.7), 336 (+2.7), 319 (−1.4), 314 (−0.7), 268 (−34.3); IR (KBr) νmax cm−1 3566, 3417, 2972, 2931, 1721, 1694, 1643, 1604, 1446, 1386, 1273, 1224, 1188, 1123, 1091, 1047, 1025, 995, 945, 835, 784, 742, 694, 670, 607; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/z 621.3791 [M + Na]+ (calcd for C38H53O7, 621.3786). Uralione E (5): colorless oil; [α]25 D −30 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 204 (4.25), 247 (4.00), 275 (3.91) nm; ECD (MeOH) λmax nm (Δε) 377 (+9.3), 317 (−9.9), 298 (−1.7), 270 (−74.3), 245 (+32.9), 223 (+14.2); IR (KBr) νmax cm−1 3441, 2972, 2925, 1726, 1694, 1623, 1448, 1379, 1225, 1177, 855, 801, 766, 689, 666; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/z 587.3727 [M + H]+ (calcd for C38H51O5, 587.3731). Uralione F (6): colorless oil; [α]25 D −32 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 204 (4.73), 249 (4.42), 281 (4.35) nm; ECD (MeOH) λmax nm (Δε) 374 (+4.4), 316 (−17.4), 291 (+12.3), 272 (−1.9), 252 (+55.9), 219 (−41.7), 206 (−12.7); IR (KBr) νmax cm−1 3433, 2971, 2926, 1724, 1700, 1619, 1446, 1384, 1224, 1186, 1123, 961, 846, 768, 690, 670; 13C and 1H NMR data (Tables 1 and 2); HRESIMS m/z 587.3732 [M + H]+ (calcd for C38H51O5, 587.3731). Uralione G (7): colorless oil; [α]25 D +17 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 204 (4.24), 250 (3.94), 280 (3.83) nm; ECD (MeOH) λmax nm (Δε) 319 (−16.1), 286 (+36.4), 269 (+16.0), 252 (+46.3), 216 (−33.2); IR (KBr) νmax cm−1 3420, 2971, 2853, 1725, 1699, 1625, 1447, 1400, 1236, 960, 846, 691; 13C and 1H NMR (see Tables 1 and 3); HRESIMS m/z 587.3732 [M + H]+ (calcd. for C38H51O5, 587.3731). Uralione H (8): colorless oil; [α]25 D −210 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 204 (4.21), 250 (3.93), 278 (3.82) nm; ECD (MeOH) λmax nm (Δε) 375 (+1.5), 319 (−27.4), 290 (−6.1), 267 (−21.6), 246 (+12.7), 224 (−34.9), 206 (+13.9); IR (KBr) νmax cm−1

Figure 4. Effects of uralodin A (14) on the immobility time in the FST (A) and TST (B) on LPS-induced mice (60 in total). Behavioral tests were performed 24 h after the injection of LPS. The immobility duration was determined during the last 4 min of the FST. The results are presented as the means ± standard deviation (SD) (n = 12). Significantly different values were detected through one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. ###p < 0.001 compared to the normal control group; ***p < 0.001 compared to the vehicle group. CH2Cl2 (3 × 3 L). The PE-soluble fraction (247 g) was subjected to a column of silica gel (100−200 mesh; 1500.0 g; ⦶ 15.0 × 80.0 cm2), and PE−EtOAc gradient elution (1:0 to 0:1) was used to afford five fractions (A−E). Fraction B (63 g) was subjected to MPLC (70% and 90% MeOH, acetone) on an ODS column (40−63 μm; 120.0 g; ⦶ 4.0 cm × 40.0 cm2) to afford three fractions (BI−BIII). Silica gel column chromatography (200−300 mesh; 300.0 g; ⦶ 4.5 × 40.0 cm2) of BII (26 g) with PE−acetone (50:1, 25:1, 10:1, and 0:1) yielded four fractions (BIIa−BIId). Fraction BIIc (4.5 g) was further chromatographed on a silica gel column (200−300 mesh; 100.0 g; ⦶ 3.0 × 30.0 cm2; PE−acetone, 15:1) to afford two fractions (BIIcA and BIIcB). Fraction BIIcA (2.9 g) was further separated by MPLC (ODS column; 40−63 μm; 40.0 g; ⦶ 2.0 cm × 40.0 cm2; 85% MeOH) and preparative HPLC eluting with CH3CN−H2O (80:20) to afford 1 (2.5 mg) and 7 (2.9 mg) and with MeOH−H2O (85:15) to obtain 5 (9.5 mg), 6 (19.8 mg), 8 (6.5 mg), and 15 (40.0 mg). Fraction BIIcB (1.6 g) was isolated by passage over a Sephadex LH-20 column (CHCl3− MeOH, 1:1) and further purified by preparative HPLC eluting with MeOH−H2O (90:10) to afford 12 (36.0 mg) and 13 (70.0 mg) and with CH3CN−H2O (80:20) to obtain 2 (2.8 mg), 3 (1.9 mg), and 14 (80.0 mg). Fraction C (21 g) was separated by MPLC (ODS RP-C18 column; 40−63 μm; 120.0 g; ⦶ 4.0 cm × 40.0 cm2; 70% and 90% MeOH, acetone) to yield three fractions (CI−CIII). Fraction CI (6.2 g) was separated using a column of silica gel (200−300 mesh; 80.0 g; ⦶ 3.0 × 30.0 cm2) eluting with PE−acetone (8:1) to produce five H



Article

culture plates (2 × 104 cells/well, in triplicate) for 24 h and then exposed to medium containing corticosterone (200 μM) in the presence or absence of various concentrations of tested samples for an additional 48 h. Cell viability was examined using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, at the final step of each treatment, phosphate-buffered saline (PBS) was employed to gently wash the cells. After washing, 10 μL of MTT (5 mg/mL) was added to each well. After being maintained at 37 °C for 4 h and removing the culture medium, 100 μL of DMSO was added to each well to dissolve the formazan crystals generated by the reaction. A SpectraMax Plus 384 Universal microplate reader (Molecular Devices, Sunnyvale, CA, USA) was utilized to measure the absorbance at 570 nm. Cell viability was indicated in the form of a percentage of the control. Test Animals. Behavioral experiments were conducted using 60 adult male ICR mice (18−22 g) acquired from the Experimental Animal Center in Jiangsu Province (Nanjing, People’s Republic of China). The test animals were randomly housed and reared in a room with a 12/12 h light/dark cycle (lights on at 7 A.M.) at 25 ± 1 °C and had free access to filtered water and a standard diet. The experimental procedures used in this study were authorized by the Science and Technology Department of Jiangsu Province and were performed according to the Provisions and General Recommendations of the Chinese Experimental Animals Administration Legislation. Treatments. Fluoxetine hydrochloride, purchased from Changzhou Siyao Pharmaceuticals Co. Ltd., and uralodin A (14) were suspended in sodium carboxymethyl cellulose (0.03%, CMC-Na). Sixty animals were divided into five groups (n = 12) randomly: (1) control group, (2) vehicle group: LPS (i.p., 0.5 mg/kg), (3) LPS (i.p., 0.5 mg/kg) + fluoxetine (i.g., 25 mg/kg) group, (4) LPS (i.p., 0.5 mg/ kg) + uralodin A (14) (i.g., 13 mg/kg) group, and (5) LPS (i.p., 0.5 mg/kg) + uralodin A (14) (i.g., 26 mg/kg) group. All drugs were given to animals via intragastric administration once daily for 5 days. On the fifth day, LPS (0.5 mg/kg) was intraperitoneally injected 30 min after the drug administration. Twenty-four hours after LPS injection, behavioral tests were performed. The doses of uralodin A (14) used in the tail suspension test and forced-swimming test were 13 and 26 mg/ kg. The vehicle (0.9% physiological saline, 10 mL/kg) and fluoxetine hydrochloride (25 mg/kg) were used as negative and positive controls, respectively. Forced-Swimming Test. This test was implemented in accordance with the reported methodology27 with minor modifications. In brief, individual mice were forced to swim in one open cylindrical container (height = 20 cm, diameter = 14 cm) containing H2O (depth = 10 cm) at 25 ± 1 °C. The duration of swimming for each animal was 6 min, and the total duration of immobility was examined in the last 4 min. Each animal was regarded as being immobile when it floated motionless or when it only performed necessary movements to keep its head above H2O. Tail Suspension Test. Behavioral despair was induced using the TST protocol as stated in a previous report.28 In brief, being both visually and acoustically isolated, mice were suspended at a height of 50 cm above the floor using adhesive tape attached approximately 1 cm from the tip of the tail. Each mouse was suspended and observed for 6 min, and the total immobility duration was measured during the last 4 min period. The mice were considered immobile only if they hung passively and completely motionless. Statistical Analysis. The data in the figures and table are presented as the mean ± standard deviation of three replicate measurements. Comparisons between the control and experimental groups were conducted via one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. A difference value of p < 0.05 was considered to be statistically significant.

3432, 2971, 2927, 1723, 1693, 1653, 1598, 1447, 1379, 1255, 1218, 1185, 983, 841, 769, 688, 671; 13C and 1H NMR (Tables 1 and 3); HRESIMS m/z 587.3734 [M + H]+ (calcd for C38H51O5, 587.3731). Uralione I (9): colorless oil; [α]25 D −139 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.07), 250 (3.98) nm; ECD (MeOH) λmax nm (Δε) 379 (+2.7), 318 (−54.0), 285 (−14.3), 266 (−38.0), 248 (+8.7), 225 (−71.0); IR (KBr) νmax cm−1 3428, 2974, 2930, 1720, 1691, 1650, 1598, 1448, 1383, 1260, 1220, 1171, 1096, 1022, 983, 840, 757, 690, 670, 602; 13C and 1H NMR (Tables 1 and 3); HRESIMS m/ z 643.3607 [M + Na]+ (calcd for C38H52NaO7, 643.3605). Uralione J (10): colorless oil; [α]25 D −48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.21), 276 (4.09) nm; ECD (MeOH) λmax nm (Δε) 385 (+25.9), 377 (+23.9), 373 (+25.3), 307 (−151.7), 274 (+144.8); IR (KBr) νmax cm−1 3442, 2970, 2927, 2855, 1725, 1654, 1600, 1448, 1380, 1248, 1163, 1098, 985, 833, 803, 757, 686, 589; 13C and 1H NMR (Tables 1 and 3); HRESIMS m/z 587.3947 [M + H]+ (calcd for C35H55O7, 587.3942). Uralione K (11): colorless oil; [α]25 D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.38), 280 (4.31) nm; ECD (MeOH) λmax nm (Δε) 395 (−12.3), 363 (−5.7), 357 (−7.9), 336 (+0.3), 305 (−121.3), 277 (+165.5); IR (KBr) νmax cm−1 3440, 2974, 2930, 1731, 1619, 1446, 1408, 1381, 1245, 1156, 1082, 961, 895, 846, 756, 681, 666, 631, 598; 13C and 1H NMR (Tables 1 and 3); HRESIMS m/z 587.3945 [M + H]+ (calcd for C35H55O7, 587.3942). X-ray Crystallographic Analysis. Colorless crystals of compound 1 were obtained from MeOH−H2O. A Bruker Smart-1000 CCD and a graphite monochromator (Cu Kα radiation, λ = 1.541 84 Å) were used to obtain the crystallographic data at 293(2) K. Using Olex2,25 the structure was determined by the ShelXS26 structure solution program using direct methods then refined through the ShelXL26 refinement package with least-squares minimization. Crystallographic data of 1: C35H52O5 (M = 552.76); orthorhombic crystal (0.37 × 0.32 × 0.24 mm); space group P212121; unit cell dimensions a = 10.5497(2) Å, b = 12.5170(2) Å, c = 25.8998(4) Å, V = 3420.08(10) Å3; Z = 4; ρcalcd = 1.074 mg/mm3; μ = 0.550 mm−1; 18 414 reflections measured (6.826 ≤ 2θ ≤ 139.302); 6286 unique (Rint = 0.0218), which were used for all calculations; the final refinement produced R1 = 0.0636 (>2σ(I)) and wR2 = 0.1876 (all data); and Flack parameter = −0.09(7). The crystallographic data of 1 were deposited at the Cambridge Crystallographic Data Centre under deposition number CCDC 917910. Copies of the crystallographic data can be obtained for free from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK [e-mail: [email protected] or fax: (+44) 1223-336-033] or by www.ccdc.cam.ac.uk/conts/retrieving. html. Absolute Configuration at C-35 in 4 and 9, at C-22 in 10, and at C-20 in 11. One milligram of Rh2(OCOCF3)4 was dissolved in CH2Cl2 (400 μL). After mixing with samples of compounds 4, 9, 10, and 11 (0.45 mg each), the first ECD spectrum was recorded immediately, and the time evolution was monitored until stationary (approximately 10 min). The intrinsic ECD spectrum was subtracted. The absolute configuration of all the vicinal deoxygenated moieties in the test compounds could be determined by the E band observed at approximately 350 nm in the ECD spectrum.18 Neuroprotective Assay. The isolates were dissolved in DMSO at a stock concentration of 200 mM. The final concentration of DMSO in the culture medium never exceeded 0.1% (v/v), and the same concentration was used in control experiments. PC12 cells were obtained from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). These cells were maintained in DMEM (GIBCO Invitrogen Corp., Carlsbad, CA, USA) supplemented with streptomycin (100 μg/mL), penicillin (100 units/mL), 5% heat-inactivated horse serum, and 5% fetal bovine serum (Sijiqing, Hangzhou, China) in 5% CO2 at 37 °C. Cells in the exponential phase of growth were used in all experiments. The experimental design included the following groups: corticosterone (200 μM), corticosterone (200 μM) plus fluoxetine (10.0, 3.0, 1.0, 0.3, and 0.1 μM), corticosterone (200 μM) plus the test compounds (10.0, 3.0, 1.0, 0.3, and 0.1 μM), and nontreated control. Unless specified otherwise, the cell suspensions were seeded in every well of 96-well

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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.5b00667. I



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HRESIMS spectra and 1D NMR and 2D NMR of 1−11 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-8327-1402. E-mail: [email protected] (J.-G. Luo). *Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (L.-Y. Kong). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the National Key Scientific and Technological Special Projects (2012ZX09103-101-007).

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