Neuroprotective Lignans from the Fruits of ... - ACS Publications

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Neuroprotective Lignans from the Fruits of Schisandra bicolor var. tuberculata Ye Liu,† Heng-Yi Yu,‡ Yan-Mei Wang,† Tian Tian,† Wen-Ming Wu,† Ming Zhou,† Xiang-Gao Meng,§ and Han-Li Ruan*,† †

School of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Department of Pharmacy, Tongji Hospital Affiliated Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China § College of Chemistry, Central China Normal University, Wuhan 430079, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new lignans (1−9) and ten known analogues (10−19) were isolated from the fruits of Schisandra bicolor var. tuberculata. The structures of compounds 1−9 were established on the basis of spectroscopic data analysis. The absolute configuration of compound 1 was determined by single-crystal X-ray diffraction analysis with Cu Kα irradiation techniques, and the absolute configurations of compounds 2−9 were deduced by comparing their experimental ECD spectra and optical rotations with those of compound 1 or similar compounds. All isolates were evaluated for their neuroprotective activities against CoCl2, H2O2, and Aβ25−35-induced SH-SY5Y cell injury, and were found to exhibit different degrees of neuroprotective effects. At a low concentration of 3.2 nM, compounds 3, 8, 9, and 14−19 in CoCl2-induced, compounds 7, 8, 13, 17, and 18 in H2O2-induced, and compounds 2, 6, 7, 9, 10, and 12−19 in Aβ25−35-induced SH-SY5Y cell injury models, showed statistically significant neuroprotective activities, when compared with each negative control group.

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The fruits of S. bicolor var. tuberculata were subjected to phytochemical purification, leading to the isolation of nine new lignans (1−9) and ten known analogues (10−19). These compounds were evaluated for their neuroprotective effects against CoCl2, H2O2, and Aβ25−35-induced cell injuries in SHSY5Y cells.

ontaining two genera Kadsura and Schisandra, plants in the Schisandraceae have been used widely in traditional Chinese medicine as sedative and tonic agents for a long time.1 Numerous lignans and triterpenoids have been isolated from plants in this family in the last few decades, of which some possess diverse biological effects, such as anti-HIV, anti-HBV, antitumor, anticholesteremic, antihepatotoxic, antioxidant, and neuroprotective activities.2−5 Recently, lignans from plants in the Schisandraceae have been reported with significant neuroprotective effects,6,7 and research from our own group on the chemical constituents of Schisandra species has also led to the isolation of a series of lignans with considerable neuroprotective effects against CoCl2, H2O2, or Aβ25−35induced cell injury in SH-SY5Y cells.8−10 Schisandra bicolor var. tuberculata (Y. W. Law) Y. W. Law is distributed mainly in Jiangxi, Hunan and Guangxi Provinces of mainland China. Its ripe berries are edible and nutritious, which are eaten by local residents to prevent disease and keep healthy. The stems of S. bicolor var. tuberculata have been studied, leading to the isolation of several lignans and triterpenoids.11−13 Until now, the chemical constituents and bioactivities of S. bicolor var. tuberculata fruits have not been reported. A preliminary bioactivity screening experiment revealed that a crude extract exhibited promising cell survival data against CoCl2-induced hypoxic injuries and H2O2 and Aβ25−35-induced oxidative injuries in SH-SY5Y cells, at the same concentration of 0.1 mg/mL (Figures S101−S103, Supporting Information). © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The air-dried fruits of S. bicolor var. tuberculata were extracted with acetone at room temperature. The solvent was evaporated in vacuo to afford a crude extract, which was subjected to a series of chromatographic steps to obtain compounds 1−19. Schibitubin A (1) was isolated as cubic crystals. Its molecular formula was assigned as C20H24O5 based on the 13C NMR data and the HRESIMS ion at m/z 367.1511 [M + Na]+ (calcd for 367.1521). The 1H NMR data (Table 1) of 1 indicated the presence of two 1,3,4-trisubstituted benzene systems [δH 6.66 (1H, overlapped, H-2), 6.68 (1H, d, J = 8.0 Hz, H-5) and 6.57 (1H, dd, J = 8.0, 1.8 Hz, H-6); 6.66 (1H, d, J = 1.4 Hz, H-2′), 6.70 (1H, d, J = 7.9 Hz, H-5′), and 6.61 (1H, dd, J = 7.9, 1.4 Hz, H-6′)], a methylenedioxy group at δH 5.85 (2H, s, -OCH2O-3′,4′), a methoxy group at δH 3.78 (3H, s, OCH3-3), an oxygenated methylene group at δH 3.46 (1H, dd, J = 11.0, Received: January 12, 2017 Published: March 23, 2017 1117

DOI: 10.1021/acs.jnatprod.7b00035 J. Nat. Prod. 2017, 80, 1117−1124

Journal of Natural Products

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two methine groups at δH 1.75 (1H, m, H-8) and 2.09 (1H, m, H-8′), and a methyl group at δH 0.86 (3H, d, J = 6.9 Hz, H-9′). The 13C NMR and DEPT spectroscopic data (Table 2) exhibited 20 carbon signals, consisting of two methyl groups (one methoxy), four methylene groups (one methylenedioxy), eight methine groups (six sp2), and six sp2 quaternary carbons, which were in agreement with the groups deduced from the 1H NMR data. The 1H−1H COSY correlations of 1 (Figure 1) showed the presence of two main units, namely, −CH2(H-7)− CH(H-8)−CH 2 (H-9)− and −CH 2 (H-7′)−CH(H-8′)− CH3(H-9′). The above spectroscopic characteristics suggested that 1 is a dibenzylbutane lignan with two 1,3,4-trisubstituted benzene systems.14 The above NMR characteristics combined with the HMBC correlations (Figure 1) of OCH3/C-3, OCH2O/C-3′, C-4′ were consistent with a 3-methoxy-4hydroxyphenyl unit and a 3′,4′-methylenedioxyphenyl moiety, respectively. Additionally, the HMBC correlations of H-7/C-2, C-6 and H-7′/C-2′, C-6′ indicated a direct linkage of C-7 to C1, and C-7′ to C-1′, respectively. According to the above data and the molecular formula of 1, a hydroxy group was also present, which was located at C-9 by the chemical shift of C-9 (δC 63.1). Accordingly, the planar structure of 1 was determined as 3-methoxy-4-hydroxy-3′,4′-methylenedioxylignan-9-ol. The absolute configuration of 1 was determined unambiguously by single-crystal X-ray diffraction analysis with Cu Kα irradiation (Figure 2). Thus, compound 1 was determined as (8S,8′R)-3-methoxy-4-hydroxy-3′,4′-methylenedioxylignan-9-ol, and given the name schibitubin A.

7.7 Hz, H-9a), 3.41 (1H, dd, J = 11.0, 5.5 Hz, H-9b), two methylene groups [δH 2.62 (1H, dd, J = 13.8, 5.8 Hz, H-7a), 2.31 (1H, dd, J = 13.8, 9.4 Hz, H-7b), and 2.66 (1H, dd, J = 13.8, 6.6 Hz, H-7′a), 2.40 (1H, dd, J = 13.8, 8.6 Hz, H-7′b)],

Table 1. 1H NMR Spectroscopic Data (δH) of Compounds 1−9 (J in Hz, 400 MHz)a position 2 5 6 7a 7b 8 9a 9b 2′ 5′ 6′ 7′a 7′b 8′ 9′ OMe-3 OMe-4 OMe-3′ OMe-4′ OCH2O OAc-9

1

2

3

4

5

6.67 d (8.0) 6.57 dd (8.0, 1.9) 2.65 dd (14.1, 7.0) 2.32 dd (14.1, 8.8) 2.05 m 0.83 d (6.9)

6.64 (overlapped) 6.72 d (7.8) 6.62 dd (7.8, 1.6) 2.72 dd (13.9, 4.9) 2.45 dd (13.9, 9.1) 1.96 m 4.02 dd (11.2, 6.7) 3.96 dd (11.2, 5.3) 6.62 (overlapped) 6.84 d (7.9) 6.62 dd (7.9, 1.6) 2.70 dd (13.6, 5.3) 2.36 dd (13.6, 8.9) 1.96 m 0.88 d (6.7)

6.62 (overlapped) 6.82 d (8.0) 6.65 dd (8.0, 1.6) 2.72 dd (13.9, 4.8) 2.40 dd (13.9, 8.1) 1.97 m 4.02 dd (11.1, 7.0) 3.96 dd (11.1, 5.2) 6.65 (overlapped) 6.73 d (7.8) 6.62 dd (7.8, 1.9) 2.69 dd (13.6, 6.2) 2.38 dd (13.6, 9.9) 1.98 m 0.89 d (6.7) 3.86 s

3.78 s

3.87 s

5.84 s

5.92 s 2.00 s

6.66 (overlapped) 6.68 d (8.0) 6.57 dd (8.0, 1.8) 2.62 dd (13.8, 5.8) 2.31 dd (13.8, 9.4) 1.75 m 3.46 dd (11.0, 7.7) 3.41 dd (11.0, 5.5) 6.66 d (1.4)

6.66 d (1.9)

6.64 d (1.5)

6.82 d (7.9) 6.66 dd (7.9, 1.9) 2.69 dd (13.8, 4.8) 2.37 dd (13.8, 9.8) 1.81 m 3.59 dd (11.2, 7.2) 3.55 dd (11.2, 6.0) 6.60 d (1.8)

6.68 d (8.0) 6.60 dd (8.0, 1.5) 2.62 dd (14.1, 5.6) 2.37 dd (14.1, 9.0) 1.75 m 3.47 dd (11.0, 7.5) 3.41 dd (11.0, 5.6) 6.67 d (1.9)

6.70 d (7.9) 6.61 dd (7.9, 1.4) 2.66 dd (13.8, 6.6) 2.40 dd (13.8, 8.6) 2.09 m 0.86 d (6.9) 3.78 s

6.82 d (7.9) 6.66 dd (7.9, 1.8) 2.72 dd (13.6, 7.2) 2.44 dd (13.6, 8.6) 2.11 m 0.90 d (6.9) 3.84 s 3.81 s

5.85 s

5.93 s 2.00 s

6 6.58 d (1.6) 6.81 d (7.7) 6.64 dd (7.7, 1.6) 2.73 dd (14.2, 4.8) 2.43 dd (14.2, 8.4) 1.99 m 4.04 dd (11.1, 6.9) 3.96 dd (11.1, 5.3) 6.61 d (1.4)

7 6.60 (overlapped) 6.72 d (7.8) 6.59 (overlapped) 3.26 d (15.4)

9 7.48 d (1.8)

6.88 br s 6.85 (overlapped) 4.80 d (9.7)

6.86 d (8.3) 7.63 dd (8.3, 1.8)

3.21 d (15.4)

4.87 br s 4.72 d (1.25) 6.55 d (1.8)

6.83 d (7.2) 6.65 dd (7.2, 1.4) 2.71 dd (14.2, 6.2) 2.41 dd (14.2, 7.2) 1.99 m 0.90 d (6.7) 3.85 s

6.80 d (8.0) 6.59 (overlapped) 2.72 dd (13.4, 6.2) 2.43 dd (13.4, 8.2) 2.30 m 0.99 d (6.8)

3.84 s

3.84 s 5.92 s

1.99 s

8 6.79 br s

2.81 m 4.20 dd (11.2, 9.1) 4.13 dd (11.2, 6.0) 6.85 (overlapped) 6.91 br s 6.79 br s 5.44 d (4.7)

2.64 m 0.67 d (7.2) 3.91 s

2.24 s

6.92 d (1.8) 6.84 d (8.1) 7.00 dd (8.1, 1.8) 5.94 d (10.1)

3.21 0.97 3.90 3.92 3.86 3.88

m d (7.1) s s s s

5.95 s 1.94 s

a

Data (δ) were measured in CD3OD for 1 and 3, and in CDCl3 for 2 and 4−9. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments. 1118



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Table 2. 13C NMR Spectroscopic Data (δC) of Compounds 1−9 (100 MHz)a position

1

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10 11 OMe-3 OMe-4 OMe-3′ OMe-4′ OCH2O

134.3 113.5 148.8 145.5 115.9 122.6 33.4 47.4 63.1 136.7 110.3 149.0 147.0 108.8 123.0 41.1 35.3 15.5

133.3 111.5 146.7 143.9 114.3 121.8 33.0 46.3 63.8 133.1 111.5 146.5 143.8 114.2 121.7 40.3 34.8 15.5

136.6 110.2 149.0 147.0 108.9 122.9 33.8 47.7 63.1 134.4 113.5 148.7 145.4 115.9 122.6 40.8 35.4 15.7

134.5 109.4 147.9 146.0 108.3 122.0 33.6 43.4 65.3 133.1 111.6 146.6 144.0 114.3 121.8 39.9 35.6 16.0 171.2 21.1

134.4 108.1 146.8 145.5 114.4 119.5 81.8 51.4 62.4 133.7 106.9 147.7 146.6 108.7 119.2 84.8 41.0 9.6 171.1 20.9 56.1

122.6 112.3 148.8 153.3 111.2 123.6 165.2 210.0 28.7 130.8 110.4 149.3 149.2 110.3 120.1 78.2 52.6 13.8

56.0

132.5 111.5 146.5 143.9 114.2 121.8 33.2 42.9 65.4 133.1 111.4 146.5 143.9 114.3 121.8 39.9 35.2 15.7 171.3 21.1 56.0

133.7 109.7 147.7 145.9 108.1 122.2 42.1 153.8 110.6 133.2 111.7 146.3 143.8 114.0 121.9 42.5 40.6 19.8

56.3

132.4 111.5 146.6 144.1 114.4 121.8 33.3 43.2 65.3 135.1 109.4 147.8 145.9 108.2 121.8 40.1 35.3 15.7 171.2 21.1 56.0

56.0

56.0

55.9 102.0

56.3

56.1

102.0

101.0

100.9

100.9

56.1 56.1 56.1 56.0

101.0

a

Data (δ) were measured in CD3OD for 1 and 3, and in CDCl3 for 2 and 4−9. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

1 and the occurrence of an additional signal at δH 3.81 in 2. The planar structure of 2 was then identified as 3,3′-dimethoxy-4,4′dihydroxylignan-9-ol. In 1977, a compound with the same planar structure was isolated from the catalytic hydrogenolysis products of compression wood of Larix leptolepis, but the NMR data and the absolute configuration were not provided.15 Also, in 2014, the (8R,8′R) isomer of 2 was reported as a synthetic product with different NMR spectroscopic and optical rotation data to 2.16 Compound 2 showed three positive Cotton effects (CEs) near 215, 227, and 274 nm in the ECD spectrum, which were in excellent agreement with those of compound 1 (Figure 3) and suggested the two compounds possess the same absolute configuration. Hence, compound 2 (schibitubin B) was assigned as (8S,8′R)-3,3′-dimethoxy-4,4′-dihydroxylignan9-ol. Schibitubin C (3), a yellow oil, gave the same molecular formula, C20H24O5, as compound 1, and was determined by the HRESIMS and 13C NMR data. Comparison of the 1H NMR and 13C NMR data (Tables 1 and 2) of 3 and 1 revealed the two compounds to be quite similar in structure. Detailed comparison of the HSQC and HMBC spectra of 3 and 1 demonstrated that the positions of the two aromatic groups of 1 were interchanged in 3, which was verified by the HMBC correlations of H-7/C-1, C-2, C-6, C-9 and H-7′/C-1′, C-2′, C6′, C-9′. Therefore, the planar structure of 3 was defined as 3,4methylenedioxy-3′-methoxy-4′-hydroxylignan-9-ol. The experimental ECD spectrum of 3 exhibited three main negative CEs near 216, 235, and 276 nm, which were opposite to those of compound 1 (Figure 3) and suggested 3 to have an opposite absolute configuration to 1. Compound 3 (schibitubin C) was thus deduced as (8R,8′S)-3,4-methylenedioxy-3′-methoxy-4′hydroxylignan-9-ol.

Figure 1. Key HMBC (→) and 1H−1H COSY (−) correlations of compounds 1, 4, and 9.

Figure 2. ORTEP drawing of compound 1.

Schibitubin B (2) was isolated as a yellow oil and its molecular formula was assigned as C20H26O5 from its 13C NMR spectroscopic data and the HRESIMS [M + Na]+ ion peak at m/z 369.1668 (calcd for 369.1678). The 1H NMR and 13C NMR data (Tables 1 and 2) of 2 were similar to those of 1. The main difference between the two compounds was that the aromatic ring substituted by a methylenedioxy group in 1 was replaced by a 3-methoxy-4-hydroxyphenyl moiety in 2. This was confirmed by the absence of a 1H NMR signal at δH 5.85 in 1119



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2) and the HMBC spectrum. The planar structure of 6 was thus determined as 9-acetoxy-3,3′-dimethoxy-4,4′-dihydroxylignan, which was reported as a synthetic product in 2014 with different NMR data and optical rotation value to 6.16 The ECD spectrum of 6 (three negative CEs at 208, 232, and 276 nm) showed an opposite pattern of CEs to those of compound 1 (Figure 3), which suggested that compound 6 has the opposite absolute configuration to compound 1. Therefore, 6 (schibitubin F) was assigned as (8R,8′S)-9-acetoxy-3,3′dimethoxy-4,4′-dihydroxylignan. Schibitubin G (7) was isolated as a white amorphous solid, with a molecular formula of C20H22O4, as deduced by its 13C NMR data and from the HRESIMS peak at m/z 349.1396 [M + Na]+ (calcd for 349.1416). Detailed comparison of the NMR data (Tables 1 and 2) of 7 and 3 indicated the two compounds to be similar, except for the occurrence of a terminal double bond at δH 4.87 (1H, br s, H-9a), δH 4.72 (1H, d, J = 1.25 Hz, H-9b), with corresponding carbon signals at δC 153.8 (C-8), 110.6 (C-9), in 7. The location of a Δ8 double bond was determined by the HMBC correlations of H-9/C-7, C-8. The planar structure of 7 was proposed as 3,4-methylenedioxy-3′methoxy-4′-hydroxylignan-8-ene. Compound 7 possessed a positive optical rotation ([α]20 D + 9.2), which was similar to the reported compound (βR)-4-hydroxy-β-[(4-hydroxy-3methoxyphenyl)methyl]-3-methoxy-γ-methylenebenzene-buta17 nol ([α]20 so 7 (schibitubin G) was elucidated as D + 18), (8′R)-3,4-methylenedioxy-3′-methoxy-4′-hydroxylignan-8-ene. Schibitubin H (8), a white amorphous solid, gave a molecular formula of C22H24O7 by HRESIMS peak at m/z 423.1403 [M + Na]+ (calcd for 423.1420). The 1H NMR data (Table 1), when combined with the HSQC and HMBC spectra of 8, showed the presence of two 1,3,4-trisubstituted aromatic systems [δH 6.79 (1H, br s, H-2), 6.88 (1H, br s, H-5), and 6.85 (1H, overlap, H6); 6.85 (1H, overlap, H-2′), 6.91 (1H, br s, H-5′), and 6.79 (1H, br s, H-6′)], a methylenedioxy group at δH 5.95 (2H, s, -OCH2O-3′,4′), four methine groups [δH 4.80 (1H, d, J = 9.7, H-7), 2.81 (1H, m, H-8), 5.44 (1H, d, J = 4.7, H-7′), 2.64 (1H, m, H-8′)], a methylene group [δH 4.20 (1H, dd, J = 11.2, 9.1 Hz, H-9a), 4.13 (1H, dd, J = 11.2, 6.0 Hz, H-9b)], a methoxy group at δH 3.91 (3H, s, OCH3-3), and two methyl groups [δH 0.67 (3H, d, J = 7.2 Hz, H-9′), δH 1.94 (3H, s, H-11)]. In addition, an ester carbonyl carbon at δC 171.1 was evident from the 13C NMR and DEPT data (Table 2). From the 1H−1H COSY correlations (Figure 4) of H-7/H-8, H-8/H-9, H-7′/H-

Figure 3. Experimental ECD spectra of compounds 1−6.

Schibitubin D (4) was obtained as white amorphous solid with the molecular formula C22H26O6, which was determined by its 13C NMR data and from the [M + Na]+ ion peak at m/z 409.1604 (calcd for 409.1627) in the HRESIMS. Comparison of the NMR data (Tables 1 and 2) of 4 with those of 3 indicated that the two compounds are related structurally, except for the occurrence of an extra acetoxy group in 4. The acetoxy group was located at C-9 based on the HMBC correlations of H-9/C-10, C-11 (Figure 1), which was confirmed by the downfield shift of C-9 from δC 63.1 in 3 to δC 65.3 in 4. Accordingly, the planar structure of 4 was elucidated as 9-acetoxy-3,4-methylenedioxy-3′-methoxy-4′-hydroxylignan. The ECD spectrum of 4 (three negative CEs at 216, 233, and 280 nm) was opposite to that of 1 (Figure 3), suggesting that the two compounds possess the opposite absolute configuration. Thus, 4 (schibitubin D) was determined as (8R,8′S)-9-acetoxy-3,4-methylenedioxy-3′-methoxy-4′-hydroxylignan. Schibitubin E (5), a white amorphous solid, was assigned the same molecular formula of C22H26O6 as 4 from its 13C NMR data and HRESIMS. The 1H NMR and 13C NMR spectra (Tables 1 and 2) of 5 were similar to those of 1. The only difference found was that the hydroxy group at C-9 in 1 is replaced by an acetoxy group in 5, which was verified by the HMBC correlations of H-9/C-10. Consequently, the planar structure of 5 was constructed as 9-acetoxy-3-methoxy-4hydroxy-3′,4′-methylenedioxylignan. The absolute configuration (8R,8′S) of 5 was established by its opposite pattern of the ECD spectrum (three negative CEs at 204, 218, and 289 nm) to 1 (Figure 3). Herein, the structure of 5 (schibitubin E) was defined as (8R,8′S)-9-acetoxy-3-methoxy-4-hydroxy-3′,4′-methylenedioxylignan. Schibitubin F (6) was isolated as a yellow oil. A molecular formula of C22H28O6 was assigned from its 13C NMR data and the HRESIMS peak at m/z 411.1768 [M + Na]+ (calcd for 411.1784). The NMR characteristics of compound 6 resembled those of 2, except for the presence of an additional acetoxy group located at C-9, as shown by the NMR data (Tables 1 and

Figure 4. Key HMBC (→), 1H−1H COSY (−), and NOESY (↔) correlations of compound 8.

8′, H-8′/H-9′ and H-8/H-8′, a −CH−CH (CH2−)−CH (CH3)−CH− unit was deduced. The above diagnostic signals suggested that compound 8 is a tetrahydrofuran lignan.8 The attachments of the acetoxy group at C-9, the methoxy group at C-3, and the methylenedioxy group at C-3′/C-4′ were disclosed according to the HMBC correlations (Figure 4) of H-9/C-10, C-11, MeO-3/C-3 and -OCH2O-/C-3′, C-4′, 1120



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identified as ganschisandrin (10),22 3′,3″-dimethoxylarreatricin (11),23 galgravin (12),22 (−)-nectandrin-A (13),24 (−)-futokadsurin A (14),25 (+)-9′-hydroxygalbelgin (15),26 austrobailignan-6 (16),27 oleiferin-F (17),28 (+)-dihydroguaiaretic acid (18),29 and (−)-isootobaphenol (19),30 respectively. In the previously reported research, several lignans from plants in the family Schisandraceae have shown activity against oxidative neuronal damage and may be useful in the treatment and prevention of neurodegenerative diseases.6−9,31 In the present work, all of the isolated lignans were tested for their neuroprotective effects against CoCl2-induced hypoxic injury and H2O2 and Aβ25−35-induced oxidative injury in SH-SY5Y cells. All the tested compounds showed neuroprotective effects in these in vitro assays to different degrees (Table 3; Tables

respectively. Moreover, the HMBC correlations of H-7/C-1, C2, C-6 and H-7′/C-1′, C-2′, C-6′ indicated the linkages of C-7 to the 3-methoxy-4-hydroxyphenyl unit and C-7′ to the 3′,4′methylenedioxyphenyl moiety, respectively. From all of the information available, the planar structure of 8 was determined as 9-acetoxy-3-methoxy-4-hydroxy-3′,4′-methylenedioxy-7,7′epoxylignan. The coupling constants of H-7/H-8 (J = 9.7 Hz) and H-7′/H-8′ (J = 4.7 Hz) indicated a 7,8-trans-7′,8′-cis relative configuration.8,18 Moreover, the obvious cross-peaks H7/H-9′ and H-7′/H-8 in the NOESY correlations (Figure 4) confirmed that H-8, H-7′, and H-8′ are cofacial. The absolute configuration of 8 was established on the basis of its ECD spectrum (positive CEs at 220, 242, and 286 nm, negative CE at 259 nm), which was similar to that of (+)-chicanine (positive CEs at 221, 236, and 285 nm, negative CE at 256 nm).18,19 According to the above evidence, the structure of 8 (schibitubin H) was defined as (7S,8R,7′R,8′R)-9-acetoxy-3-methoxy-4hydroxy-3′,4′-methylenedioxy-7,7′-epoxylignan. Schibitubin I (9) was obtained as a colorless oil and its molecular formula was assigned as C22H26O7 by HRESIMS peak at m/z 425.1558 [M + Na]+ (calcd for 425.1576). The 1H NMR data (Table 1) of 9, along with the DEPT and HSQC spectra, showed the presence of two 1,3,4-trisubstituted aromatic systems [δH 7.48 (1H, d, J = 1.8 Hz, H-2), 6.86 (1H, d, J = 8.3 Hz, H-5), and 7.63 (1H, dd, J = 8.3, 1.8 Hz, H6); 6.92 (1H, d, J = 1.8 Hz, H-2′), 6.84 (1H, d, J = 8.1 Hz, H5′), and 7.00 (1H, dd, J = 8.1, 1.8 Hz, H-6′)], two methine groups [δH 5.94 (1H, d, J = 10.1 Hz, H-7′) and 3.21 (1H, m, H8′)], four methoxy groups [δH 3.90 (3H, s, OCH3-3), 3.92 (3H, s, OCH3-4), 3.85 (3H, s, OCH3-3′), and 3.88 (3H, s, OCH34′)], and two methyl groups [δH 2.24 (3H, s, H-9) and 0.97 (3H, d, J = 7.1 Hz, H-9′)]. The 13C NMR data (Table 2) exhibited 22 carbon signals, including a ketone carbonyl at δC 210.0 (C-8) and an ester carbonyl at δC 165.2 (C-7). The 1D NMR characteristics suggested that compound 9 is a 7,8-secolignan.4 The 1H−1H COSY correlations (Figure 1) were supportive of a −CH (H-7′)−CH (H-8′)−CH3 (H-9′) unit being present. The HMBC correlations (Figure 1) of H-9/C-8, C-8′ indicated that a methyl ketone unit (δC 210.0 and 28.7) is linked to C-8′. Additionally, HMBC correlations of H-7′/C-1′, C-2′, and C-6′ required the direct linkage of C-7′ to C-1′. Furthermore, the HMBC cross-peak of H-7′ to the ester carbonyl carbon C-7 indicated that the latter carbon is linked to the 3,4-dimethoxyphenyl unit by an ester bond. From all the above evidence, the planar structure of 9 was established as 3,4,3′,4′-tetramethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione. This is the same as 3′,4′-dimethoxy-benzoic acid-(3″,4″dimethoxyphenyl)-2-methyl-3-oxobutyl ester with the opposite optical rotation, for which the absolute configuration was not clearly determined and the ECD data were not provided.20,21 The coupling constant (J = 10.1 Hz) between H-7′ and H-8′ in 9 indicated them to be in a trans configuration.9 In turn, the optical rotation ([α]20 D + 35.5) and ECD spectrum (positive CEs at 210, 261, 289 nm and negative CE at 233 nm) of 9 were opposite to those of (7′R,8′S)-3,4-dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-epoxylignan-7,8-dione ([α]20 D − 39.0; ECD: negative CEs at 208, 259, 294 nm and positive CE at 234 nm),9 which suggested the two compounds possess the opposite absolute configuration. Therefore, 9 (schibitubin I) was determined as (7′S,8′R)-3,4,3′,4′-tetramethoxy-7,8-seco-7,7′epoxylignan-7,8-dione. On comparing their measured spectroscopic data with values reported in the literature, the known compounds isolated were

Table 3. Neuroprotective Effects of Compounds 1−19 against 0.5 μM Aβ25‑35-Induced Neuronal Cell Death in Dopaminergic Neuroblastoma SH-SY5Y Cellsa 0.5 μM

test concentration (μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 vitamin Ee

0.0032 70.8 77.4 75.8 73.1 68.3 74.1 84.7 68.6 78.4 75.1 64.5 76.9 72.1 81.7 71.3 73.5 81.6 79.2 84.5 72.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.62 0.40d 0.35b 0.75c 0.91 2.26d 1.00d 0.59 1.88d 0.57d 1.43c 0.31d 0.53d 0.62d 0.75d 0.29d 2.55d 1.15d 2.02d 3.35d

0.08 83.6 74.3 88.2 80.2 80.8 81.0 78.5 88.6 85.1 85.3 77.1 92.9 85.8 75.3 94.3 77.1 83.3 85.1 71.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.82d 0.58d 1.61d 0.59d 2.54d 0.93d 1.21d 1.17d 2.70d 1.48d 0.70d 2.46d 1.07d 0.93c 3.70d 0.35d 1.65d 1.59d 2.25b

2 84.2 69.2 82.4 87.0 69.0 83.2 78.1 79.3 80.4 79.8 71.0 86.7 77.6 75.3 87.3 72.0 76.6 80.8 75.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Aβ25−35 2.23d 0.68d 1.89d 0.74d 1.15 1.91d 1.74d 1.35d 1.39d 2.32d 1.75d 1.30d 1.87d 1.01c 2.69d 1.31d 0.75d 2.29d 1.15b

68.1 63.1 73.5 68.2 68.2 64.5 64.5 71.8 65.4 69.4 60.2 69.4 65.4 70.6 65.8 63.1 68.2 68.1 68.1 60.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.10 2.48 0.85 2.41 2.41 1.24 1.24 1.16 0.20 1.57 1.86 1.57 0.20 1.70 2.15 2.48 2.41 1.10 1.10 1.86

a

The data (cell viability, measured by MTT assay) were normalized and expressed as a percentage of the control group, which was set to 100%. Data expressed as means ± SEM. Three independent experiments were carried out. bp < 0.05. cp < 0.01. dp < 0.001. e Vitamin E was used as the positive control at 50 μM.

S1−S2, Supporting Information). Compounds 10, 17, 18 in the CoCl2-induced hypoxic injury model, and compounds 2, 4, 6, 8, 12, 13, 16, 17 in the H2O2-induced oxidative injury model, and compounds 3, 4, 6-13, 15, 17, 19 in the Aβ25−35-induced oxidative injury assay, improved cell viabilities by more than 15% compared with the negative control group, respectively. Additionally, at a low concentration of 3.2 nM, compounds 3, 8, 9, 14−19 in CoCl2-induced, compounds 7, 8, 13, 17, 18 in H2O2-induced, and compounds 2, 6, 7, 9, 10, 12−19 in Aβ25−35-induced SH-SY5Y cell injury models, showed statistically significant neuroprotective activities compared with the negative control group in each case. The isolated lignan types were representative of the dibenzylbutanes (1−7, 16−18), tetrahydrofurans (8, 10−15), a 7,8-seco-lignan (9), and an aryltetralin (19). Among them, compounds 1−6 belong to a series of new dibenzylbutanes with 1121



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min), 5 (2.6 mg, tR 28 min), and 16 (60.4 mg, tR 31 min). Fraction A2 was fractioned via silica gel CC (petroleum ether-acetone, 19:1) and semipreparative RP-HPLC (MeOH−H2O, 80:20, flow rate: 2.0 mL/ min) to afford compounds 10 (30.3 mg, tR 23 min) and 12 (33.3 mg, tR 27 min) and subfraction A2−4, which was further purified by HPLC (MeOH-H2O, 70:30, flow rate: 2.0 mL/min) to yield compounds 19 (19.2 mg, tR 37 min) and 7 (9.4 mg, tR 48 min); Fraction B was subjected to CC over Sephadex LH-20 eluted with CH2Cl2−CH3OH (1:1) to give subfractions B1−B2. Subfraction B1 was separated by normal-phase silica gel CC (petroleum ether-acetone, 4.5:1) and semipreparative HPLC (250 × 10 mm i.d., eluted with MeOH−H2O, 70:30, flow rate: 2.0 mL/min) to obtain compounds 8 (2.8 mg, tR 21 min) and 13 (26.5 mg, tR 26 min). Subfraction B2 was separated over ODS (S-50 μm) CC, eluted with a gradient of increasing amount of MeOH (60−100%) in H2O and semipreparative HPLC (250 × 10 mm i.d., eluted with MeOH−H2O, 75:25, flow rate: 2.0 mL/min) to obtain compounds 11 (18.7 mg, tR 18 min), 15 (26.0 mg, tR 24 min), 17 (14.8 mg, tR 25 min), and 18 (32.9 mg, tR 28 min). Fraction C was separated over Sephadex LH-20 eluted with CH2Cl2−CH3OH (1:1) to yield subfractions C1 and C2. Then, subfraction C1 was further fractionated over ODS (S-50 μm), eluted with a gradient of increasing amount of MeOH (50−100%) in H2O to give C1−1 and C1−2, which were purified by RP-HPLC using a mobile phase of MeOH−H2O, 57.5:42.5 and 65:35, respectively, to obtain compounds 9 (5.7 mg, tR 29 min) from C1−1 and 6 (5.9 mg, tR 38 min), 3 (57.2 mg, tR 42 min), and 1 (26.6 mg, tR 45 min) from C1−2. Then, subfraction C2 was separated over Sephadex LH-20 eluted with CH2Cl2−CH3OH (1:1) to yield subfractions C2−1−C2−3. Subfraction C2−2 was purified by RPHPLC by using a mobile phase of MeOH−H2O (65:35) to afford compounds 2 (34.1 mg, tR 24 min) and 14 (23.2 mg, tR 27 min). Schibitubin A (1). Cubic crystals; [α]20 D + 2.9 (c 0.35, CDCl3); UV (MeOH) λmax (log ε) 230 (3.95, sh), 285 (3.69), 370 (3.03) nm; ECD (c 0.08, MeOH) 211 (Δε + 13.37), 234 (Δε + 9.13), 275 (Δε + 1.58) nm; IR (film) νmax 3444, 2927, 1606, 1513, 1489, 1443, 1377, 1246, 1153, 1123, 1036, 931, 813, 679 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 367.1511 (calcd for C20H24O5Na, 367.1521, Δ − 2.7 ppm). Schibitubin B (2). Yellow oil; [α]20 D − 2.2 (c 0.31, CDCl3); UV (MeOH) λmax (log ε) 230 (4.02, sh), 285 (3.62) nm; ECD (c 0.17, MeOH) 215 (Δε + 33.78), 227 (Δε + 20.22), 274 (Δε + 14.30) nm; IR (film) νmax 3406, 2931, 1604, 1515, 1455, 1430, 1373, 1270, 1236, 1152, 1124, 1032, 937, 819, 796, 740, 631 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 369.1668 (calcd for C20H26O5Na, 369.1678, Δ − 2.7 ppm). Schibitubin C (3). Yellow oil; [α]20 D − 8.4 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 205 (4.26), 230 (3.97), 285 (3.66), 370 (2.86) nm; ECD (c 0.06, MeOH) 216 (Δε − 2.12), 235 (Δε − 1.54), 276 (Δε − 0.35) nm; IR (film) νmax 3406, 2957, 2930, 1606, 1512, 1489, 1443, 1372, 1269, 1244, 1190, 1153, 1124, 1036, 929, 814, 738, 629, 564 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 367.1511 (calcd for C20H24O5Na, 367.1521, Δ − 2.7 ppm). Schibitubin D (4). White amorphous solid; [α]20 D − 8.4 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 205 (3.70), 285 (2.91), 370 (2.39) nm; ECD (c 0.11, MeOH) 216 (Δε − 1.00), 233 (Δε − 2.34), 280 (Δε − 0.58) nm; IR (film) νmax 3437, 2958, 2920, 2852, 1735, 1602, 1512, 1489, 1445, 1378, 1243, 1154, 1094, 1036, 932, 862, 803 cm−1; 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 409.1604 (calcd for C22H26O6Na, 409.1627, Δ − 5.6 ppm). Schibitubin E (5). White amorphous solid; [α]20 D − 7.7 (c 0.17, CHCl3); UV (MeOH) λmax (log ε) 230 (3.22, sh), 285 (2.96), 370 (2.24) nm; ECD (c 0.05, MeOH) 204 (Δε − 13.09), 218 (Δε − 2.28), 289 (Δε − 0.62) nm; IR (film) νmax 3449, 2958, 2919, 2851, 1732, 1605, 1512, 1489, 1443, 1368, 1245, 1153, 1123, 1098, 1036, 931, 862, 803, 605 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 409.1608 (calcd for C22H26O6Na, 409.1627, Δ − 4.6 ppm).

selective oxidation of C-9. Dibenzylbutanes are a group of wellknown lignans that contain two shikimate-derived biogenetic subunits connected by a carbon−carbon linkage (8−8′), having two chiral centers at C-8 and C-8′ in a general way.32,33 Although quite a few methods have been reported to determine the absolute configurations of dibenzylbutanes, such as biosynthetic pathway analysis and comparison of the optical rotation values or circular dichroism spectra with similar compounds,14,33−35 there is still a lack of strong structural evidence because of the flexibility of the butane portion of these molecules. In this work, the absolute configuration of compound 1 was determined unambiguously by single-crystal X-ray diffraction analysis with Cu Kα irradiation. The absolute configurations of other new dibenzylbutanes (2−6) were deduced by comparison of their experimental ECD spectra with that of compound 1. The absolute configurations of compounds 7−9 were determined by comparing their experimental ECD spectra or optical rotation values with those of similar compounds. Moreover, the neuroprotective assay results elucidate the bioactivity of S. bicolor var. tuberculata fruits and the potential of these isolates as neuroprotective precursors and, in particular, provide some supporting evidence for the use of these fruits in traditional Chinese medicine.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV spectra were carried out on a Varian Cary 50 scan UV/vis spectrophotometer. ECD spectra were recorded on a JASCO J-810 spectropolarimeter. 1D and 2D NMR spectra were recorded on a Bruker-AM-400 NMR spectrometer. Chemical shifts (δ) are expressed in ppm and coupling constants are given in Hz. HRESIMS was performed on a Thermo Scientific LTQOrbitrap XL mass spectrometer. The X-ray crystallographic data was collected on a Bruker SMART APEX-II CCD diffractometer equipped with graphite monochromatized Cu Kα radiation (λ = 1.54178 Å). TLC analysis was carried out on silica gel GF254 plates (Yantai Institute of Chemical Technology). Visualization of the TLC plates were achieved under UV at 254 nm and sprayed with 5% H2SO4−EtOH followed by heating on a hot plate. MPLC was performed using a Buchi pump module C-605. Column silica gel (200−300 or 300−400 mesh; Qingdao Marine Chemical Inc.), Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden), and ODS gel (YMC, S-50 μm) were used for column chromatography. Reversed-phase HPLC was performed on Agilent 1260 system and Agilent 1100 system. YMCPack ODS-A C18 5 μm (250 × 10 mm i.d.; YMC, Tokyo, Japan) columns were used for analytical and semipreparative purposes. MTT assays were all performed on a BioTek Synergy 2 multimode microplate reader. Plant Material. The fruits of S. bicolor var. tuberculata were collected in Xinning county of Hunan Province, People’s Republic of China, in August 2012 and authenticated by Mr. Xu-Ai Liu and Mr. Yibo Luo of the Xinning Forestry Bureau. A voucher specimen (i.d. 20120831) has been deposited in the Herbarium of Materia Medica Faculty of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China. Extraction and Isolation. The air-dried fruits of S. bicolor var. tuberculata (220 g) were extracted with acetone (800 mL × 5) at room temperature. The solvent was evaporated in vacuo to afford a crude extract (43.7 g), which was subjected to MPLC [46 × 3.6 cm i.d., 200−300 mesh silica, 200 g, eluted with a mixture of petroleum ether and acetone gradient system, 1:0, 20:1, 10:1, 4:1, 1.5:1, 1:1.5, 1:4, 0:1, and CH3OH] to afford three fractions A-C. Fraction A was chromatographed over Sephadex LH-20 eluted with CH2Cl2− CH3OH (1:1) to give subfractions A1−A2. Fraction A1 was further purified by silica gel (petroleum ether−acetone, 4.5:1) and semipreparative HPLC (250 × 10 mm i.d., eluted with MeOH−H2O, 75:25, flow rate: 2.0 mL/min) to yield compounds 4 (2.7 mg, tR 26 1122



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Schibitubin F (6). Yellow oil; [α]20 D − 12.2 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 210 (4.12), 285 (3.58), 370 (2.83) nm; ECD (c 0.19, MeOH) 208 (Δε − 18.10), 232 (Δε − 31.91), 276 (Δε − 2.72) nm; IR (film) νmax 3434, 2921, 1727, 1601, 1514, 1455, 1429, 1367, 1265, 1236, 1152, 1124, 1033, 818, 797 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 411.1768 (calcd for C22H28O6Na, 411.1784, Δ − 3.9 ppm). Schibitubin G (7). Amorphous solid; [α]20 D + 9.2 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 230 (3.54), 285 (3.29), 370 (2.41) nm; ECD (c 0.25, MeOH) 206 (Δε − 30.61), 227 (Δε + 12.86), 289 (Δε − 1.61) nm; IR (film) νmax 3476, 2960, 2924, 2776, 2063, 1855, 1729, 1641, 1607, 1512, 1488, 1441, 1366, 1267, 1243, 1151, 1123, 1037, 930, 895, 804, 739, 626, 560 cm−1; 1H NMR (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 349.1396 (calcd for C20H22O4Na, 349.1416, Δ − 5.7 ppm). Schibitubin H (8). Amorphous solid; [α]20 D + 18.7 (c 0.08, CHCl3); UV (MeOH) λmax (log ε) 230 (3.35), 285 (2.93), 370 (2.15) nm; ECD (c 0.13, MeOH) 220 (Δε + 3.81), 242 (Δε + 17.62), 286 (Δε + 9.04) nm; IR (film) νmax 3448, 2958, 2925, 2851, 1727, 1647, 1513, 1488, 1438, 1371, 1253, 1243, 1162, 1095, 1033, 934, 872, 811, 764, 722, 608 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 423.1403 (calcd for C22H24O7Na, 423.1420, Δ − 4.0 ppm). Schibitubin I (9). Colorless oil; [α]20 D + 35.5 (c 3.66, CHCl3); UV (MeOH) λmax (log ε) 205 (4.45), 265 (3.78), 370 (2.88) nm; ECD (c 0.11, MeOH) 210 (Δε + 14.13), 233 (Δε − 2.01), 261 (Δε + 5.48), 289 (Δε + 4.12) nm; IR (film) νmax 3449, 2964, 2938, 2838, 2616, 1713, 1597, 1515, 1461, 1418, 1352, 1269, 1223, 1175, 1138, 1106, 1025, 962, 876, 815, 764, 629, 562 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; (+)-HRESIMS [M + Na]+ m/z 425.1558 (calcd for C22H26O7Na, 425.1576, Δ − 4.2 ppm). X-ray Crystal Structure Analysis of Compound 1. Suitable crystal of compound 1 was obtained by slowly evaporating methanol solution at ambient temperature. High-quality colorless crystal was mounted on a glass fiber in a random orientation. The data was collected by a Bruker SMART APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å) by using ω scan mode. The structure was solved by direct methods using Olex2 software,36 and the non-hydrogen atoms were located from the trial structure and then refined anisotropically with SHELXL-2016 using a full-matrix least-squares procedure based on F2.37 The weighted R factor, wR, and goodness-of-fit S values were obtained based on F2.The hydrogen atom positions were fixed geometrically at the calculated distances and allowed to ride on the parent atoms. Crystallographic data excluding structure factors has been deposited (number 1533203) at the Cambridge Crystallographic Data Center. Crystal data for 1. C20H24O5, MW = 344.39, crystal size 0.28 × 0.25 × 0.20 mm3, monoclinic, space group P21, a = 9.76560 (10) Å, b = 6.16280 (10) Å, c = 15.4441 (2) Å, V = 910.72(2) Å3, Z = 2, T = 273.15 K, α = γ = 90.00°, β = 101.5300(10)°, ρ (calcd) = 1.252 g· cm−3, λ (Cu Kα) = 1.54178 Å, μ (Cu Kα) = 0.732 mm−1, F(000) = 366.0, θ range for data collection 2.92° to 67.082°, reflections collected 14171, independent reflections 3028, R(int) = 0.0232, h (−11/11), k (−7/6), l (−18/18), final R indices [I > 2σ(I)] R1 = 0.0388, wR2 = 0.1121, R indices (all data) R1 = 0.0394, wR2 = 0.1128, goodness-of-fit on F2 1.063, data/restraints/parameters 3028/1/230, largest diff. peak and hole 0.18 and −0.16 e.Å−3, Flack parameter 0.05 (5), Hooft parameter 0.06 (5). The crystallographic data has been deposited in the Cambridge Crystallographic Data Centre with the deposition number of 1533203. Assays to Determine Potential Neuroprotective Activity. The neuroprotective activities of the crude extract and isolated compounds were measured according to a published procedure by the MTT method.9,38 In brief, human dopaminergic neuroblastoma SHSY5Y cells were cultured in DMEM culture medium plus 10% (v/v) fetal bovine serum (Sijiqing, Hangzhou, People’s Republic of China) with 100 U/mL penicillin and 100 μg/mL streptomycin, and

maintained at 37 °C in 5% CO2 and in a 95% humidified air incubator (Heal Force HF-90, Hong Kong). Hydrogen peroxide solution (30% H2O2), cobalt(II) chloride (CoCl2, purity >98%), Aβ25−35 (purity >97%), and 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl2-tetrazolium bromide (MTT) were purchased from Sigma. Then, 350 μM H2O2, 300 μM CoCl2, and 0.5 μM Aβ25−35 (cell viability 50% to 70% compared with the control group) were added. SH-SY5Y cells were seeded in 96-well culture plates 15 000 cells/well (92 μL of cell suspension/well). After 24 h of being incubated in the incubator, cells were pretreated with 11.5 μL of various concentrations of the crude extract (0.004, 0.02, 0.1 mg/mL) or test compounds (0.0032, 0.08, 2 μM) for 2 h before incubation in medium containing H2O2 (350 μM) or CoCl2 (300 μM) or Aβ25−35 (0.5 μM). Next, 15 μL MTT (5 mg/ mL) was added after a 14 h treatment. For the MTT assay, the supernatant was discarded and DMSO (100 μL/well) was added. Then, the 96-well plate was vibrated on a microvibrator for 5 min, and the optical density at 570 nm was measured on an enzymeimmunoassay instrument (BioTek Synergy 2 reader). All samples were cultured in triplicate. Statistical analysis was performed by oneway analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Newman-Keuls Multiple Comparison method. The data were expressed as means ± SEM of three replicates.

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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.7b00035. 1D and 2D NMR spectra, HRESIMS, UV, IR and ECD spectra of compounds 1−9, Neuroprotective effects of the crude extract of S. bicolor var. tuberculata against CoCl2, H2O2, and Aβ25−35-induced cell injuries in SHSY5Y cells. Neuroprotective effects of compounds 1−19 against 300 μM CoCl2-induced and 350 μM H2O2induced SH-SY5Y cell deaths (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-13339986848. Fax: 86 27 83692739. ORCID

Han-Li Ruan: 0000-0003-0882-1009 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (No. 31270394, 21572073, and 31500282) and the Fundamental Research Funds for the Central Universities (No. 2016YXMS150). We are grateful to the staff at the analytical and testing center of Huazhong University of Science and Technology for collecting the spectroscopic data.

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