Fitoterapia 103 (2015) 63–70
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New lignans from the roots of Schisandra sphenanthera Kan Jiang a, Qiu-Yan Song a, Shou-Jiao Peng a, Qian-Qian Zhao a, Guang-Da Li a, Ya Li a,⁎, Kun Gao a,b,⁎⁎ a b
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People's Republic of China School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University; Ningbo 315100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Accepted in revised form 13 March 2015 Accepted 14 March 2015 Available online 19 March 2015 Keywords: Schisandra sphenanthera Lignans Antioxidant activity RBCs
a b s t r a c t Nine new lignans (1–8, 13) and five known ones (9–12, 14) have been isolated from the roots of Schisandra sphenanthera and were tested for their capacity to scavenge 2,2-diphenyl-1-Picrylhydrazyl (DPPH). Of these lignans tested, compounds 1, 7, 8 and 13 exhibited noteworthy antioxidant activity with IC50 values of 92, 115, 35 and 48 μg/mL, respectively. The anti-oxidative haemolysis of human red blood cells (RBCs) activity of the most active compound 8, which is similar to that of vitamin C, was evaluated. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Schisandra belonging to the plant family Schisandraceae has long been used in traditional Chinese medicine for the treatment of hepatitis, diabetes, diarrhea, cough, etc [1,2]. It has been reported that many kinds of lignans had been isolated from this genus, such as dibenzocyclooctadiene, dibenzylbutane and tetrahedrofurane types, and some of them exhibited antimicrobic, antiviral, herbicidal, antifeedant and insulin sensitivity-improving activities [3–9]. Schisandra sphenanthera is widely distributed in the southwest region of China [10]. Previous phytochemical studies on the fruits, stems and leaves of S. sphenanthera have led to the isolation of a variety of lignans and triterpenoids [11,12], while the chemical components of the roots of S. sphenanthera have never been reported. In this paper, we conducted a systematic bioactivity-guided isolation of the 70% aqueous acetone extract from the roots of S. sphenanthera and new compounds with ⁎ Corresponding author. Tel.: +86 931 8912592; fax: +86 931 8912582. ⁎⁎ Correspondence to: K. Gao, State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People's Republic of China. Tel.: +86 931 8912592; fax: +86 931 8912582. E-mail addresses: [email protected] (Y. Li), [email protected] (K. Gao).
http://dx.doi.org/10.1016/j.fitote.2015.03.015 0367-326X/© 2015 Elsevier B.V. All rights reserved.
natural anti-oxidative activity were discovered for the first time. The systematic isolation led to the identification of eight new tetrahydrofuran lignans (1–8), one new butane-type lignan (13) and five known ones (schiglaucin B, 9 [13]; schiglaucin A, 10 [12]; epoxyzuonin A, 11 [14]; talaumidin, 12 [15]; myristargenol A, 14 [16]) from the anti-oxidative active EtOAc-soluble fraction of the 70% aqueous acetone extract. The antioxidant activity of these compounds was assessed by DPPH scavenging experiments. Of these lignans tested, compound 8 exhibited noteworthy antioxidant activity and its capacity to protect human red blood cells (RBCs) from oxidative haemolysis was evaluated, which is similar to that of vitamin C. 2. Experimental 2.1. General Column chromatography: silica gel (200–300 mesh; Qingdao Marine Chemical Factory, China); macroporous resin (HP-20, Kabuskiki Kaisha); Sephadex LH-20 (Amersham Pharmacia Biotech). TLC (Thin layer chromatography): silica gel GF254 plates (10–40 μm; Qingdao Marine Chemical Factory, China). A Waters 1525 series instrument equipped with a YMC-Pack ODSA column (250 × 10 mm, 5 μm) was used for semipreparative
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K. Jiang et al. / Fitoterapia 103 (2015) 63–70
HPLC. 1H and 13C NMR Spectra: Bruker AM-400BB (400 MHz) and a Varian Mercury-600BB (600 MHz) NMR spectrometer; recorded in CDCl3; δ in ppm relative to TMS; J values in Hz. HRESI-MS: Bruker APEX-II mass spectrometer; in m/z. Optical rotations: Perkin-Elmer 341 polarimeter. IR Spectra: Nicolet FTIR-360 spectrometer. CD spectra were obtained on an Olis DSM 1000 spectrometer.
was separated on semipreparative HPLC (MeOH: H2O, 1: 3, v:v) to yield compounds 9 (1.3 mg) and 10 (2.5 mg). In the same way, compounds 11 (3.6 mg) and 12 (2.7 mg) were obtained from FrC.7.2 (0.21 g). Further separation of FrC.7.3 (0.13 g) by reversed-phase C18 silica gel column, eluted with MeOH: H2O (4: 1, v:v), led to the appearance of compounds 13 (2.3 mg) and 14 (4.2 mg).
2.2. Plant material
2.3.1. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)8-hydroxyl-7′-(5′-hydroxy-3′-methoxyphenyl)-8′-methyltetrahydrofuran (1) Yellow oil; [α]25 D +60 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 228 (0.15) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 389.1977 [M + H]+ (calcd for C21H24O7).
Roots of S. sphenanthera were collected from Taibai Mountain in Shaanxi Province of China, in the summer of 2011. The plant was identified by Guo-Liang Zhang, at the School of Life Sciences, Lanzhou University, China. A voucher (No. 11-07) specimen was deposited with the Natural Organic Academy of Lanzhou University. 2.3. Extraction and isolation The air-dried and powdered roots of (7 kg) were extracted three times (each time for 7 days) with 70% aqueous acetone at room temperature. The solvent was evaporated under reduced pressure, producing an extract (685 g), which was found to have antioxidant effect in the DPPH scavenging assay. The crude extract was dissolved in hot H2O (60 °C, 2 L) to accelerate dissolution and then partitioned with petroleum ether (PE), EtOAc and n-BuOH at room temperature, respectively. The EtOAc-soluble fraction was found to be more anti-oxidative than the PE and n-BuOH-soluble fraction, exhibiting that the EtOAc-soluble fraction included the majority of the active components. Thus, the EtOAc-soluble fraction was further purified and investigated. Initially, the EtOAc-soluble fraction was applied to macroporous resin (HP-20,3 L) with a gradient of H2O: EtOH (70: 30, 50: 50, 20: 80, v:v) as eluent, and three fractions (A, B, and C) were collected according to the TLC analysis. Fraction C (248 g) was subjected to silica gel column chromatography (200–300 mesh, 2000 g) eluted with a gradient of PE (petroleum ether): acetone (100: 0, 50: 1, 20: 1, 10: 1, 5: 1, 2: 1, 1: 1, 0: 100, v:v) to give eight fractions, FrC.1– FrC.8. Using H2O: MeOH (1: 1, 2: 5, 3: 10, 1: 5, 0: 1, v:v) as eluent, FrC.6 (6.8 g) was separated on a column of reversedphase C18 silica gel, thus resulting in five subfractions. FrC.6.1– FrC.6.5. FrC.6.1 (1.3 g) was separated on a Sephadex LH–20 (CH3OH) to obtain three fractions (FrC.6.1.1–FrC.6.1.3). FrC.6.1.1 (0.1 g) and FrC.6.1.3 (0.12 g) were further fractionated on semipreparative HPLC (MeOH: H2O, 1: 3, v:v) to yield compounds 1 (3 mg) and 2 (2.1 mg). Compounds 3 (2.2 mg) and 4 (1.8 mg) were obtained from FrC.6.1.2 (0.15 g) by silica gel column chromatography (PE: acetone, 10: 1 to 5: 1, v:v). FrC.6.3 (2.1 g) was separated on a silica gel column and eluted with gradient mixtures of PE: acetone (from 8: 1 to 5: 1, v:v) to obtain compound 5 (3.2 mg) and FrC.6.3.1 (0.2 g). The separation of FrC.6.3.1 was done on semipreparative HPLC (MeOH: H2O, 1: 4, v:v) to obtain compounds 6 (2.8 mg) and 7 (1.9 mg). FrC.6.4 (0.15 g) was separated on silica gel column chromatography (PE: acetone, 20: 1 to 5: 1, v:v), and then on Sephadex LH-20 (CHCl3: MeOH, 1: 1, v:v) to give compound 8 (2.3 mg). FrC.7 (2.8 g) was applied to a silica gel column and eluted with various gradients of PE: acetone (15: 1, 10: 1, 5: 1, v:v) to give three fractions (FrC.7.1–FrC.7.3). FrC.7.1 (0.15 g)
2.3.2. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxy-phenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′,5′-trimethoxyphenyl)tetrahydrofuran (2) Yellow oil; [α]25 D +50 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 232 (0.42) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 433.1613 [M + H]+ (calcd for C23H28O8). 2.3.3. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)8-hydroxyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)tetrahydrofuran (3) Yellow oil; [α]25 D +50 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 226 (0.25) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 387.1824 [M + H]+ (calcd for C21H22O7). 2.3.4. (7S, 8S, 7′R, 8′S)-(7, 8-trans-8, 8′-trans-7′, 8′-trans)-7methoxyl-7-(3,4- methylenedioxy-phenyl)-8 methyl -8′-methyl7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran (4) Yellow oil; [α]25 D +20 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 232 (0.25) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 409.1712 [M + H]+ (calcd for C22H26O6). 2.3.5. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4,5-trimethoxyphenyl)8methyl -8′-methyl-7′-(3′,4′- methylenedioxyphenyl)tetrahydrofuran (5) Yellow oil; [α]25 D +40 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 227 (0.24) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 417.1823 [M + H]+ (calcd for C23H28O7). 2.3.6. (7S, 8S, 7′R, 8′S)-7,8-epoxide-7-(3,4-methylenedioxyphenyl)8 methyl -8′-methyl-7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran (6) Yellow oil; [α]25 D +20 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 227 (0.32) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 371.1629 [M + H]+ (calcd for C21H22O6). 2.3.7. (7S, 8S, 7′R, 8′S)-7,8-epoxide -7-(3,4-methylenedioxyphenyl)8-methyl -8′-methyl-7′-(4′-hydroxy-3′-methoxyphenyl)tetrahydrofuran (7) Yellow oil; [α]25 D +40 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 229 (0.18) nm; 1H and 13C NMR data, see Tables 1
5.41 5.38, 5.37
5.96 3.81 5.93 5.99 3.91 5.99 3.88 3.90
5.58
1.57(m) 0.81(d,6.8)
6.78(d,8.0) 6.52(dd,8;0,1.6) 2.74(dd,13.2,3.6) 2.11(dd,13.2,10.8)
6.84(d,8.0) 6.79(dd,8.0) 4.59(d,8.8) 2.41(m) 0.99(d,6.8) 6.90(d,8.0) 6.96(d,8.0) 4.87(d,10.4) 2.47 (dd,10.4,6.8) 1.01(d,6.8)
6.82(d,8.0) 7.02(d,8.0) 4.59(d,9.6) 2.18(m) 0.98(d,6.8) 3.26 5.96 3.85 3.89 3.89 6.81(d,8.0) 6.95(d,8.0) 4.61(d,9.6) 2.18(m) 0.96(d,6.8) 3.24 5.96 3.89 3.92 6.80(d,8.0) 6.85(d,8.0) 4.83(d,10.4) 2.44 (dd,10.4,6.8) 0.95(d,6.8) 3.20 5.99;5.96
1.70(dd,11.6,6.8) 1.08(d,6.8) 7.00(s)
6.67(s) 4.84(d,10.0) 2.48 (dd,10,6.8) 0.99(d,6.8) 3.25 6.00 3.85 3.89 3.89 6.91(s) 4.84(d,10.0) 2.47 (dd,9.6,6.8) 0.96(d,6.8) 3.22 5.99 3.92
5.60
1.31(s) 6.67(s) 1.31(s) 6.91(s) 6.98(s)
1.29(s) 6.94(s)
1.70(dd,11.6,6.8) 1.07(d,6.8) 7.01(s)
1.32(s) 7.08(d,1.6)
1.31(s) 7.04(s)
6.86(d,8.0) 6.99(dd,8.0,1.6) 4.89(d,10.0) 2.49(m) 0.99(d,6.8)
6.81(s) 4.64(d,7.6) 1.78(m) 1.07(d,6.8) 6.43(d,1.6) 6.84(d,8.0) 6.93(dd,8.0,1.6) 4.04 (d,9.2) 1.71 (m) 0.61(d,6.8) 6.77(s) 6.78(s) 6.86(d,8.0) 7.07(d,8.0) 6.85(d,8.0) 7.10(d,8.0)
6.88(d,8.0) 7.06(d,8.0)
6.86(d,8.0) 7.02(d,8.0)
6.84(d,8.0) 7.20(d,8.0)
6.84(d,8.0) 7.17(d,8.0)
6.88(d,1.2) 6.82(d,1.6) 6.86(d,1.6)
8
7.14(s)
7
6.78(s) 7.00(s)
6 5 4
7.05(s) 7.05(s)
7.04(s)
2.3.9. (7, 8-trans-8, 8′-cis)-7-(3, 4-dihydroxyphenyl)-7′-(3, 4methylenedioxyphenyl)-8, 8′-dimethylbutan-7-ol (13) Pale white amorphous solid; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 367.1618 [M + H]+ (calcd for C20H24O5).
A 2,2-diphenyl-1-Picryl‐hydrazyl (DPPH) assay was performed as previously described. L-Ascorbic acid (33 μg/mL) was used as the positive control. Reaction mixtures containing an methanol solution of 200 μM DPPH (100 μL) and two fold serial dilutions of the sample (dissolved in 100 μL of methanol, with sample concentrations in the range of 2–500 μg/mL) were placed in a 96-well micro-plate and incubated at 30 °C for 30 min in the dark. After incubation, the absorbance was read at 517 nm with a Tecan Infinite 200 Pro Micro-plate Reader, and the means of three readings were calculated. The radicalscavenging activity (RSA) was calculated as a percentage of DPPH˙ discoloration using the equation: RSA % = [Abs. of control-(Abs. of sample-Abs. of blank) / Abs. of control] × 100%. The IC50 value is the concentration of the sample required to scavenge 50% of the DPPH radicals and was obtained by extrapolation from a linear regression analysis. Tests were carried out in triplicate.
2.5. Oxidative haemolysis of human red blood cells (RBC) assay
1
3
2.3.8. (7S, 8S, 7′R, 8′S)-7-(3,4- methylenedioxyphenyl)-8methyl -8′-methyl-7′-(3′,4′- dihydroxy-phenyl)-tetrahydrofuran (8) Pale yellow amorphous solid; [α]25 D +10 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 228 (0.15) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 329.1713 [M + H]+ (calcd for C19H20O5).
2.4. DPPH radical-scavenging assay
2 4 5 6 7 8 9 2′ 4′ 5′ 6′ 7′ 8′ 9′ 7-OMe −OCH2O− OMe OMe OMe OH
2
65
and 2; positive-ion HRESIMS m/z 357.1624 [M + H]+ (calcd for C20H20O6).
Position
Table 1 1 H NMR spectroscopic data of compounds 1–8, 13 [400 MHz, CDCl3, δ (ppm)]. Proton coupling constants (J) in Hz are given in parentheses.
7.18(s)
13
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
The 5% suspension of RBCs in phosphate-buffered saline (PBS, pH 7.4) was incubated under air atmosphere at 37 °C for 5 min; into this a PBS solution of AAPH was added to initiate haemolysis. The reaction mixture was shaken gently while being incubated at 37 °C. The extent of haemolysis was determined spectrophotometrically as described previously. Briefly, aliquots of the reaction mixture were taken at appropriate time intervals, diluted with 0.15 M NaCl, and centrifuged at 2000 rpm for 10 min to separate the RBCs. The percentage haemolysis was determined by measuring the absorbance of the supernatant at 540 nm and compared with that of a complete haemolysis by treating the same RBC suspension with distilled water. In the case of anti-haemolysis experiments, lignans and Vc dissolved in dimethyl sulfoxide (DMSO) were added and incubated before addition of AAPH. The final concentration of DMSO was 0.1% (v/v) and did not interfere with the determination. Every experiment was repeated three times and the results were reproducible within 10% deviation.
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K. Jiang et al. / Fitoterapia 103 (2015) 63–70
Table 2 13 C NMR spectroscopic data of compounds 1–8, 13 [100 MHz, CDCl3, δ (ppm)]. Carbon
1
2
3
4
5
6
7
8
13
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 7-OMe −OCH2O− −OMe −OMe −OMe
133.4 108.2 148.2 148.1 108.2 121.2 111.8 82.6 20.1 130.3 114.3 146.9 110.1 145.7 114.3 88.0 48.9 8.6 50.5 101.5 56.1
136.9 107.9 147.9 147.8 111.7 120.3 111.8 82.3 19.8 134.2 112.5 143.7 153.2 143.7 107.7 88.0 48.7 8.4 50.2 101.2 58.8 56.0 58.9
133.4 107.6 149.4 148.9 108.1 120.9 111.2 82.5 19.9 135.4 107.2 147.7 147.1 107.9 121.2 87.5 49.2 8.3 50.3 101.5;101.0
135.4 107.6 149.4 149.0 108.2 120.2 111.0 54.9 14.3 134.4 110.4 147.3 147.9 109.2 120.2 89.7 47.6 10.5 50.1 101.3 56.0 56.2
132.5 107.5 152,4 137.8 152.3 108.2 111.2 52.6 14.5 133.2 109.3 146.5 147.1 109.5 120.1 88.5 49.2 10.2 50.2 101.5 55.9 56.1
134.2 110.3 149.4 149.0 108.7 119.9 111.0 82.2 20.2 133.4 108.3 148.5 147.9 108.1 121.5 87.7 49.5 8.7
133.3 108.0 148.2 147.6 108.4 120.4 114.0 81.9 19.9 133.1 107.8 146.6 145.3 109.4 121.3 87.5 49.2 8.4
131.5 113.3 147.5 146.8 108.1 119.8 85.7 47.8 12.0 129.8 115.2 144.5 146.2 107.0 120.5 84.9 43.6 9.7
138.5 111.5 148.2 113.2 143.8 114.1 75.8 45.6 10.9 133.7 108.2 147.2 146.4 107.0 120.3 37.8 36.9 17.8
101.6 56.1 56.2
101.3 55.9
101.0
101.3 56.0
3. Results and discussion The EtOAc-soluble fraction from air-dried and powdered roots of S. sphenanthera was repeatedly separated by column chromatography (silica gel and Sephadex LH-20) and semipreparative HPLC yielded nine new compounds (1–8, 13) and five known compounds (9–12, 14) (Fig. 1). Compound 1 was isolated as a yellow oil and its molecular formula was established as C21H24O7 by HR-ESI-MS at m/z 389.1977 [M + H]+, indicating 10° of unsaturation. The signals in the 1H-NMR spectrum for aromatic protons (δH 7.10, s, 1H; 6.85, d, J = 8.0 Hz, 1H; 7.05, d, J = 8.0 Hz, 1H; 6.91, s, 2H; 6.98, s, 1H), in conjunction with the signals corresponding to a methoxyl group (δH 3.92, 3H), a hydroxyl group (δH 5.60) and a methylenedioxyl group (δH 5.95 br s) indicated that the
substitution pattern of the two aromatic rings was both trisubstituted (5′-hydroxy-3′-methoxyphenyl and 3,4methylene-dioxyphenyl for the two aromatic rings). Other characteristic proton signals showed a methine group (δH 4.82,d, J = 10.0 Hz), a tertiary methyl group (δH 1.31, s) attached to a carbon carrying oxygen atom, a secondary methyl group (δH 0.94, d, J = 6.8 Hz), and another upfield methoxyl group. The above information, along with the analysis of its 13C-NMR spectroscopic data (Table 2) suggested that 1 was an asymmetric tetrahydrofuran lignan [17–19]. Extensive analysis of 1H-1H COSY and HMBC spectroscopic data led to the establishment of the planar structure of compound 1. The HMBC correlations (Fig. 2) from the methoxyl protons at δH 3.92 and hydroxyl proton at δH 5.60 to C-3′ and C-5′, and from the methylenedioxyl protons to C-3 and C-4 indicated that the
Fig. 1. The structures of compounds 1–14.
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
Fig. 2. Key HMBC correlations of compound 1.
methoxyl group and hydroxyl group were attached at C-3′ and C-5′, respectively while the methylenedioxyl group was attached at C-3 and C-4. The upfield shifted methoxyl
67
group (δH 3.22 and δC 50.46) was deduced to be attached at C-7 established by HMBC correlation (Fig. 2) from the methoxyl protons to C-7. In addition, the HMBC correlations from H3-9, H3-9′, H-7′ to the oxygenated quaternary carbon C-8 (δC 82.55), along with the analysis of its molecular formula, indicated that a hydroxyl group was attached to C-8 of 1. The relative configuration of 1 was established on the NOE cross-peaks of H-8′ with Me-9, H-2′, H-6′ and OMe-7, which suggested that H-8′, Me-9, OMe-7 were cofacial, and H-7′, Me-9′ and OH-8 were on the other side. The absolute configuration of 1 was established on the basis of the circular dichroism (CD) curve from 200 to 400 nm in its CD (Fig. 3) spectrum and the optical rotation. Compound 1 showed a positive Cotton effect at 209, 237 nm and a negative Cotton effect at 251 nm, consistent with those of schisphenlignan H
Fig. 3. CD and UV spectra of 1–8.
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[10] and the optical rotation value of 1 ([α]25 D + 60) is in agreement with that of (+)-neo-olivil ([α]25 D + 49) [20]. Thus, the structure of 1 was established as (7S, 8S 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)-8hydroxyl-7′-(5′-hydroxy-3′-methoxyphenyl)-8′-methyltetrahyd-ofuran [21]. Compounds 2 and 3 were both obtained as a yellow oil and showed molecular ions at m/z 433.1613 [M + H]+ and 387.1824 [M + H]+ in the HRESIMS, corresponding to the molecular formula C23H28O8 and C21H22O7, respectively. The 1H and 13C NMR spectra of 2 and 3 were very similar to those of 1, and the obvious chemical shift differences resulted from the substituents variations in the aromatic rings, which suggested that compounds 2 and 3 were both asymmetric tetrahydrofuran lignans. The 1H and 13C NMR spectroscopic data revealed the presence of three methoxyl groups located at C-3′, C-4′ and C-5′ in 2 instead of the methoxyl group and the hydroxyl group located at C-3′ and C-5′ in 1, which could be deduced from the HMBC correlations from the three methoxyl protons to C-3′, C4′ and C-5′, respectively. For compound 3, a methylenedioxyl group located at C-3′ and C-4′ replaced the methoxyl group and the hydroxyl group located at C-3′ and C-5′ in 1, which was determined by the HMBC correlations from the methylenedioxyl protons to C-3′ and C-4′. Thus, the structures of 2 and 3 were elucidated as 7-methoxyl-7-(3,4-methylenedioxy-phenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′,5′-trimethoxyphenyl)-tetrahydrofuran and 7-methoxyl-7-(3,4-methylenedioxyphenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)-tetrahydrofuran, respectively [22]. On the basis of NOE cross-peaks, the relative configurations of 2 and 3 were determined as 7,8-trans-8,8′-trans-7,8-trans. Compound 4 was also obtained as a yellow oil and its molecular formula of C22H26O6 was established from HR-ESIMS ([M + Na]+, m/z 409.1712) and 13C-NMR spectroscopic data (Table 2), indicating 10° of unsaturation. Through a comparison of its 1H and 13C NMR spectral data with those of 1, compound 4 was also assigned as an asymmetric tetrahydrofuran lignin and its 1H NMR spectroscopic data exhibited two 1,3,4-trisubstituted aromatic systems. Besides the signals of the two aromatic rings and the tetrahydrofuran ring, the signals of three methoxyl protons and a methylenedioxyl proton could also be observed in the 1H and 13C NMR spectra. The location of the three methoxyl groups was deduced at C-3′, C-4′ and C-7, and the methylenedioxyl group at C-3 and C-4 by the HMBC correlations from the three methoxyl protons to C3′, C-4′ and C-7, and the methylenedioxyl protons to C-3 and C4. With an analysis of the NOE correlation experiment, compound 4 was elucidated as (7, 8-trans-8, 8′-trans-7′, 8′trans)-7-methoxyl-7-(3,4- methylene-dioxyphenyl)-8 methyl -8′-methyl-7′-(3′,4′- dimethoxyphenyl)-tetrahydrofuran. Compound 5 gave a molecular formula of C23H28O7 as determined by the HR-ESI-MS at m/z 417.1823 [M + H]+. The 1 H and 13C NMR spectroscopic data (Tables 1 and 2) implied that compound 5 had a tetrahydrofuran lignan skeleton possessing four methoxyl and one methylenedioxyl substitutions. The HMBC correlations from the four methoxyl protons to C-3, C-4, C-5 and C-7 verified that they were located at C-3, C-4, C-5 and C-7, respectively. The HMBC correlations from the methylenedioxyl protons to C-3′ and C-4′ revealed that the methylenedioxyl group was located at C-3′ and C-4′. Compound 5 was thus assigned as 7-methoxyl-7-(3,4,5-trimethoxyphenyl)-
8-methyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)-tetrahydrofuran. The relative configuration of 5 was deduced from NOE correlations as 7,8-trans-8,8′-trans-7′,8′-trans. Compounds 6 and 7 were both obtained as yellow oil and showed molecular ions at m/z 371.1629 and 357.1624 in the HRESIMS (both found [M + H]+), corresponding to the molecular formula C21H22O6 and C20H20O6, respectively. A comparison of 1H and 13C NMR spectroscopic data of 6 and 7 with those of 11 disclosed that the main structural differences between these compounds are related to the substituents in the aromatic rings. Two methoxyl groups at C-3′ and C-4′ in 6 replaced the methylenedioxyl group in 11. Meanwhile the methylenedioxyl group at C-3′ and C-4′ in 11 was substituted by a hydroxyl group and a methoxyl group in 7. Thus, the structures of 6 and 7 were determined as 7,8-epoxide -7-(3,4-methylenedioxyphenyl)-8 methyl8′-methyl-7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran and 7,8-epoxide-7-(3,4- methylenedioxy phenyl)-8-methyl-8′methyl-7′-(4′-hydroxy-3′-methoxyphenyl)-tetrahydrofuran, respectively. The 7′,8′-trans-8′,8-cis orientation of 6 was elucidated from the NOE cross-peaks of H-7′/H-8′; H-9′/H-9. Compound 7 had the same relative configuration as compound 6 on the basis of NOE cross-peaks. Compound 8 was obtained as a pale yellow, amorphous solid. Its molecular formula of C19H20O5 was established from HR-ESI-MS (found [M + H]+ 329.1715) and 13C NMR spectroscopic data, indicating 10° of unsaturation. The 1H and 13 C NMR spectra of 8 resembled those of 12. The only difference was the occurrence of a hydroxyl group at C-3′ in 8 replacing the methoxy group in 12, which was confirmed by the correlation from the proton of 3′-OH with C-3′. As previously reported, the signal at δ H 4.89 (1H, d, J = 10.0 Hz) for H-7 indicates that this hydrogen is in trans configuration with the adjacent H-8′, and H-7′ (δ H 4.57, 1H, d, J = 10.0 Hz) also has a trans configuration with the adjacent H-8′. Thus, the structure of 8 was established as (7, 8-trans-8, 8′-trans-7′, 8′-trans)-7(3,4-methylenedioxyphenyl)-8-methyl-8′-methyl-7′-(3′,4′dihydroxyphenyl)-tetrahydrofuran. The CD curves of compounds 2–8 were similar to that of 1 (Fig. 3). Based on biogenetic considerations and comparisons with the CD spectrum and the optical rotation value, the absolute configurations of compounds 2, 3, 4, 5 and 8 were defined as (7S, 8S, 7′R, 8′S), and compounds 6 and 7 were established as (7S, 8S, 7′R, 8′S) (Fig. 1). Compound 13 was obtained as a pale white, amorphous solid. Its molecular formula of C20H24O5 was established from HR-ESI-MS (found [M + Na]+ 367.1618) and 13C NMR spectroscopic data (Table 2), indicating 9° of unsaturation. The 1H NMR spectroscopic data (Table 1) of 13 showed proton signals corresponding to two trisubstituted aromatic rings, two methyl groups, a methylene group, three methine groups, a methoxyl group and a methylenedioxyl group, which suggested that the skeleton of 13 was a 1,4-bisphenyl-2,3dimethylbutane-type lignan. A detailed comparison of the 1H and 13C NMR spectroscopic data of 13 with those of 14 disclosed that the main structural difference between them was to be the locations of the substitutions in aromatic rings. The HMBC correlations from the methoxyl proton and hydroxyl proton to C-3 and C-5 indicated that the two groups in 13 were attached at C-3 and C-5, respectively. On the basis of NOE cross-peaks, the relative configuration of 13 was
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
69
Scheme 1. Proposed biogenetic pathway of the asymmetric tetrahydrofuran lignan with substitutions at C-7 and C-8.
established as (7,8-trans-8,8′-cis)-7-(3,4-dihydroxyphenyl)7′-(3,4-methylenedioxy-phenyl)-8,8′-dimethyl-butan-7-ol (Fig. 1). Although, the presence of additional hydroxyl or methoxyl groups at C-7 and C-8 positions of the asymmetric tetrahydrofuran lignan is very uncommon in plants, compounds of this type have been reported from the Schisandra sphenanthera [10] and other plants belonging to the genus Schisandra [7–9]. And a plausible biosynthetic pathway of these lignans is proposed in Scheme 1. All the lignans were tested for their capacity to scavenge 2,2-diphenyl-1-Picryl‐hydrazyl (DPPH). The IC50 values of lignans were given in Table 3. Within the series of lignans tested, compound 8 exhibited the highest antioxidative activity with IC50 values of 35 μg/mL, which is similar to that of vitamin C. It was clearly seen that the activity of these compounds depends significantly on the number of phenolic groups in the molecules. Compounds 1–8 were all asymmetric tetrahydrofuran lignans. The main structural differences between these compounds refer to the substituents in the aromatic rings. Compound 8, which possesses two phenolic groups, was the most active one (IC50 = 35 μg/mL), followed by compounds 1 and 7, which possess one phenolic group and exhibited similar activity (IC50 = 92 μg/mL, 115 μg/mL). Compounds 2, 3, 5, 9 and 10 which bear no phenolic group, showed weak antioxidative activity (IC50 ~ 200 μg/mL).
Although DPPH assay has been widely used to conveniently test the antioxidative activity of phenolic compounds, the method is only of chemical relevance and the system used is a homogenous solution. Human RBCs are heterogeneous media, and the RBC model has been extensively studied both as a source of free radicals and as a target for oxidative damage. Therefore, the antioxidative effect of compound 8 was investigated in RBC model [23,24]. In the absence of AAPH, the RBCs were stable and little haemolysis took place within 3.5 h. An addition of AAPH induced haemolysis (Fig. 4). It could be seen that compound 8 protected RBCs in a significant way from oxidative-AAPH induced hamolysis at a concentration of 40 μM. The inhibition time produced by 40 μM compound 8 was 90 min as shown in Fig. 4. At a lower concentration (20 μM) compound 8 still had protective activity similar to that of vitamin C (Fig. 4). This is the first report to demonstrate the antioxidant activities of the asymmetric tetrahydrofuran lignans with hydroxyl or methoxyl groups at C-7 and C-8 positions. Components from traditional Chinese medicine are different from drugs and their actions on human physiology are usually moderate. The isolated compounds did not show potent
Table 3 Antioxidative activity of compounds 1–10 and 13. Compound
IC50 (μg/mL)
1 2 3 5 7 8 9 10 13 EtOAc-soluble fraction Crude extract Vitamin C (positive control)
92 233 182 210 115 35 195 261 48 622 781 33
Fig. 4. Inhibitory effect of compound 8 and Vc against AAPH-induced haemolysis of 5% human RBCs in 0.15 M PBS (pH 7.4) in an aerobic atmosphere at 37 °C.
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antioxidant activities, however whether these compounds have the benefits to the health of the human beings should be further studied. Conflict of interest The authors of the present manuscript have declared that no competing interests exist. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 31270396) and the Fundamental Research Funds for the Central Universities (No. lzujbky-2014176). References [1] Huyke C, Engel K, Simon HB, Quirin KW, Schempp CM. Composition and biological activity of different extracts from Schisandra sphenanthera and Schisandra chinensis. Planta Med 2007;73:1116. [2] Xin HW, Wu XC, Li Q, Yu AR, Zhu M, Shen Y, et al. Effects of Schisandra sphenanthera extract on the pharmacokinetics of tacrolimus in healthy volunteers. Br J Clin Pharmacol 2007;64:469. [3] Song QY, Zhang CJ, Li Y, Wen J, Zhao XW, Liu ZL, et al. Lignans from the fruit of Schisandra sphenanthera, and their inhibition of HSV-2 and adenovirus. Phytochem Lett 2013;6:174. [4] Sun J, Yu J, Zhang PC, Tang F, Yang YN, Yue YD, et al. Isolation and identification of lignans from Caulis Bambusae in Taenia with antioxidant properties. J Agric Food Chem 2013;61:4556. [5] Lou ZH, Li HM, Gao LH, Li RT. Antioxidant lignans from the seeds of Vitex negundo var Cannabifolia. J Asian Nat Prod Res 2014;16:963. [6] Xiao WL, Huang SX, Wang RR, Zhong JL, Gao XM, Sun HD, et al. Nortriterpenoids and lignans from Schisandra sphenanthera. Phytochemistry 2008;69:2862. [7] Pu JX, Gao XM, Wang RR, Yang LB, Lei C, Sun HD, et al. Three new compounds from Kadsura longipedunculata. Chem Pharm Bull 2008;56: 1143. [8] Liu HT, Xu LJ, Peng Y, Yang XW, Xiao PG. Two new lignans from Schisandra henryi. Chem Pharm Bull 2009;57:405. [9] Huang RM, Huang HJ, Zhang NL, Zhua YH, Jiang XF, Qiu SX, et al. Two new lignans from the stems of Schisandra bicolor. J Asian Nat Prod Res 2012;14: 1116.
[10] Liang CQ, Hu J, Luo HR, Shang SZ, Du X, Sun HD, et al. Schisphenlignans A– E: five new dibenzocyclooctadiene lignans from Schisandra sphenanthera. Fitoterapia 2013;86:171. [11] He F, Pu JX, Huang SX, Wang YY, Xiao WL, Li LM, et al. Schinalactone A, a new cytotoxic triterpenoid from Schisandra sphenanthera. Org Lett 2010; 12:1208. [12] He F, Li XY, Yang GY, Li XN, Luo X, Zou J, et al. Nortriterpene constituents from Schisandra sphenanthera. Tetrahedron 2012;68:440. [13] Yu HY, Hao C, Meng FY, Li X, Chen ZY, Liang X, et al. Neuroprotective lignans from the stems of Schisandra glaucescens. Planta Med 2012;78: 1962. [14] Kuo YH, Chen CH, Lin YL. New lignans from the heartwood of Chamaecyparis obtusa var formosana. Chem Pharm Bull 2002;50:978. [15] Matcha K, Ghosh S. A stereocontrolled approach for the synthesis of 2, 5diaryl-3, 4-disubstituted furano lignans through a highly diastereoselective aldol condensation of an ester enolate with an a-chiral center: total syntheses of (−)-talaumidin and (−)-virgatusin. Tetrahedron Lett 2008; 49:3433. [16] Nakatani N, Ikeda K, Kikuzaki H, Kido M, Yuzo Y. Diaryldimethylbutane lignans from Myristica Argentea and their antimicrobial action against Streptococcus mutans. Phytochemistry 1988;27:3127. [17] Martins RCC, Lago JHG, Albuquerque S, Kato MJ. Trypanocidal tetrahydrofuran lignans from inflorescences of Piper solmsianum. Phytochemistry 2003;64:667. [18] Martins RCC, Latorre LR, Sartorelli PC, Kato MJ. Phenylpropanoids and tetrahydrofuran lignans from Piper solmsianum. Phytochemistry 2000;55: 843. [19] Da Silva Filho AA, Albuquerque S, Silva MLAE, Eberlin MN, Tomazela DM, Bastos JK. Tetrahydrofuran lignans from Nectandra megapotamica with trypanocidal activity. J Nat Prod 2004;67:42. [20] Schottner M, Reiner J, Tayman Francis SK. (+)-Neo-olivil from roots of Urtica dioica. Phytochemistry 1997;46:1107. [21] Wu JL, Li N, Hasegawa T, Sakai JI, Kakuta S, Ando M, et al. Bioactive tetrahydrofuran lignans from Peperomia dindygulensis. J Nat Prod 2005;68: 1656. [22] Yang GY, Li YK, Wang RR, Li XN, Xiao WL, Sun HD, et al. Dibenzocyclooctadiene Lignans from Schisandra wilsoniana and Their anti-HIV-1 activities. J Nat Prod 2010;73:915. [23] Deng SL, Chen WF, Bo Zhou, Yang L, Liu ZL. Protective effects of curcumin and its analogues against free radical-induced oxidative haemolysis of human red blood cells. Food Chem 2006;98:112. [24] Shang YJ, Jin XL, Shang XL, Tang JJ, Liu GY, Zhou B, et al. Antioxidant capacity of curcumin-directed analogues: structure–activity relationship and influence of microenvironment. Food Chem 2010;119:1435.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New lignans from the roots of Schisandra sphenanthera Kan Jiang a, Qiu-Yan Song a, Shou-Jiao Peng a, Qian-Qian Zhao a, Guang-Da Li a, Ya Li a,⁎, Kun Gao a,b,⁎⁎ a b
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People's Republic of China School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University; Ningbo 315100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Accepted in revised form 13 March 2015 Accepted 14 March 2015 Available online 19 March 2015 Keywords: Schisandra sphenanthera Lignans Antioxidant activity RBCs
a b s t r a c t Nine new lignans (1–8, 13) and five known ones (9–12, 14) have been isolated from the roots of Schisandra sphenanthera and were tested for their capacity to scavenge 2,2-diphenyl-1-Picrylhydrazyl (DPPH). Of these lignans tested, compounds 1, 7, 8 and 13 exhibited noteworthy antioxidant activity with IC50 values of 92, 115, 35 and 48 μg/mL, respectively. The anti-oxidative haemolysis of human red blood cells (RBCs) activity of the most active compound 8, which is similar to that of vitamin C, was evaluated. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The genus Schisandra belonging to the plant family Schisandraceae has long been used in traditional Chinese medicine for the treatment of hepatitis, diabetes, diarrhea, cough, etc [1,2]. It has been reported that many kinds of lignans had been isolated from this genus, such as dibenzocyclooctadiene, dibenzylbutane and tetrahedrofurane types, and some of them exhibited antimicrobic, antiviral, herbicidal, antifeedant and insulin sensitivity-improving activities [3–9]. Schisandra sphenanthera is widely distributed in the southwest region of China [10]. Previous phytochemical studies on the fruits, stems and leaves of S. sphenanthera have led to the isolation of a variety of lignans and triterpenoids [11,12], while the chemical components of the roots of S. sphenanthera have never been reported. In this paper, we conducted a systematic bioactivity-guided isolation of the 70% aqueous acetone extract from the roots of S. sphenanthera and new compounds with ⁎ Corresponding author. Tel.: +86 931 8912592; fax: +86 931 8912582. ⁎⁎ Correspondence to: K. Gao, State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People's Republic of China. Tel.: +86 931 8912592; fax: +86 931 8912582. E-mail addresses: [email protected] (Y. Li), [email protected] (K. Gao).
http://dx.doi.org/10.1016/j.fitote.2015.03.015 0367-326X/© 2015 Elsevier B.V. All rights reserved.
natural anti-oxidative activity were discovered for the first time. The systematic isolation led to the identification of eight new tetrahydrofuran lignans (1–8), one new butane-type lignan (13) and five known ones (schiglaucin B, 9 [13]; schiglaucin A, 10 [12]; epoxyzuonin A, 11 [14]; talaumidin, 12 [15]; myristargenol A, 14 [16]) from the anti-oxidative active EtOAc-soluble fraction of the 70% aqueous acetone extract. The antioxidant activity of these compounds was assessed by DPPH scavenging experiments. Of these lignans tested, compound 8 exhibited noteworthy antioxidant activity and its capacity to protect human red blood cells (RBCs) from oxidative haemolysis was evaluated, which is similar to that of vitamin C. 2. Experimental 2.1. General Column chromatography: silica gel (200–300 mesh; Qingdao Marine Chemical Factory, China); macroporous resin (HP-20, Kabuskiki Kaisha); Sephadex LH-20 (Amersham Pharmacia Biotech). TLC (Thin layer chromatography): silica gel GF254 plates (10–40 μm; Qingdao Marine Chemical Factory, China). A Waters 1525 series instrument equipped with a YMC-Pack ODSA column (250 × 10 mm, 5 μm) was used for semipreparative
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HPLC. 1H and 13C NMR Spectra: Bruker AM-400BB (400 MHz) and a Varian Mercury-600BB (600 MHz) NMR spectrometer; recorded in CDCl3; δ in ppm relative to TMS; J values in Hz. HRESI-MS: Bruker APEX-II mass spectrometer; in m/z. Optical rotations: Perkin-Elmer 341 polarimeter. IR Spectra: Nicolet FTIR-360 spectrometer. CD spectra were obtained on an Olis DSM 1000 spectrometer.
was separated on semipreparative HPLC (MeOH: H2O, 1: 3, v:v) to yield compounds 9 (1.3 mg) and 10 (2.5 mg). In the same way, compounds 11 (3.6 mg) and 12 (2.7 mg) were obtained from FrC.7.2 (0.21 g). Further separation of FrC.7.3 (0.13 g) by reversed-phase C18 silica gel column, eluted with MeOH: H2O (4: 1, v:v), led to the appearance of compounds 13 (2.3 mg) and 14 (4.2 mg).
2.2. Plant material
2.3.1. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)8-hydroxyl-7′-(5′-hydroxy-3′-methoxyphenyl)-8′-methyltetrahydrofuran (1) Yellow oil; [α]25 D +60 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 228 (0.15) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 389.1977 [M + H]+ (calcd for C21H24O7).
Roots of S. sphenanthera were collected from Taibai Mountain in Shaanxi Province of China, in the summer of 2011. The plant was identified by Guo-Liang Zhang, at the School of Life Sciences, Lanzhou University, China. A voucher (No. 11-07) specimen was deposited with the Natural Organic Academy of Lanzhou University. 2.3. Extraction and isolation The air-dried and powdered roots of (7 kg) were extracted three times (each time for 7 days) with 70% aqueous acetone at room temperature. The solvent was evaporated under reduced pressure, producing an extract (685 g), which was found to have antioxidant effect in the DPPH scavenging assay. The crude extract was dissolved in hot H2O (60 °C, 2 L) to accelerate dissolution and then partitioned with petroleum ether (PE), EtOAc and n-BuOH at room temperature, respectively. The EtOAc-soluble fraction was found to be more anti-oxidative than the PE and n-BuOH-soluble fraction, exhibiting that the EtOAc-soluble fraction included the majority of the active components. Thus, the EtOAc-soluble fraction was further purified and investigated. Initially, the EtOAc-soluble fraction was applied to macroporous resin (HP-20,3 L) with a gradient of H2O: EtOH (70: 30, 50: 50, 20: 80, v:v) as eluent, and three fractions (A, B, and C) were collected according to the TLC analysis. Fraction C (248 g) was subjected to silica gel column chromatography (200–300 mesh, 2000 g) eluted with a gradient of PE (petroleum ether): acetone (100: 0, 50: 1, 20: 1, 10: 1, 5: 1, 2: 1, 1: 1, 0: 100, v:v) to give eight fractions, FrC.1– FrC.8. Using H2O: MeOH (1: 1, 2: 5, 3: 10, 1: 5, 0: 1, v:v) as eluent, FrC.6 (6.8 g) was separated on a column of reversedphase C18 silica gel, thus resulting in five subfractions. FrC.6.1– FrC.6.5. FrC.6.1 (1.3 g) was separated on a Sephadex LH–20 (CH3OH) to obtain three fractions (FrC.6.1.1–FrC.6.1.3). FrC.6.1.1 (0.1 g) and FrC.6.1.3 (0.12 g) were further fractionated on semipreparative HPLC (MeOH: H2O, 1: 3, v:v) to yield compounds 1 (3 mg) and 2 (2.1 mg). Compounds 3 (2.2 mg) and 4 (1.8 mg) were obtained from FrC.6.1.2 (0.15 g) by silica gel column chromatography (PE: acetone, 10: 1 to 5: 1, v:v). FrC.6.3 (2.1 g) was separated on a silica gel column and eluted with gradient mixtures of PE: acetone (from 8: 1 to 5: 1, v:v) to obtain compound 5 (3.2 mg) and FrC.6.3.1 (0.2 g). The separation of FrC.6.3.1 was done on semipreparative HPLC (MeOH: H2O, 1: 4, v:v) to obtain compounds 6 (2.8 mg) and 7 (1.9 mg). FrC.6.4 (0.15 g) was separated on silica gel column chromatography (PE: acetone, 20: 1 to 5: 1, v:v), and then on Sephadex LH-20 (CHCl3: MeOH, 1: 1, v:v) to give compound 8 (2.3 mg). FrC.7 (2.8 g) was applied to a silica gel column and eluted with various gradients of PE: acetone (15: 1, 10: 1, 5: 1, v:v) to give three fractions (FrC.7.1–FrC.7.3). FrC.7.1 (0.15 g)
2.3.2. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxy-phenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′,5′-trimethoxyphenyl)tetrahydrofuran (2) Yellow oil; [α]25 D +50 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 232 (0.42) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 433.1613 [M + H]+ (calcd for C23H28O8). 2.3.3. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)8-hydroxyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)tetrahydrofuran (3) Yellow oil; [α]25 D +50 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 226 (0.25) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 387.1824 [M + H]+ (calcd for C21H22O7). 2.3.4. (7S, 8S, 7′R, 8′S)-(7, 8-trans-8, 8′-trans-7′, 8′-trans)-7methoxyl-7-(3,4- methylenedioxy-phenyl)-8 methyl -8′-methyl7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran (4) Yellow oil; [α]25 D +20 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 232 (0.25) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 409.1712 [M + H]+ (calcd for C22H26O6). 2.3.5. (7S, 8S, 7′R, 8′S)-7-methoxyl-7-(3,4,5-trimethoxyphenyl)8methyl -8′-methyl-7′-(3′,4′- methylenedioxyphenyl)tetrahydrofuran (5) Yellow oil; [α]25 D +40 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 227 (0.24) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 417.1823 [M + H]+ (calcd for C23H28O7). 2.3.6. (7S, 8S, 7′R, 8′S)-7,8-epoxide-7-(3,4-methylenedioxyphenyl)8 methyl -8′-methyl-7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran (6) Yellow oil; [α]25 D +20 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 227 (0.32) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 371.1629 [M + H]+ (calcd for C21H22O6). 2.3.7. (7S, 8S, 7′R, 8′S)-7,8-epoxide -7-(3,4-methylenedioxyphenyl)8-methyl -8′-methyl-7′-(4′-hydroxy-3′-methoxyphenyl)tetrahydrofuran (7) Yellow oil; [α]25 D +40 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 229 (0.18) nm; 1H and 13C NMR data, see Tables 1
5.41 5.38, 5.37
5.96 3.81 5.93 5.99 3.91 5.99 3.88 3.90
5.58
1.57(m) 0.81(d,6.8)
6.78(d,8.0) 6.52(dd,8;0,1.6) 2.74(dd,13.2,3.6) 2.11(dd,13.2,10.8)
6.84(d,8.0) 6.79(dd,8.0) 4.59(d,8.8) 2.41(m) 0.99(d,6.8) 6.90(d,8.0) 6.96(d,8.0) 4.87(d,10.4) 2.47 (dd,10.4,6.8) 1.01(d,6.8)
6.82(d,8.0) 7.02(d,8.0) 4.59(d,9.6) 2.18(m) 0.98(d,6.8) 3.26 5.96 3.85 3.89 3.89 6.81(d,8.0) 6.95(d,8.0) 4.61(d,9.6) 2.18(m) 0.96(d,6.8) 3.24 5.96 3.89 3.92 6.80(d,8.0) 6.85(d,8.0) 4.83(d,10.4) 2.44 (dd,10.4,6.8) 0.95(d,6.8) 3.20 5.99;5.96
1.70(dd,11.6,6.8) 1.08(d,6.8) 7.00(s)
6.67(s) 4.84(d,10.0) 2.48 (dd,10,6.8) 0.99(d,6.8) 3.25 6.00 3.85 3.89 3.89 6.91(s) 4.84(d,10.0) 2.47 (dd,9.6,6.8) 0.96(d,6.8) 3.22 5.99 3.92
5.60
1.31(s) 6.67(s) 1.31(s) 6.91(s) 6.98(s)
1.29(s) 6.94(s)
1.70(dd,11.6,6.8) 1.07(d,6.8) 7.01(s)
1.32(s) 7.08(d,1.6)
1.31(s) 7.04(s)
6.86(d,8.0) 6.99(dd,8.0,1.6) 4.89(d,10.0) 2.49(m) 0.99(d,6.8)
6.81(s) 4.64(d,7.6) 1.78(m) 1.07(d,6.8) 6.43(d,1.6) 6.84(d,8.0) 6.93(dd,8.0,1.6) 4.04 (d,9.2) 1.71 (m) 0.61(d,6.8) 6.77(s) 6.78(s) 6.86(d,8.0) 7.07(d,8.0) 6.85(d,8.0) 7.10(d,8.0)
6.88(d,8.0) 7.06(d,8.0)
6.86(d,8.0) 7.02(d,8.0)
6.84(d,8.0) 7.20(d,8.0)
6.84(d,8.0) 7.17(d,8.0)
6.88(d,1.2) 6.82(d,1.6) 6.86(d,1.6)
8
7.14(s)
7
6.78(s) 7.00(s)
6 5 4
7.05(s) 7.05(s)
7.04(s)
2.3.9. (7, 8-trans-8, 8′-cis)-7-(3, 4-dihydroxyphenyl)-7′-(3, 4methylenedioxyphenyl)-8, 8′-dimethylbutan-7-ol (13) Pale white amorphous solid; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 367.1618 [M + H]+ (calcd for C20H24O5).
A 2,2-diphenyl-1-Picryl‐hydrazyl (DPPH) assay was performed as previously described. L-Ascorbic acid (33 μg/mL) was used as the positive control. Reaction mixtures containing an methanol solution of 200 μM DPPH (100 μL) and two fold serial dilutions of the sample (dissolved in 100 μL of methanol, with sample concentrations in the range of 2–500 μg/mL) were placed in a 96-well micro-plate and incubated at 30 °C for 30 min in the dark. After incubation, the absorbance was read at 517 nm with a Tecan Infinite 200 Pro Micro-plate Reader, and the means of three readings were calculated. The radicalscavenging activity (RSA) was calculated as a percentage of DPPH˙ discoloration using the equation: RSA % = [Abs. of control-(Abs. of sample-Abs. of blank) / Abs. of control] × 100%. The IC50 value is the concentration of the sample required to scavenge 50% of the DPPH radicals and was obtained by extrapolation from a linear regression analysis. Tests were carried out in triplicate.
2.5. Oxidative haemolysis of human red blood cells (RBC) assay
1
3
2.3.8. (7S, 8S, 7′R, 8′S)-7-(3,4- methylenedioxyphenyl)-8methyl -8′-methyl-7′-(3′,4′- dihydroxy-phenyl)-tetrahydrofuran (8) Pale yellow amorphous solid; [α]25 D +10 (c 0.1, MeOH); UV (CH3CH2OH) λmax (log ε) 228 (0.15) nm; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 329.1713 [M + H]+ (calcd for C19H20O5).
2.4. DPPH radical-scavenging assay
2 4 5 6 7 8 9 2′ 4′ 5′ 6′ 7′ 8′ 9′ 7-OMe −OCH2O− OMe OMe OMe OH
2
65
and 2; positive-ion HRESIMS m/z 357.1624 [M + H]+ (calcd for C20H20O6).
Position
Table 1 1 H NMR spectroscopic data of compounds 1–8, 13 [400 MHz, CDCl3, δ (ppm)]. Proton coupling constants (J) in Hz are given in parentheses.
7.18(s)
13
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
The 5% suspension of RBCs in phosphate-buffered saline (PBS, pH 7.4) was incubated under air atmosphere at 37 °C for 5 min; into this a PBS solution of AAPH was added to initiate haemolysis. The reaction mixture was shaken gently while being incubated at 37 °C. The extent of haemolysis was determined spectrophotometrically as described previously. Briefly, aliquots of the reaction mixture were taken at appropriate time intervals, diluted with 0.15 M NaCl, and centrifuged at 2000 rpm for 10 min to separate the RBCs. The percentage haemolysis was determined by measuring the absorbance of the supernatant at 540 nm and compared with that of a complete haemolysis by treating the same RBC suspension with distilled water. In the case of anti-haemolysis experiments, lignans and Vc dissolved in dimethyl sulfoxide (DMSO) were added and incubated before addition of AAPH. The final concentration of DMSO was 0.1% (v/v) and did not interfere with the determination. Every experiment was repeated three times and the results were reproducible within 10% deviation.
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K. Jiang et al. / Fitoterapia 103 (2015) 63–70
Table 2 13 C NMR spectroscopic data of compounds 1–8, 13 [100 MHz, CDCl3, δ (ppm)]. Carbon
1
2
3
4
5
6
7
8
13
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 7-OMe −OCH2O− −OMe −OMe −OMe
133.4 108.2 148.2 148.1 108.2 121.2 111.8 82.6 20.1 130.3 114.3 146.9 110.1 145.7 114.3 88.0 48.9 8.6 50.5 101.5 56.1
136.9 107.9 147.9 147.8 111.7 120.3 111.8 82.3 19.8 134.2 112.5 143.7 153.2 143.7 107.7 88.0 48.7 8.4 50.2 101.2 58.8 56.0 58.9
133.4 107.6 149.4 148.9 108.1 120.9 111.2 82.5 19.9 135.4 107.2 147.7 147.1 107.9 121.2 87.5 49.2 8.3 50.3 101.5;101.0
135.4 107.6 149.4 149.0 108.2 120.2 111.0 54.9 14.3 134.4 110.4 147.3 147.9 109.2 120.2 89.7 47.6 10.5 50.1 101.3 56.0 56.2
132.5 107.5 152,4 137.8 152.3 108.2 111.2 52.6 14.5 133.2 109.3 146.5 147.1 109.5 120.1 88.5 49.2 10.2 50.2 101.5 55.9 56.1
134.2 110.3 149.4 149.0 108.7 119.9 111.0 82.2 20.2 133.4 108.3 148.5 147.9 108.1 121.5 87.7 49.5 8.7
133.3 108.0 148.2 147.6 108.4 120.4 114.0 81.9 19.9 133.1 107.8 146.6 145.3 109.4 121.3 87.5 49.2 8.4
131.5 113.3 147.5 146.8 108.1 119.8 85.7 47.8 12.0 129.8 115.2 144.5 146.2 107.0 120.5 84.9 43.6 9.7
138.5 111.5 148.2 113.2 143.8 114.1 75.8 45.6 10.9 133.7 108.2 147.2 146.4 107.0 120.3 37.8 36.9 17.8
101.6 56.1 56.2
101.3 55.9
101.0
101.3 56.0
3. Results and discussion The EtOAc-soluble fraction from air-dried and powdered roots of S. sphenanthera was repeatedly separated by column chromatography (silica gel and Sephadex LH-20) and semipreparative HPLC yielded nine new compounds (1–8, 13) and five known compounds (9–12, 14) (Fig. 1). Compound 1 was isolated as a yellow oil and its molecular formula was established as C21H24O7 by HR-ESI-MS at m/z 389.1977 [M + H]+, indicating 10° of unsaturation. The signals in the 1H-NMR spectrum for aromatic protons (δH 7.10, s, 1H; 6.85, d, J = 8.0 Hz, 1H; 7.05, d, J = 8.0 Hz, 1H; 6.91, s, 2H; 6.98, s, 1H), in conjunction with the signals corresponding to a methoxyl group (δH 3.92, 3H), a hydroxyl group (δH 5.60) and a methylenedioxyl group (δH 5.95 br s) indicated that the
substitution pattern of the two aromatic rings was both trisubstituted (5′-hydroxy-3′-methoxyphenyl and 3,4methylene-dioxyphenyl for the two aromatic rings). Other characteristic proton signals showed a methine group (δH 4.82,d, J = 10.0 Hz), a tertiary methyl group (δH 1.31, s) attached to a carbon carrying oxygen atom, a secondary methyl group (δH 0.94, d, J = 6.8 Hz), and another upfield methoxyl group. The above information, along with the analysis of its 13C-NMR spectroscopic data (Table 2) suggested that 1 was an asymmetric tetrahydrofuran lignan [17–19]. Extensive analysis of 1H-1H COSY and HMBC spectroscopic data led to the establishment of the planar structure of compound 1. The HMBC correlations (Fig. 2) from the methoxyl protons at δH 3.92 and hydroxyl proton at δH 5.60 to C-3′ and C-5′, and from the methylenedioxyl protons to C-3 and C-4 indicated that the
Fig. 1. The structures of compounds 1–14.
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
Fig. 2. Key HMBC correlations of compound 1.
methoxyl group and hydroxyl group were attached at C-3′ and C-5′, respectively while the methylenedioxyl group was attached at C-3 and C-4. The upfield shifted methoxyl
67
group (δH 3.22 and δC 50.46) was deduced to be attached at C-7 established by HMBC correlation (Fig. 2) from the methoxyl protons to C-7. In addition, the HMBC correlations from H3-9, H3-9′, H-7′ to the oxygenated quaternary carbon C-8 (δC 82.55), along with the analysis of its molecular formula, indicated that a hydroxyl group was attached to C-8 of 1. The relative configuration of 1 was established on the NOE cross-peaks of H-8′ with Me-9, H-2′, H-6′ and OMe-7, which suggested that H-8′, Me-9, OMe-7 were cofacial, and H-7′, Me-9′ and OH-8 were on the other side. The absolute configuration of 1 was established on the basis of the circular dichroism (CD) curve from 200 to 400 nm in its CD (Fig. 3) spectrum and the optical rotation. Compound 1 showed a positive Cotton effect at 209, 237 nm and a negative Cotton effect at 251 nm, consistent with those of schisphenlignan H
Fig. 3. CD and UV spectra of 1–8.
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K. Jiang et al. / Fitoterapia 103 (2015) 63–70
[10] and the optical rotation value of 1 ([α]25 D + 60) is in agreement with that of (+)-neo-olivil ([α]25 D + 49) [20]. Thus, the structure of 1 was established as (7S, 8S 7′R, 8′S)-7-methoxyl-7-(3,4-methylenedioxyphenyl)-8hydroxyl-7′-(5′-hydroxy-3′-methoxyphenyl)-8′-methyltetrahyd-ofuran [21]. Compounds 2 and 3 were both obtained as a yellow oil and showed molecular ions at m/z 433.1613 [M + H]+ and 387.1824 [M + H]+ in the HRESIMS, corresponding to the molecular formula C23H28O8 and C21H22O7, respectively. The 1H and 13C NMR spectra of 2 and 3 were very similar to those of 1, and the obvious chemical shift differences resulted from the substituents variations in the aromatic rings, which suggested that compounds 2 and 3 were both asymmetric tetrahydrofuran lignans. The 1H and 13C NMR spectroscopic data revealed the presence of three methoxyl groups located at C-3′, C-4′ and C-5′ in 2 instead of the methoxyl group and the hydroxyl group located at C-3′ and C-5′ in 1, which could be deduced from the HMBC correlations from the three methoxyl protons to C-3′, C4′ and C-5′, respectively. For compound 3, a methylenedioxyl group located at C-3′ and C-4′ replaced the methoxyl group and the hydroxyl group located at C-3′ and C-5′ in 1, which was determined by the HMBC correlations from the methylenedioxyl protons to C-3′ and C-4′. Thus, the structures of 2 and 3 were elucidated as 7-methoxyl-7-(3,4-methylenedioxy-phenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′,5′-trimethoxyphenyl)-tetrahydrofuran and 7-methoxyl-7-(3,4-methylenedioxyphenyl)-8-hydroxyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)-tetrahydrofuran, respectively [22]. On the basis of NOE cross-peaks, the relative configurations of 2 and 3 were determined as 7,8-trans-8,8′-trans-7,8-trans. Compound 4 was also obtained as a yellow oil and its molecular formula of C22H26O6 was established from HR-ESIMS ([M + Na]+, m/z 409.1712) and 13C-NMR spectroscopic data (Table 2), indicating 10° of unsaturation. Through a comparison of its 1H and 13C NMR spectral data with those of 1, compound 4 was also assigned as an asymmetric tetrahydrofuran lignin and its 1H NMR spectroscopic data exhibited two 1,3,4-trisubstituted aromatic systems. Besides the signals of the two aromatic rings and the tetrahydrofuran ring, the signals of three methoxyl protons and a methylenedioxyl proton could also be observed in the 1H and 13C NMR spectra. The location of the three methoxyl groups was deduced at C-3′, C-4′ and C-7, and the methylenedioxyl group at C-3 and C-4 by the HMBC correlations from the three methoxyl protons to C3′, C-4′ and C-7, and the methylenedioxyl protons to C-3 and C4. With an analysis of the NOE correlation experiment, compound 4 was elucidated as (7, 8-trans-8, 8′-trans-7′, 8′trans)-7-methoxyl-7-(3,4- methylene-dioxyphenyl)-8 methyl -8′-methyl-7′-(3′,4′- dimethoxyphenyl)-tetrahydrofuran. Compound 5 gave a molecular formula of C23H28O7 as determined by the HR-ESI-MS at m/z 417.1823 [M + H]+. The 1 H and 13C NMR spectroscopic data (Tables 1 and 2) implied that compound 5 had a tetrahydrofuran lignan skeleton possessing four methoxyl and one methylenedioxyl substitutions. The HMBC correlations from the four methoxyl protons to C-3, C-4, C-5 and C-7 verified that they were located at C-3, C-4, C-5 and C-7, respectively. The HMBC correlations from the methylenedioxyl protons to C-3′ and C-4′ revealed that the methylenedioxyl group was located at C-3′ and C-4′. Compound 5 was thus assigned as 7-methoxyl-7-(3,4,5-trimethoxyphenyl)-
8-methyl-8′-methyl-7′-(3′,4′-methylenedioxyphenyl)-tetrahydrofuran. The relative configuration of 5 was deduced from NOE correlations as 7,8-trans-8,8′-trans-7′,8′-trans. Compounds 6 and 7 were both obtained as yellow oil and showed molecular ions at m/z 371.1629 and 357.1624 in the HRESIMS (both found [M + H]+), corresponding to the molecular formula C21H22O6 and C20H20O6, respectively. A comparison of 1H and 13C NMR spectroscopic data of 6 and 7 with those of 11 disclosed that the main structural differences between these compounds are related to the substituents in the aromatic rings. Two methoxyl groups at C-3′ and C-4′ in 6 replaced the methylenedioxyl group in 11. Meanwhile the methylenedioxyl group at C-3′ and C-4′ in 11 was substituted by a hydroxyl group and a methoxyl group in 7. Thus, the structures of 6 and 7 were determined as 7,8-epoxide -7-(3,4-methylenedioxyphenyl)-8 methyl8′-methyl-7′-(3′,4′-dimethoxyphenyl)-tetrahydrofuran and 7,8-epoxide-7-(3,4- methylenedioxy phenyl)-8-methyl-8′methyl-7′-(4′-hydroxy-3′-methoxyphenyl)-tetrahydrofuran, respectively. The 7′,8′-trans-8′,8-cis orientation of 6 was elucidated from the NOE cross-peaks of H-7′/H-8′; H-9′/H-9. Compound 7 had the same relative configuration as compound 6 on the basis of NOE cross-peaks. Compound 8 was obtained as a pale yellow, amorphous solid. Its molecular formula of C19H20O5 was established from HR-ESI-MS (found [M + H]+ 329.1715) and 13C NMR spectroscopic data, indicating 10° of unsaturation. The 1H and 13 C NMR spectra of 8 resembled those of 12. The only difference was the occurrence of a hydroxyl group at C-3′ in 8 replacing the methoxy group in 12, which was confirmed by the correlation from the proton of 3′-OH with C-3′. As previously reported, the signal at δ H 4.89 (1H, d, J = 10.0 Hz) for H-7 indicates that this hydrogen is in trans configuration with the adjacent H-8′, and H-7′ (δ H 4.57, 1H, d, J = 10.0 Hz) also has a trans configuration with the adjacent H-8′. Thus, the structure of 8 was established as (7, 8-trans-8, 8′-trans-7′, 8′-trans)-7(3,4-methylenedioxyphenyl)-8-methyl-8′-methyl-7′-(3′,4′dihydroxyphenyl)-tetrahydrofuran. The CD curves of compounds 2–8 were similar to that of 1 (Fig. 3). Based on biogenetic considerations and comparisons with the CD spectrum and the optical rotation value, the absolute configurations of compounds 2, 3, 4, 5 and 8 were defined as (7S, 8S, 7′R, 8′S), and compounds 6 and 7 were established as (7S, 8S, 7′R, 8′S) (Fig. 1). Compound 13 was obtained as a pale white, amorphous solid. Its molecular formula of C20H24O5 was established from HR-ESI-MS (found [M + Na]+ 367.1618) and 13C NMR spectroscopic data (Table 2), indicating 9° of unsaturation. The 1H NMR spectroscopic data (Table 1) of 13 showed proton signals corresponding to two trisubstituted aromatic rings, two methyl groups, a methylene group, three methine groups, a methoxyl group and a methylenedioxyl group, which suggested that the skeleton of 13 was a 1,4-bisphenyl-2,3dimethylbutane-type lignan. A detailed comparison of the 1H and 13C NMR spectroscopic data of 13 with those of 14 disclosed that the main structural difference between them was to be the locations of the substitutions in aromatic rings. The HMBC correlations from the methoxyl proton and hydroxyl proton to C-3 and C-5 indicated that the two groups in 13 were attached at C-3 and C-5, respectively. On the basis of NOE cross-peaks, the relative configuration of 13 was
K. Jiang et al. / Fitoterapia 103 (2015) 63–70
69
Scheme 1. Proposed biogenetic pathway of the asymmetric tetrahydrofuran lignan with substitutions at C-7 and C-8.
established as (7,8-trans-8,8′-cis)-7-(3,4-dihydroxyphenyl)7′-(3,4-methylenedioxy-phenyl)-8,8′-dimethyl-butan-7-ol (Fig. 1). Although, the presence of additional hydroxyl or methoxyl groups at C-7 and C-8 positions of the asymmetric tetrahydrofuran lignan is very uncommon in plants, compounds of this type have been reported from the Schisandra sphenanthera [10] and other plants belonging to the genus Schisandra [7–9]. And a plausible biosynthetic pathway of these lignans is proposed in Scheme 1. All the lignans were tested for their capacity to scavenge 2,2-diphenyl-1-Picryl‐hydrazyl (DPPH). The IC50 values of lignans were given in Table 3. Within the series of lignans tested, compound 8 exhibited the highest antioxidative activity with IC50 values of 35 μg/mL, which is similar to that of vitamin C. It was clearly seen that the activity of these compounds depends significantly on the number of phenolic groups in the molecules. Compounds 1–8 were all asymmetric tetrahydrofuran lignans. The main structural differences between these compounds refer to the substituents in the aromatic rings. Compound 8, which possesses two phenolic groups, was the most active one (IC50 = 35 μg/mL), followed by compounds 1 and 7, which possess one phenolic group and exhibited similar activity (IC50 = 92 μg/mL, 115 μg/mL). Compounds 2, 3, 5, 9 and 10 which bear no phenolic group, showed weak antioxidative activity (IC50 ~ 200 μg/mL).
Although DPPH assay has been widely used to conveniently test the antioxidative activity of phenolic compounds, the method is only of chemical relevance and the system used is a homogenous solution. Human RBCs are heterogeneous media, and the RBC model has been extensively studied both as a source of free radicals and as a target for oxidative damage. Therefore, the antioxidative effect of compound 8 was investigated in RBC model [23,24]. In the absence of AAPH, the RBCs were stable and little haemolysis took place within 3.5 h. An addition of AAPH induced haemolysis (Fig. 4). It could be seen that compound 8 protected RBCs in a significant way from oxidative-AAPH induced hamolysis at a concentration of 40 μM. The inhibition time produced by 40 μM compound 8 was 90 min as shown in Fig. 4. At a lower concentration (20 μM) compound 8 still had protective activity similar to that of vitamin C (Fig. 4). This is the first report to demonstrate the antioxidant activities of the asymmetric tetrahydrofuran lignans with hydroxyl or methoxyl groups at C-7 and C-8 positions. Components from traditional Chinese medicine are different from drugs and their actions on human physiology are usually moderate. The isolated compounds did not show potent
Table 3 Antioxidative activity of compounds 1–10 and 13. Compound
IC50 (μg/mL)
1 2 3 5 7 8 9 10 13 EtOAc-soluble fraction Crude extract Vitamin C (positive control)
92 233 182 210 115 35 195 261 48 622 781 33
Fig. 4. Inhibitory effect of compound 8 and Vc against AAPH-induced haemolysis of 5% human RBCs in 0.15 M PBS (pH 7.4) in an aerobic atmosphere at 37 °C.
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antioxidant activities, however whether these compounds have the benefits to the health of the human beings should be further studied. Conflict of interest The authors of the present manuscript have declared that no competing interests exist. Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 31270396) and the Fundamental Research Funds for the Central Universities (No. lzujbky-2014176). References [1] Huyke C, Engel K, Simon HB, Quirin KW, Schempp CM. Composition and biological activity of different extracts from Schisandra sphenanthera and Schisandra chinensis. Planta Med 2007;73:1116. [2] Xin HW, Wu XC, Li Q, Yu AR, Zhu M, Shen Y, et al. Effects of Schisandra sphenanthera extract on the pharmacokinetics of tacrolimus in healthy volunteers. Br J Clin Pharmacol 2007;64:469. [3] Song QY, Zhang CJ, Li Y, Wen J, Zhao XW, Liu ZL, et al. Lignans from the fruit of Schisandra sphenanthera, and their inhibition of HSV-2 and adenovirus. Phytochem Lett 2013;6:174. [4] Sun J, Yu J, Zhang PC, Tang F, Yang YN, Yue YD, et al. Isolation and identification of lignans from Caulis Bambusae in Taenia with antioxidant properties. J Agric Food Chem 2013;61:4556. [5] Lou ZH, Li HM, Gao LH, Li RT. Antioxidant lignans from the seeds of Vitex negundo var Cannabifolia. J Asian Nat Prod Res 2014;16:963. [6] Xiao WL, Huang SX, Wang RR, Zhong JL, Gao XM, Sun HD, et al. Nortriterpenoids and lignans from Schisandra sphenanthera. Phytochemistry 2008;69:2862. [7] Pu JX, Gao XM, Wang RR, Yang LB, Lei C, Sun HD, et al. Three new compounds from Kadsura longipedunculata. Chem Pharm Bull 2008;56: 1143. [8] Liu HT, Xu LJ, Peng Y, Yang XW, Xiao PG. Two new lignans from Schisandra henryi. Chem Pharm Bull 2009;57:405. [9] Huang RM, Huang HJ, Zhang NL, Zhua YH, Jiang XF, Qiu SX, et al. Two new lignans from the stems of Schisandra bicolor. J Asian Nat Prod Res 2012;14: 1116.
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