Lignans from the Fruit of Schisandra glaucescens with

Article pubs.acs.org/jnp

Lignans from the Fruit of Schisandra glaucescens with Antioxidant and Neuroprotective Properties Heng-Yi Yu,†,# Zu-Yu Chen,†,‡,# Bin Sun,† Junjun Liu,† Fan-Yu Meng,† Ye Liu,† Tian Tian,† An Jin,† and Han-Li Ruan*,† †

Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430000, People’s Republic of China ‡ College of Environment Engineering, Wuhan Textile University, Wuhan 430000, People’s Republic of China S Supporting Information *

ABSTRACT: Two rare 7,8-seco-lignans (1, 2), three new lignan glycosides (3, 4a, 4b), and 10 known lignans (5−14) were isolated from the fruit of Schisandra glaucescens Diels. The absolute configurations of 1 and 2 were determined by comparing their experimental and calculated electronic circular dichroism spectra. The molecular structures of the new compounds (3, 4a, and 4b), including their absolute configurations, were determined using various spectroscopic methods and hydrolysis reactions. The antioxidant activities of the isolated compounds were tested using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays. Compounds 4, 7, 8, 10, 11, and 12 exhibited antioxidant activities of varying potential in both assays. Of these compounds, 7 showed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, with IC50 values of 15.7 (150 μM DPPH) and 34.6 μM (300 μM DPPH), respectively, and 4, 12, and 7 displayed higher total antioxidant activities than Trolox in the ferric reducing antioxidant power assay. The neuroprotective effects of these compounds against Aβ25−35-induced cell death in SH-SY5Y cells were also investigated. Compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aβ25−35-induced SH-SY5Y cell death compared with the group treated only with Aβ25−35.

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basis for the antioxidant and neuroprotective effects of S. glaucescens fruit. As a result, 15 lignans, including two rare 7,8seco-lignans and three new lignan glycosides, were isolated, identified, and tested for their antioxidant and neuroprotective activities. 7,8-Seco-lignans (or 1-phenylbutyl benzoates) are a group of rare lignans that possess various bioactivities, including antiHIV,8,9 anti-HSV,10 antiadenovirus,10 and cytotoxic activities.11 These compounds were first isolated from the leaves of Ocotea foetens (Lauraceae) in 199512 and were then found in Holostylis reniformis (Aristolochiaceae) and Beilschmiedia tsangii (Lauraceae).11,13 In 2009, these compounds were identified for the first time in a plant in the Schisandraceae family when two 7,8seco-lignans were isolated from the fruit of S. sphenanthera.14 Since then, a number of analogues have been isolated from plants in the Schisandraceae family.8−10,15

lants of the genus Schisandra are economically valuable and widely used in traditional Chinese medicine. Schisandra glaucescens Diels is a vine primarily found in western Hubei and southeastern Sichuan Provinces in China. In traditional Chinese medicine, the stems of this plant have been used for the treatment of various diseases, including contusions, rheumatism, and arthritis.1 In recent years, the stems of S. glaucescens have been studied extensively, and a number of triterpenoids and lignans have been isolated.2−7 The ripe fruit of S. glaucescens is a sweet red berry that is consumed as a health food by villagers in the mountains around Shennongjia, China. These berries are believed to be beneficial for the lungs and kidneys, relieve the symptoms of asthma, reduce sweating and night sweats, alleviate chronic diarrhea, and reduce neurasthenia. Preliminary bioactivity screening revealed that the ethanol extract of S. glaucescens fruit had 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and provided neuroprotection against Aβ25−35-induced SHSY5Y cell death. Phytochemical studies and antioxidant and neuroprotective assays were used to investigate the chemical © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 15, 2013

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dx.doi.org/10.1021/np4010536 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of compounds 1−14.

Table 1. NMR Spectroscopic Data (400 MHz, CDCl3) for Compounds 1 and 2 1 position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH3 4-OCH3 3′,4′-OCH2O−

δC, type 122.6, 112.3, 148.9, 153.4, 110.5, 123.7, 165.1, 209.7, 28.8, 132.2, 107.4, 148.1, 147.9, 108.4, 121.5, 78.1, 52.6, 13.7, 56.2, 56.1, 101.3,

C CH C C CH CH C C CH3 C CH C C CH CH CH CH CH3 CH3 CH3 CH2

2 δH (J in Hz)

position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3′-OCH3 4′-OCH3 3,4-OCH2O−

7.48, d (1.9)

6.86, d (8.5) 7.62, dd (8.4, 1.9)

2.24, s 6.90, brs

6.78, 6.91, 5.92, 3.18, 0.98, 3.90, 3.92, 5.94,

d (8.2) overlap d (10.1) dq (10.1, 7.1) d (7.1) s s dd (4.5, 1.2)

Most structural studies of 7,8-seco-lignans have focused on 7,8-seco-holostylone B.13 One- and two-dimensional NMR experiments have been used to determine the relative

δC, type 124.2, 109.6, 147.9, 151.9, 108.2, 125.6, 164.7, 209.9, 28.8, 130.8, 110.5, 149.2, 149.3, 111.3, 120.2, 78.2, 52.5, 13.8, 56.1, 56.0, 101.9,

C CH C C CH CH C C CH3 C CH C C CH CH CH CH CH3 CH3 CH3 CH2

δH (J in Hz) 7.40, d (1.6)

6.81, d (8.2) 7.60, dd (8.2, 1.7)

2.24, s 6.91, d (1.9)

6.84, 6.99, 5.94, 3.20, 0.96, 3.88, 3.86, 6.02,

d (8.2) dd (8.2, 1.9) d (10.1) dq (10.1, 7.1) d (7.1) s s s

configuration of 7,8-seco-holostylone B, but its absolute configuration has not been determined.13 Herein, the absolute configurations of two rare 7,8-seco-lignans (1 and 2, Figure 1) B



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H-2,6/H-9 were observed (Figure 2). However, no correlation between H-2′,6′/H-9 or between H-2,6/H-9′ was detected in the NOESY spectrum. The above evidence suggested that 1 has the same relative configuration as 7,8-seco-holostylone B13 and schisandlignan B.15 The absolute configuration of 1 was determined by comparing its experimental and calculated ECD spectra. As shown in Figure 3, the sequence and signs of the Cotton effects

were defined by comparing their experimental and calculated electronic circular dichroism (ECD) spectra.

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RESULTS AND DISCUSSION The air-dried fruit of S. glaucescens were extracted with 95% ethanol at room temperature, and the extract was concentrated in vacuo. The resulting crude extract was sequentially partitioned with petroleum ether, EtOAc, and n-BuOH. The petroleum ether- and EtOAc-soluble fractions were subjected to silica gel, Sephadex LH-20, and MCI column chromatography followed by repeated RP C18 HPLC to afford compounds 1−14 (Figure 1). Compound 1 was obtained as a colorless oil with a negative specific rotation, [α]20 D −39 (c 0.9, CHCl3), and its molecular formula was assigned as C21H22O7 based on the 13C NMR spectroscopic data and an HRESIMS ion at m/z 409.1258 [M + Na]+ (calcd 409.1263). The IR spectrum exhibited absorption bands from phenyl (1598 cm−1) and carbonyl (1713 cm−1) moieties. The 1H NMR data for 1 (Table 1) indicated the presence of two 1,2,4-trisubstituted benzene systems [δH 7.48 (1H, d, J = 1.9 Hz, H-2), 6.86 (1H, d, J = 8.5 Hz, H-5) and 7.62 (1H, dd, J = 8.4, 1.9 Hz, H-6); 6.90 (1H, brs, H-2′), 6.78 (1H, d, J = 8.2 Hz, H-5′) and 6.91 (1H, overlapped, H-6′)], one methylenedioxy [δH 5.94 (2H, dd, J = 4.5, 1.2 Hz, 3′,4′-OCH2O−)], two methoxy groups [δH 3.90 (3H, s, 3-OCH3) and 3.92 (3H, s, 4-OCH3)], two methyls [δH 2.24 (3H, s, H-9) and 0.98 (3H, d, J = 7.1 Hz, H-9′)], and two methines [δH 5.92 (1H, d, J = 10.1 Hz, H-7′) and 3.18 (1H, dq, J = 10.1, 7.1 Hz, H-8′)]. The 13 C NMR data (Table 1) indicated two carbonyl carbon resonances at δC 209.7 (C-8) and 165.1 (C-7). The above 1D NMR characteristics suggested that 1 is a 7,8-seco-lignan.13 In the 1H−1H COSY spectrum, the presence of a −CH(H-7′)− CH(H-8′)−CH3(H-9′)− unit was evident. In the HMBC spectrum (Figure 2), H-9 [δH 2.24 (3H, s)] was correlated to

Figure 3. Experimental ECD spectrum of compound 1 (A) and calculated ECD spectra for the enantiomers (7′R,8′S)- and (7′S,8′R)-1 (B).

Figure 2. Key HMBC (→) and NOESY (↔) correlations of compound 1.

in the experimental ECD spectrum were consistent with those in the calculated ECD curve of (7′R,8′S)-1. Thus, the structure of 1 was determined to be (7′R,8′S)-3,4-dimethoxy-3′,4′methylenedioxy-7,8-seco-7,7′-epoxylignan-7,8-dione. Compound 2 was obtained as a colorless oil with a negative specific rotation, [α]20 D −73 (c 1.5, CHCl3), and its molecular formula was assigned as C21H22O7, the same as that of 1, based on the 13C NMR data and an HRESIMS ion at m/z 409.1256 [M + Na]+ (calcd 409.1263). The 1H and 13C NMR data for 2 (Table 1) indicated that 2 has all of the functional groups of 1. Analysis of the HSQC and HMBC spectra of 2 suggested that the positions of the two aromatic groups are interchanged compared to 1. In the HMBC spectrum (Figure 4), H-7′ [δH 5.94 (1H, d, J = 10.1 Hz)] was correlated to C-1′ (δC 130.8), C-2′ (δC 110.5), C-6′ (δC 120.2), and C-7 (δC 164.7), which indicated the linkage of

C-8 (δC 209.7) and C-8′ (δC 52.6), which indicated that a methyl ketone moiety (δC 209.7 and 28.8) is linked to C-8′. Additionally, H-7′ [δH 5.91 (1H, d, J = 10.1 Hz)] was correlated to C-1′ (δC 132.2), C-2′ (δC 107.4), C-6′ (δC 121.5), and C-7 (δC 165.1) in the HMBC spectrum, which indicated the linkage of C-7′ to the 3,4-methylenedioxyphenyl unit and the linkage of C-7′ to the 3,4-dimethoxyphenyl unit via an ester oxygen. On the basis of the above evidence, the planar structure of 1 is 3,4-dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-epoxylignan-7,8-dione, which is the same as those of the known compounds 7,8-seco-holostylone B13 and schisandlignan B.15 The coupling constant (J = 10.1 Hz) between H-7′ [δH 5.92 (1H, d, J = 10.1 Hz)] and H-8′ [δH 3.18 (1H, dq, J = 10.1, 7.1 Hz)] indicated that the preferred conformation of these two protons is anti.13 Key NOESY correlations of H-2′,6′/H-9′ and C



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agreed with the experimental data. Therefore, the structure of 2 was established as (7′R,8′S)-3,4-methylenedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione. Compound 3 was obtained as a white, amorphous powder with a negative specific rotation, [α]20 D −44 (c 0.2, CH3OH), and its molecular formula was assigned as C31H40O15 based on the 13C NMR data and an HRESIMS ion at m/z 675.2252 [M + Na]+ (calcd 675.2265). The IR spectrum exhibited absorption bands from hydroxy (3392 cm−1) and phenyl (1610 cm−1) groups. The 1H NMR data for 3 (Table 2) indicated the presence of two 1,2,4-trisubstituted benzene systems [δH 6.61−6.62 (2H, overlap, H-2 and H-2′), 6.68 (2H, d, J = 8.0 Hz, H-5 and H-5′), and 6.59 (2H, overlap, H-6 and H-6′)], two methylenedioxy groups [δH 5.88 (4H, s, 3,4-OCH2O− and 3′,4′-OCH2O−)], two methines [δH 2.02 (1H, m, H-8) and 1.95 (1H, m, H-8′)], and four methylenes (two of which bear oxygen functional groups). In addition, protons from two monosaccharide moieties were observed at δH 3.22−5.00 [two anomeric protons at δH 4.20 (1H, d, J = 7.8 Hz, H-1″) and 5.00 (1H, brs, H-1‴)]. These 1H NMR characteristics suggested that 3 is a lignan glycoside. Because the aglycone of 3 was not stable under acidic conditions,16 enzymatic hydrolysis of 3 with β-cellulase was performed, and tetracentronside B (5) and (−)-dihydrocubebin (aglycone) were obtained (Figure S67, Supporting Information). The presence of tetracentronside B was confirmed by TLC and HPLC analysis compared with 5, and (−)-dihydrocubebin was identified from its 1D NMR spectra and 17 Additionally, specific rotation, [α]20 D −34 (c 0.2, CHCl3). negative Cotton effects at approximately 234 and 276 nm were observed in the ECD spectra of 3, 5, and (−)-dihydrocubebin (Figure 6), which confirmed the R configurations of C-8 and C8′ in these three compounds.18 To identify the monosaccharide moieties in 3, acid hydrolysis of 3 with 4 mol/L aqueous TFA was performed and yielded Dglucose and L-arabinose, which were identified through gas chromatography after treatment with L-cysteine methyl ester hydrochloride and TMS derivatization (Figure 7). The coupling constant (J = 7.8 Hz) of the anomeric proton [δH 4.20 (1H, d, J = 7.8 Hz, H-1″)] of D-glucose and the characteristic carbon resonances [δC 109.8 (C-1‴), 83.1 (C2‴), and 85.8 (C-4‴)] of L-arabinose suggest that the two monosaccharide moieties are β-D-glucopyranosyl and α-Larabinofuranosyl, respectively. Moreover, the correlations of H-1‴ [δH 5.00 (1H, brs)] to C-6″ (δC 68.0) and H-1″ [δH 4.20 (1H, d, J = 7.8 Hz)] to C-9 (δC 70.2) in the HMBC spectrum revealed the connections of (−)-dihydrocubebin and two monosaccharide moieties in 3. Finally, the structure of 3 was determined to be (8R,8′R)-9-O-(6′-O-α-L-arabinofuranosyl)-βD-glucopyranosyldihydrocubebin. Compound 4 was obtained as a white, amorphous powder with a negative specific rotation, [α]20 D −18 (c 0.2, CH3OH), and its molecular formula was assigned as C26H34O11 based on the 13C NMR data and HRESIMS ions at m/z 545.1984 [M + Na]+ (calcd 545.1999) and m/z 1067.4086 [2 M + Na]+ (calcd 1067.4100). The 1H NMR spectrum of 4 was similar to that of 5, except that one methylenedioxy group in 5 was replaced by a hydroxy and a methoxy group in 4. The 1H and 13C NMR spectra of 4 revealed two sets of resonances, suggesting that 4 was a mixture of isomers 4a and 4b.

Figure 4. Key HMBC (→) and NOESY (↔) correlations of compound 2.

C-7′ to the 3,4-dimethoxyphenyl unit and the linkage of C-7′ to the 3,4-methylenedioxyphenyl unit via an ester oxygen. Thus, the planar structure of 2 was established as 3,4-methylenedioxy3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione. The coupling constant (J = 10.1 Hz) between H-7′ [δH 5.94 (1H, d, J = 10.1 Hz)] and H-8′ [δH 3.20 (1H, dq, J = 10.1, 7.1 Hz)] and the NOESY correlations of H-2′,6′/H-9′ and H-2,6/H-9 suggested that 2 has the relative configuration shown in Figure 4, which is the same as those of rel-(7′R,8′S)-3,4-methylenedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione14 and schisandlignan A.15 The absolute configuration of 2 was also determined by comparing its experimental and calculated ECD spectra. As shown in Figure 5, the calculated ECD spectrum of (7′R,8′S)-2

Figure 5. Experimental ECD spectrum of compound 2 (A) and calculated ECD spectra for the enantiomers (7′R,8′S)- and (7′S,8′R)-2 (B). D



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Table 2. NMR Spectroscopic Data (400 MHz, CD3OD) for Compound 3 position 1 2 3 4 5 6 7a 7b 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′a 7′b 8′ 9′ 3,4-OCH2O− 3′,4′-OCH2O−

δC, type 136.1, 110.4, 148.9, 147.0, 108.8, 123.1, 35.7,

C CH C C CH CH CH2

41.8, CH 70.2, CH 3.52, m 136.2, C 110.4, CH 148.9, C 147.0, C 108.8, CH 123.2, CH 35.7, CH2 44.3, 62.5, 101.9, 101.9,

CH CH2 CH2 CH2

δH (J in Hz) 6.62, 2″ 3″ 6.68, 6.59, 2.70, 2.63, 2.02, 3.87, 1‴ 2‴ 6.61, 4‴ 5‴a 6.68, 6.59, 2.70, 2.63, 1.95, 3.59, 5.88, 5.88,

position

overlap

d (8.0) overlap m m m overlap

overlap

d (8.0) overlap m m m overlap s s

δC, type

δH (J in Hz)

9-O-glucose 1″ 2″ 3″ 4″ 5″ 6″a 6″b

104.4, CH 75.1, CH 78.0, CH 72.0, CH 76.7, CH 68.0, CH2

4.20, 3.22, 3.38, 3.29, 3.44, 4.03, 3.63,

d (7.8) t (8.1) m m m m m

6″-O-arabinose 1‴ 2‴ 3‴ 4‴ 5‴a 5‴b

109.8, CH 83.1, CH 78.8, CH 85.8, CH 63.0, CH2

5.00, 4.01, 3.84, 3.98, 3.73, 3.64,

brs m m m dd (12.0, 3.2) m

and 2D NMR spectra, it was not possible to assign either set to 4a or 4b because of the overlap resonances. The structures of compounds 5−14 were respectively confirmed as tetracentronside B (5),16 5-[(2S,3R)-4-(3,4dimethoxyphenyl)-2,3-dimethylbutyl]-1,3-benzodioxole (6),20 meso-dihydroguaiaretic acid (7),21 (+)-anwulignan (8),22 (−)-galcatin (9),23 (+)-isootobaphenol (10),23 4′,5-O-didemethylcyclogalgravin (11),24 (+)-chicanine (12),25 (+)-zuihonin C (13),26 and (+)-zuihonin A (14),26 based on comparisons with the corresponding literature data. The free radical scavenging activity of each isolated lignan was tested using a DPPH assay from 6.25 to 100 μM. Compounds 4, 7, 8, 10, 11, and 12 exhibited free radical scavenging activities with varying potential, whereas the other compounds did not exhibit any activity. The IC50 values of compounds 4, 7, 8, 10, 11, and 12 and the positive controls (vitamins C and E) are presented in Table 4. The compounds exhibited DPPH radical scavenging activities in the following descending order: 7 > 12 > vitamin C > 4 > vitamin E > 8 > 11 > 10. Notably, compounds 7 and 12 showed higher DPPH radical scavenging capacities than vitamin C, and compounds 7, 12, and 4 showed higher DPPH radical scavenging capacities than vitamin E. The ferric reducing antioxidant power (FRAP) assay is a method for measuring the total antioxidant activity of a sample. At low pH, the reduction of the TPTZ-Fe(III) complex to TPTZ-Fe(II), which has an intense blue color, can be monitored by measuring the change in absorption at 593 nm. The corresponding FeSO4 values are calculated using standard curves and regression equations, and a higher FeSO4 value indicates higher ferric reducing power. In this experiment, all of the isolated lignans were tested for total antioxidant activity using the FRAP assay from concentrations of 0.15 to 1.5 mM. Similarly to the results in the DPPH assay, compounds 4, 7, 8, 10, 11, and 12 showed concentration-dependent reducing capabilities at the tested concentrations, whereas the other

Figure 6. ECD spectra of compounds 3, 5, and (−)-dihydrocubebin.

Enzymatic hydrolysis of 4 with β-cellulase yielded only one aglycone, piperphilippinin VI (Figure S68, Supporting Information), which was identified from its 1D NMR spectra 19 and specific rotation, [α]20 Negative D −28 (c 0.1, CHCl3). Cotton effects at approximately 232 and 287 nm were observed in the ECD spectra of 4 and piperphilippinin VI (Figure 8), which confirmed the R configurations of C-8 and C-8′ in the two compounds.18 Acid hydrolysis of 4 with 4 mol/L aqueous TFA yielded only D-glucose, which was identified by gas chromatography after treatment with L-cysteine methyl ester hydrochloride and TMS derivatization (Figure 7). Thus, we propose that D-glucose is linked to either 9-OH or 9′-OH in piperphilippinin VI, which explains the two sets of resonances observed for 4 in the 1H and 13C NMR spectra. Thus, compound 4 was a mixture of (8R,8′R)-9-O-β-D-glucopyranosylpiperphilippinin VI (4a) and (8R,8′R)-9′-O-β-D-glucopyranosylpiperphilippinin VI (4b). Although the two sets of NMR resonances (Table 3) are distinguishable in both the 1D E



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Table 3. NMR Spectroscopic Data (400 MHz, CD3OD) for Compounds 4a and 4b 4aa position

δC, type

1 2 3 4 5 6 7a 7b 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′a 7′b 8′ 9′a 9′b 4-OCH3 3′,4′-OCH2O−

132.5, C 112.0, CH 147.4, C 144.0, C 114.4, CH 121.4, CH 34.1, CH2 2.60, m 40.3, CH 68.9, CH 3.53, m 134.8, C 109.0, CH 147.5, C 145.6, C 107.4, CH 121.8, CH 34.0, CH2 2.60, m 42.5, CH 61.3, CH2 3.56, m 54.9, CH3 100.6, CH2 9-O-glucose 103.2, CH 73.8, CH 76.7, CH 70.3, CH 76.5, CH 61.4, CH2

1″ 2″ 3″ 4″ 5″ 6″a 6″b

Figure 7. GC analysis of the aqueous layer of the acid hydrolysis reaction mixtures of compounds 3 and 4. (A) Separation of enantiomers of arabinose and glucose after treatment with L-cysteine methyl ester hydrochloride and TMS derivatization. (B) Gas chromatogram of component monosaccharides of 3 after treatment with L-cysteine methyl ester hydrochloride and TMS derivatization. (C) Gas chromatogram of component monosaccharide of 4 after treatment with L-cysteine methyl ester hydrochloride and TMS derivatization.

a

4ba

δH (J in Hz) 6.62, overlap

6.68, overlap 6.59, overlap 2.69, m 2.60, m 2.08, m 3.89, overlap 3.56, m 134.9, C 6.61, brs 147.5, C 145.6, C 6.68, overlap 6.59, overlap 2.69, m 2.60, m 1.99, m 3.62, m 3.53, m 3.79, s 5.89, brs 4.19, 3.20, 3.35, 3.26, 3.25, 3.86, 3.64,

d (7.6) m m m m m m

δC, type

δH (J in Hz)

132.5, C 112.2, CH 147.4, C 144.0, C 114.4, CH 121.3, CH 34.0, CH2

6.68, d (8.0) 6.59, overlap 2.69, m

42.9, CH 61.2, CH2

1.99, m 3.62, m

109.0, CH

6.61, s

107.4, CH 121.7, CH 34.2, CH2

6.68, d (8.0) 6.59, m 2.69, m

40.3, CH 69.0, CH

2.08, m 3.89, overlap

55.0, CH3 100.6, CH2 9′-O-glucose 103.2, CH 73.8, CH 76.7, CH 70.3, CH 76.5, CH 61.4, CH2

3.79, s 5.89, brs

6.62, overlap

4.19, 3.20, 3.35, 3.26, 3.25, 3.86, 3.64,

d (7.6) m m m m m m

The NMR data of compounds 4a and 4b might be interchangeable.

Table 4. IC50 Values of Compounds 4, 7, 8, 10, 11, and 12, Vitamin C, and Vitamin E in DPPH Assaya DPPH

compound 4 7 8 10 11 12 vitamin C vitamin E

150 μM

300 μM

IC50 ± SD (μM)

IC50 ± SD (μM)

26.51 15.70 33.17 181.10 37.46 23.33 24.68 26.94

± ± ± ± ± ± ± ±

0.98 1.10 1.12 11.10 0.35 1.66 0.04 0.99

51.41 34.64 75.09 359.70 83.39 44.45 59.33 62.09

± ± ± ± ± ± ± ±

2.44 0.46 1.51 20.45 0.86 0.89 0.52 0.29

a

IC50 values were calculated using the software Graph Pad Prism 5.0. The data are expressed as the means ± SD. Three independent experiments were performed. Vitamins C and E were used as the positive controls.

compounds did not exhibit such activities. As shown in Figure 9, compounds 4, 7, 8, 10, 11, and 12 and Trolox exhibited total antioxidant abilities in the following descending order at a concentration of 1.5 mM: 4 > 12 > 7 > Trolox > 8 > 11 > 10.

Figure 8. ECD spectra of compound 4 and piperphilippinin VI.

F



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various spectroscopic methods and hydrolysis reactions. The antioxidant activities of the isolated compounds were also investigated. Compounds 4, 7, 8, 10, 11, and 12, all of which contain phenolic hydroxy groups capable of behaving as hydrogen atom donors, exhibited antioxidant activities with varying potential in both the DPPH and FRAP assays, whereas the other compounds, which do not contain any phenolic hydroxy groups in their structures, did not show activity in these assays. Several different types of lignans,6,28−32 especially dibenzocyclooctenes, obtained from plants belonging to the Schisandraceae family have been reported to exhibit neuroprotective effects in various neurocytotoxic models, including those involving hydrogen peroxide, tert-butyl hydroperoxide, cobalt chloride, glutamate, homocysteine, and β-amyloid. In this study, compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aβ25−35induced SH-SY5Y cell death compared with the Aβ25−35-treated group. Although additional studies, including in vivo animal experiments in particular, are required to provide further evidence for the use of S. glaucescens fruit as a health food, the results of the current study further elucidate the bioactivity of S. glaucescens fruit.

Figure 9. Antioxidant activities of compounds 4, 7, 8, 10, 11, and 12 and Trolox in the FRAP assay.

Compounds 4, 12, and 7 exhibited higher total antioxidant activities than Trolox in this test. Alzheimer’s disease is a devastating neurodegenerative disorder that is estimated to affect more than 35 million people worldwide, and no effective treatment is available.27 Substantial evidence indicates that the amyloid β-protein (Aβ) plays a key role in the disease and may be a potential therapeutic target. Herein, all of the isolated lignans were tested for their neuroprotective effects against Aβ25−35-induced cell death in SH-SY5Y cells from 0.08 to 10 μM. As shown in Table 5, compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aβ25−35-induced SHSY5Y cell death compared with the Aβ25−35-treated group. In conclusion, two rare 7,8-seco-lignans, three new lignan glycosides, and 10 known lignans were isolated from the fruit of S. glaucescens Diels. The absolute configurations of the two 7,8seco-lignans were determined for the first time through comparison of their experimental and calculated ECD spectra. Compounds 1 and 2 were determined to have the same structures as the known compounds schisandlignans B and A, respectively, based on a comparison of their spectroscopic data and specific rotations. The molecular structures and absolute configurations of the new compounds were determined using

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV spectra were measured on a Varian Cary 50 Scan UV/vis spectrophotometer. ECD spectra were recorded on a Jasco J-810 spectropolarimeter. IR spectra were recorded on a Bruker VERTEX 70 FT-IR microscopic spectrometer. NMR spectra were recorded on a Bruker-AM-400 spectrometer. HRESIMS was performed on a Thermo Scientific LTQOrbitrap XL mass spectrometer. MPLC was performed using a Buchi pump module C-605. Column chromatography was performed on silica gel (200−300 or 300−400 mesh; Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden), or MCI gel (CHP20P, 75−150 μm; Mitsubishi Chemical Industries Ltd., Tokyo, Japan). HPLC was performed on an Agilent 1260 system. A reversed-phase HPLC column (Outstand C18, 250 × 4.6 mm i.d.; Intramax, Wuhan, China), a normal-phase HPLC column (SP-120−5-APS, 250 × 4.6 mm i.d.; Global Chromatography Co.,

Table 5. Neuroprotective Effects of Compounds 1−14 against Aβ25‑35-Induced Neuronal Cell Death in Dopaminergic Neuroblastoma SH-SY5Y Cellsa concentrations (μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 vitamin C

0.08

0.4

2

10

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

65.4 ± 2.4d 62.0 ± 2.9c 59.2 ± 1.4 62.1 ± 0.6 64.0 ± 1.1 62.2 ± 0.1c 63.8 ± 1.6 61.5 ± 2.0c 58.2 ± 1.1 59.9 ± 0.4 60.7 ± 1.6 63.1 ± 2.1c 58.1 ± 1.3 59.2 ± 0.4 55.6 ± 0.9

68.9 ± 2.2d 63.5 ± 3.0c 62.0 ± 0.7 61.5 ± 0.9 63.1 ± 2.1 61.1 ± 0.7b 69.2 ± 1.3c 61.8 ± 3.0c 59.8 ± 1.4 60.5 ± 0.5 62.1 ± 0.7 68.6 ± 1.4d 58.6 ± 0.9 60.3 ± 0.4 58.5 ± 1.0c

66.8 ± 2.1d 63.4 ± 3.0c 58.3 ± 1.3 61.0 ± 1.4 65.2 ± 1.7 61.9 ± 1.4c 68.3 ± 0.7c 64.4 ± 3.1c 58.7 ± 0.2 60.5 ± 0.2 65.2 ± 1.7d 73.3 ± 1.8d 60.4 ± 2.2 60.1 ± 1.1 58.0 ± 1.7c

58.3 53.2 58.0 60.6 65.0 58.1 62.2 57.0 57.4 57.7 58.6 55.4 58.2 60.0 54.1

0.9b 2.8 0.4 2.0 1.0 0.6 0.7 1.8 0.9 0.2 0.8 0.9 1.0 1.0 0.4

Aβ25−35 49.2 49.2 56.9 56.9 61.7 57.1 61.7 49.4 57.1 57.1 57.3 49.4 57.3 57.3 51.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 0.4 1.2 1.2 0.5 1.1 1.2 1.5 1.1 1.1 0.3 1.5 0.3 0.3 0.9

a

The data (cell viability, measured by MTT assay) were normalized and are expressed as a percentage of the control group, which is set to 100%. The data are expressed as the means ± SEM. Three independent experiments were performed. Vitamin C was used as the positive control. bp < 0.05. c p < 0.01. dp < 0.001, compared with the Aβ25−35-induced group. G



Article

8.65 × 10−3, CH3OH) λmax (θ) 220 (−10817, tr), 240 (730, pk), 265 (−1415, tr), 281 (−167, pk), 298 (−1183, tr) nm; IR (KBr) νmax 3423, 2963, 2921, 1714, 1595, 1513, 1443, 1366, 1259, 1158, 1106, 1068, 1033, 806, 763, 679 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 409.1256 [M + Na]+ (calcd for C21H22O7Na, 409.1263). (8R,8′R)-9-O-(6′-O-α-L-Arabinofuranosyl)-β-D-glucopyranosyldihydrocubebin (3): white, amorphous powder; [α]20 D −44 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 236 (3.97), 288 (3.96) nm; ECD (c 1.63 × 10−3, CH3OH) λmax (θ) 234 (−1852, tr), 260 (−218, pk), 275 (−380, tr), 296 (344, pk) nm; IR (KBr) νmax 3392, 2928, 2892, 1633, 1610, 1489, 1442, 1360, 1317, 1247, 1190, 1038, 927, 861, 810, 776, 719, 636 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 675.2252 [M + Na]+ (calcd for C31H40O15Na 675.2265). (8R,8′R)-9-O-β-D-Glucopyranosylpiperphilippinin VI (4a) and (8R,8′R)-9′-O-β-D-glucopyranosylpiperphilippinin VI (4b): white, amorphous powder; [α]20 D −18 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 231 (4.10), 284 (3.90) nm; ECD (c 1.578 × 10−3, CH3OH) λmax (θ) 214 (1729, tr), 218 (2312, pk), 232 (−1290, tr), 243 (−690, pk), 244 (−693, tr), 258 (−271, pk), 283 (−599, tr), 312 (−101, pk) nm; IR (KBr) νmax 3417, 2920, 2852, 1644, 1608, 1513, 1490, 1443, 1369, 1247, 1157, 1124, 1076, 1035, 926, 864, 811, 739 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 545.1984 [M + Na]+ (calcd for C26H34O11Na, 545.1999) and 1067.4086 [2 M + Na]+ (calcd for C52H68O22Na, 1067.4100). ECD Calculations. The conformational spaces for the 7′R,8′S and 7′S,8′R isomers of compounds 1 and 2 were explored using the BALLOON program with a genetic algorithm.33 The conformations generated by BALLOON were subjected to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program.34 Duplicate conformations were identified and removed when the root-mean-square (RMS) distance was less than 0.5 Å for any two geometry-optimized conformations. The remaining conformations were further optimized at the B3LYP/6-31G* level of theory in MeOH with the IEFPCM solvation model using Gaussian 09,35 and the duplicate conformations emerging after these calculations were removed according to the same RMS criteria above. The harmonic vibrational frequencies were calculated to confirm the stability of the final conformers (Figures S69 and S70, Supporting Information). The oscillator strengths and rotational strengths of the 20 weakest electronic excitations of each conformer were calculated using the TDDFT methodology at the B3LYP/6311G** level of theory with MeOH as the solvent with the IEFPCM solvation model implemented in Gaussian 09. The ECD spectra for each conformer were simulated using a Gaussian function with a bandwidth σ of 0.45 eV. The spectra were combined after Boltzmann weighting according to their population contributions (Tables S1 and S2, Supporting Information). Enzymatic Hydrolysis. To obtain the aglycones without the occurrence of structural changes, compounds 3 and 4 (each 5 mg) were treated with β-cellulase (from Trichoderma viride, 0.5 mg for both 3 and 4) in aqueous solution (2 mL for both 3 and 4) at 50 °C for 3 h. After extraction with EtOAc (5 mL × 5), the combined EtOAc layer was evaporated to dryness and analyzed by TLC and HPLC. Semipreparative HPLC was used to isolate the aglycones of the two compounds. Acid Hydrolysis and GC Analysis. GC was utilized to determine the absolute configurations of the saccharides of compounds 3 and 4, according to a slightly modified literature procedure.36 Briefly, 3 and 4 (each 2 mg) were heated at 90 °C with 4 mol/L aqueous TFA (2 mL) for 3 h. The reaction mixture was freeze-dried overnight and diluted with H2O (5 mL). After extraction with EtOAc (5 mL × 4), the aqueous layer was evaporated to dryness, and the residue was dissolved in pyridine (1 mL) and reacted with 5 mg of L-cysteine methyl ester hydrochloride at 60 °C for 1 h. Subsequently, 0.9 mL of hexamethyl disilazane−trimethylchlorosilane (2:1) was added, and the mixture was heated at 60 °C for 1 h. The mixture was washed with H2O (5 mL × 2) and extracted with n-hexane, and the n-hexane layer was analyzed by GC-FID using an OV-17 capillary column (30 m × 0.32 mm × 0.5

Ltd., Suzhou, China), and a chiral HPLC column (Chiralcel OD-H, 150 × 4.6 mm i.d.; Daicel Chemical Industries, Ltd., Tokyo, Japan) were used for analytical purposes. A YMC-Pack ODS-A HPLC column (C18, 250 × 10 mm i.d.; YMC, Tokyo, Japan) was used for semipreparative purposes. Gas chromatography was conducted on an Agilent 7820A system. An OV-17 capillary column (30 m × 0.32 mm × 0.5 μm; Lanzhou Zhongke Antai Analysis Technology Co., Ltd., Lanzhou, China) was used for the GC analysis. The DPPH, FRAP, and MTT assays were all performed on a BioTek Synergy 2 multimode microplate reader. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT), amyloid β-protein fragment 25−35 (Aβ25−35), 2,2-diphenyl-1-picrylhydrazyl, L-ascorbic acid (vitamin C), α-tocopherol (vitamin E), L-cysteine methyl ester hydrochloride, hexamethyl disilazane, trimethylchlorosilane, D-glucose, L-glucose, D-arabinose, and L-arabinose were purchased from Aladdin Industry Corporation, Shanghai, China. β-Cellulase (from Trichoderma viride, activity ≥35 u/mg) was manufactured by Shanghai YuanYe Biotechnology Co., Ltd., Shanghai, China. Trifluoroacetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. A FRAP assay kit was purchased from the Beyotime Institute of Biotechnology, Nantong, China. Plant Material. The fruit of S. glaucescens was collected in the Shennongjia Mountains of Hubei Province, China, in September 2011 and was identified by Mr. Shi-Gui Shi (Shennongjia Institute for Drug Control). A voucher specimen (ID 20110905) was deposited in the Herbarium of Materia Medica, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. Extraction and Isolation. The air-dried fruit of S. glaucescens (15 kg) was extracted with 95% ethanol at room temperature, and the solution was concentrated in vacuo to yield a crude extract (750 g). The extract was added to 1.5 L of deionized H2O and sequentially partitioned with petroleum ether (PE), EtOAc, and n-BuOH. The petroleum ether-soluble fraction (210 g) was chromatographed on a silica gel column eluting with PE−EtOAc (from 1:0 to 6:4) to afford 10 fractions. Fraction 6 (18.5 g) was chromatographed on silica gel (CHCl3−EtOAc, from 1:0 to 3:1) and Sephadex LH-20 (CH2Cl2− MeOH, 1:1) followed by semipreparative HPLC (MeOH−H2O, 80:20) to yield compounds 1 (11 mg) and 2 (8 mg). The EtOAcsoluble fraction (150 g) was chromatographed on a silica gel column eluting with PE−EtOAc (from 99:1 to 1:2) to afford 14 fractions. Fraction 2 (12.5 g) was chromatographed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, from 15:1 to 10:1) followed by semipreparative HPLC (MeOH−H2O, 85:15) to yield compounds 6 (16 mg), 8 (560 mg), 9 (18 mg), and 14 (26 mg). Fraction 3 (4.8 g) was chromatographed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, 6:1) columns to yield compound 13 (70 mg). Fraction 4 (15.3 g) was chromatographed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, from 9:1 to 3:1) columns to yield compounds 7 (240 mg) and 12 (110 mg). Fraction 6 (5.3 g) was chromatographed on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and silica gel (PE−EtOAc, 4:1) followed by semipreparative HPLC (MeOH−H2O, 77.5:22.5) to yield compounds 10 (15 mg) and 11 (22 mg). Fraction 10 (19.7 g) was chromatographed on MCI gel (MeOH−H2O, from 40:60 to 100:0) and silica gel (CH2Cl2−MeOH, from 85:15 to 60:40) followed by semipreparative HPLC (MeOH−H2O, 55:45) to yield compounds 3 (42 mg), 4 (11 mg), and 5 (350 mg). (7′R,8′S)-3,4-Dimethoxy-3′,4′-methylenedioxy-7,8-seco-7,7′-epoxylignan-7,8-dione (1): colorless oil; [α]20 D −39 (c 0.9, CHCl3); UV (CH3OH) λmax (log ε) 228 (4.73), 257 (4.35), 288 (4.03) nm; ECD (c 9.48 × 10−3, CH3OH) λmax (θ) 208 (−5231, tr), 234 (3957, pk), 259 (−1864, tr), 281 (697, pk), 294 (−550, tr) nm; IR (KBr) νmax 3433, 2922, 1713, 1598, 1512, 1446, 1416, 1270, 1248, 1221, 1175, 1137, 1100, 1037, 814, 764, 678 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 409.1258 [M + Na]+ (calcd for C21H22O7Na, 409.1263). (7′R,8′S)-3,4-Methylenedioxy-3′,4′-dimethoxy-7,8-seco-7,7′-epoxylignan-7,8-dione (2): colorless oil; [α]20 D −73 (c 1.5, CHCl3); UV (CH3OH) λmax (log ε) 226 (4.56), 259 (4.13), 288 (3.85) nm; ECD (c H



Article

μm) under the following conditions. The injector temperature was 250 °C. The column temperature was initially 200 °C and gradually increased at a rate of 5 °C/min up to 250 °C and kept at 250 °C for 10 min. For detection, a flame ionization detector was used at 250 °C. Helium was used as the carrier gas at a constant flow rate of 1 mL/ min. The derivatives of D-glucose, L-glucose, D-arabinose, and Larabinose were analyzed simultaneously. DPPH Radical Scavenging Activity. The abilities of the compounds to scavenge DPPH radical were evaluated as previously reported with minor modifications.37 Briefly, reaction mixtures containing DPPH (final concentrations of 150 or 300 μM) and the compounds (final concentrations of 6.25, 12.5, 25, 50, 75, and 100 μM) in EtOH (with 5% DMSO) were placed in the wells of 96-well plates. An EtOH solution (with 5% DMSO) was used as a control. Vitamins C and E were used as the positive controls. The absorbance of the reaction mixture at 517 nm was measured at steady state after 30 min of incubation at room temperature in the dark using a microplate reader. All tests were performed in triplicate, and the results were averaged. IC50 is defined as the concentration of compound that scavenges 50% of the DPPH radical and was calculated using the software program Graph Pad Prism 5.0 through nonlinear regression (curve fitting) and the equation [log (inhibitor) vs normalized response] for dose response (variable slope). Ferric Reducing Antioxidant Power Assay. The FRAP assay was performed according to the manufacturer’s instruction. Briefly, a fresh working solution was prepared by mixing TPTZ solution, TPTZ diluent, and detective buffer in a ratio of 1:10:1 (v/v). The working solution was warmed to 37 °C before use. Then, 5 μL samples at concentrations from 0.15 to 1.5 mM were mixed with 180 μL of FRAP working solution and kept for 5 min at 37 °C. The absorbances of the reaction mixtures were recorded at 593 nm. A standard curve was prepared using FeSO4 at concentrations from 0.15 to 5.00 mM, and the total antioxidant activities of the tested compounds are expressed in terms of FeSO4 values, which were calculated using the standard curves. Neuroprotective Activity. The neuroprotective activities of the isolated compounds were measured according to a published procedure.38 Human dopaminergic neuroblastoma SH-SY5Y cells purchased from the cell bank of the Basic Medical College of Huazhong University of Science and Technology were maintained in DMEM culture medium (Solarbio, Beijing, China) with 10% calf serum (Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 75 U/ mL streptomycin in a 37 °C incubator (Heal Force HF-90, Hong Kong) in 5% CO2. Cells were trypsinized with 0.25% trypsin, counted, and seeded in 96-well culture plates (1 × 104 cells/well). After 24 h of incubation, the cells were pretreated with compounds 1−14 (0.08, 0.4, 2, and 10 μM) for 2 h before incubation in medium containing Aβ25−35 (1 μM). After 24 h of treatment, 15 μL of MTT (5 mg/mL) was added. For the MTT assays, the supernatant was discarded, and DMSO (100 μL/well) was added. The 96-well plate was vibrated on a microvibrator for 10 min, and the optical density at 570 nm was measured using a microplate reader. All samples were cultured in triplicate. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Newman−Keuls multiple comparison method. The data are expressed as the means ± SEM of three assays.

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details of isomers 7′R,8′S and 7′S,8′R of compounds 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86 27 83657870. Fax: 86 27 83692739. E-mail: ruanhl@ mails.tjmu.edu.cn. Author Contributions #

H.-Y. Yu and Z.-Y. Chen contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff at the analytical and testing center of Huazhong University of Science and Technology for collecting the spectroscopic data. This work was supported by the Natural Science Foundation of China (Nos. 31270394, 81072547, and 21102050), the Ministry of Science and Technology of the People’s Republic of China (International Cooperative Project, Grant No. 2010DFA32430), and the Wuhan Program for Science and Technology (No. 2013060501010158).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13 C, DEPT135, HSQC, HMBC, COSY, NOESY, HRESIMS, IR, UV, and ECD spectra of compounds 1−4, 1 H/13C NMR and ECD spectra of (−)-dihydrocubebin and piperphilippinin VI, DPPH radical scavenging activity of the ethanol extract of the fruit of Schisandra glaucescens Diels, neuroprotective effect of the ethanol extract of S. glaucescens fruit against Aβ25−35-induced SH-SY5Y cell death, HPLC analyses of the EtOAc layers of the enzymatic hydrolysis reaction mixtures of compounds 3 and 4, ECD calculation I



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Lignans from the Fruit of Schisandra glaucescens with

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