Lanostane Triterpenoids from the Stems of Schisandra glaucescens

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Extraction and isolation
• X-ray crystal structure analysis
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Ten new triterpenoids, schiglausins A–J (1–10), as well as four known compounds, were isolated from the stems of Schisandra glaucescens. Their structures were elucidated on the basis of spectroscopic methods, including extensive NMR spectra and CD experiment. Compound 8 was determined to be a norlanostane triterpenoid. The crystal structure of compound 1 has been determined using single-crystal X-ray analysis while its absolute configuration was assigned on the basis of the CD spectrum. All isolates were tested for their FXR agonistic and antagonistic effects.

Introduction

Wuweizi, the fruits of genus Schisandra (Schisandraceae), have been widely used in traditional Chinese medicine (TCM) as tonic, sedative, and astringent agents for a long time [1]. Modern phytochemical and pharmacological studies have shown that this family is a rich source of lignans and lanostane- and cycloartane-type triterpenoids, which possess various beneficial pharmacological effects such as antihepatitis, antitumor, and anti-HIV-1 activities [2], [3], [4], [5], [6]. Wuweizi can reduce the levels of stress in rats' CORT and Glu, and protect the structure of the adrenal cortex [7]. When used together with dexamethasone, Wuweizi can cure intraheptic cholestasis of pregnancy (ICP) [8]. Schisandra glaucescens Diels is a climbing plant distributed in the northwestern part of mainland China, which is used in folk medicine for treating cough with dyspnea, spontaneous sweat, night sweat, chronic diarrhea, and neurasthenia, and its fruits are eaten locally. There has been only one report on the chemical constituents of S. glaucescens, which was collected from the Shennongjia mountain area of Hubei province [9]. Motivated by a search for bioactive metabolites from plants of Schisandra, we investigated the stems of S. glaucescens collected from Shanxi Qinling Mountain. As a result, fourteen triterpenoids were isolated, including ten new substances, schiglausins A–J (1–10), along with four known compounds (11–14) ([Fig. 1]). The structures of compounds 1–10 were established by their spectroscopic data. The crystal structure of compound 1 has been determined using single-crystal X-ray analysis while its absolute configuration was assigned on the basis of the CD spectrum.

Materials and Methods

General

Melting points were obtained on an XRC-1 micro melting point apparatus and are uncorrected. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. CD spectra were tested using a Chirascan circular dichroism spectrometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. X-ray data was determined using a Bruker APEX DUO instrument. 1D and 2D NMR spectra were recorded on Bruker AM-400, DRX-500, and AVANCE III-600MHz spectrometers. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Mass spectra were performed on a VG Autospec-3000 spectrometer at 70 eV. Column chromatography was performed on silica gel (200–300 mesh; Qingdao Marine Chemical, Inc.), Lichroprep RP-18 gel (40–63 µm; Merck), and MCI-gel CHP 20P (75–150 µm; Mitsubishi Chemical Corp.). Thin-layer chromatography (TLC) was carried out on silica gel 60 F₂₅₄ on glass plates (Qingdao Marine Chemical, Inc.) using various solvent systems, and spots were visualized by heating the silica gel plates sprayed with 95–98 % H₂SO₄-EtOH (V/V = 10 : 90).

Plant material

The stems of S. glaucescens were collected in Qinling Mountain, Shanxi Province, People's Republic of China, in August 2009. The specimen was identified by Prof. Xi-Wen Li, and a voucher specimen (No. KIB 2009–08–08) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and isolation

The plant material of S. glaucescens (6.5 kg) was powdered and exhaustively extracted with Me₂CO-H₂O (V/V = 7 : 3, 3 × 25 L) at room temperature. The solvent was evaporated in vacuo, and the crude extract was partitioned between H₂O and EtOAc. The EtOAc extract (238.2 g) was chromatographed on a silica gel column (80–100 mesh, 15 × 120 cm, 1.0 kg) eluting with CHCl₃-Me₂CO (V/V = 1 : 0, 9 : 1, 8 : 2, 2 : 1, 1 : 1, and 0 : 1, each 20 L) to afford fractions I–VII. Fraction III (8.7 g) was applied to RP-18 (3 × 40 cm), eluted with an MeOH-H₂O (V/V = 9 : 11, 13 : 7, 17 : 3, 100 : 0, each 10 L) gradient system, to afford four fractions. Fraction III-3 (1.5 g) was subjected to semipreparative HPLC (MeOH-H₂O, V/V = 17 : 3) to yield compound 9 (6.0 mg, t_R = 11.29 min, purity > 95 %). Fraction IV (13.2 g) was chromatographed over silica gel (200–300 mesh, 6 × 80 cm, 200 g), developed with petroleum ether-EtOAc (V/V = 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5, each 2 L) to afford five fractions. Fraction IV-4 (2.9 g) was subjected to semipreparative HPLC (MeOH-H₂O, V/V = 4 : 1, 2 L) to yield compounds 1 (24.1 mg, t_R = 11.34 min, purity > 95 %) and 3 (38.0 mg, t_R = 12.86 min, purity > 95 %). Fraction IV-5 (1.7 g) was purified over silica gel (petroleum ether-Me₂CO, V/V = 6 : 1, 4 L) to furnish 14 (10.1 mg, purity > 95 %) and 11 (5.4 g, purity > 98 %). Fraction V (18.6 g) was purified by repeated chromatography over silica gel (200–300 mesh, 6 × 80 cm, 300 g), followed by semipreparative HPLC (MeOH-H₂O, V/V = 9 : 1) to yield 8 (10.5 mg, t_R = 32.62 min, purity > 95 %). Fraction VI (30.1 g) was chromatographed on a silica gel column (200–300 mesh, 8 × 80 cm, 400 g), eluting with CHCl₃-Me₂CO (V/V = 9 : 1, 5 : 1, 2 : 1, 1 : 1, each 2 L) to afford four fractions. Fraction VI-2 (8.6 g) was repeatedly chromatographed on silica gel (200–300 mesh) and Sephadex LH-20 (CHCl₃-MeOH, 4 × 30 cm), and finally by semipreparative HPLC (CH₃CN : H₂O, V/V = 2 : 3 and CH₃CN : H₂O, V/V = 7 : 3) to yield compounds 2 (8.2 mg, t_R = 48.96 min, purity > 95 %) and 10 (6.5 mg, t_R = 21.59 min, purity > 96 %). Fraction VI-4 (15.4 g) was subjected to semipreparative HPLC (MeOH-H₂O, V/V = 4 : 1) to yield compound 4 (62.9 mg, t_R = 8.51 min, purity > 98 %). Fraction VII (16.9 g) was chromatographed on a silica gel column (200–300 mesh, 6 × 80 cm, 300 g), eluting with CHCl₃-Me₂CO (V/V = 10 : 1, 5 : 1, 2 : 1, 1 : 1, each 2 L) to afford four fractions. Fraction VII-2 (8.4 g) was purified by recrystallization and repeated chromatography over silica gel, RP-18, and Sephadex LH-20 (MeOH), followed by preparative and semipreparative HPLC (CH₃CN : H₂O, V/V = 7 : 3) to yield compounds 5 (4.8 mg, t_R = 23.33 min, purity > 95 %), 7 (12.1 mg, t_R = 33.81 min, purity > 95 %), 12 (2.8 mg, t_R = 30.74 min, purity > 95 %), and 13 (9.2 mg, t_R = 24.82 min, purity > 97 %). Similarly, fraction VII-4 (2.9 g) was purified using the above-mentioned chromatography methods to yield compound 6 (8.5 mg, t_R = 22.43 min, purity > 94 %).

Schiglausin A (1): colorless crystals (MeOH); m. p. 209–212 °C; [α]_D ^24.3 + 118.2 (c 0.10, MeOH); UV (MeOH) λ _max (log ε): 205 (4.35) nm; CD (CH₃OH, c 0.12) nm (Δε) 257 (+ 2.31); IR (KBr) ν _max 3507, 3430, 2976, 2943, 2867, 1724, 1706, 1460, 1451, 1394, 1375, 1263, 1119, 1091, 1029, 952, 856, 754, 589 cm⁻¹; ¹³C NMR (CDCl₃, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CDCl₃, 500 MH_Z) spectroscopic data: see [Table 2]; positive ESIMS m/z [M + Na]⁺ 507; HRESIMS m/z [M + H]⁺ 485.3253 (calcd. 485.3267 for C₃₀H₄₅O₅).

Schiglausin B (2): white powder (MeOH); m. p. 168–170 °C; [α]_D ^24.4 + 109.4 (c 0.12, MeOH); UV (MeOH) λ _max (log ε) 204 (4.45), 250 (3.08) nm; CD (CH₃OH, c 0.25) nm (Δε) 257 (+ 2.97); IR (KBr) ν _max 3440, 2938, 2871, 1731, 1452, 1374, 1248, 1126, 1088, 1053, 955, 855, 582 cm⁻¹; ¹³C NMR (C₅D₅ N, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (C₅D₅ N, 400 MH_Z) spectroscopic data: see [Table 2]; positive ESIMS m/z [M + Na]⁺ 523; HRESIMS m/z [M + Na]⁺ 523.3033 (calcd. 523.3035 for C₃₀H₄₄O₆Na).

Schiglausin C (3): white powder (MeOH); m. p. 121–123 °C; [α]_D ^24.1 + 104.4 (c 0.10, MeOH); UV (MeOH) λ _max (log ε) 205 (4.48) nm; CD (CH₃OH, c 0.10) nm (Δε) 254 (+ 1.58); IR (KBr) ν _max 3449, 2968, 2925, 1708, 1453, 1392, 1374, 1245, 1122, 1028, 97, 952, 855 cm⁻¹; ¹³C NMR (CDCl₃, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CDCl₃, 500 MH_Z) spectroscopic data: see [Table 2]; negative ESIMS m/z [M + Cl]⁻ 551; HRESIMS m/z [M + Cl]⁻ 551.3134 (calcd. 551.3189 for C₃₁H₄₈O₆Cl).

Schiglausin D (4): white powder (MeOH); m. p. 120–123 °C; [α]_D ^24.4 + 24.1 (c 0.17, MeOH); UV (MeOH) λ _max (log ε) 204 (4.51) nm; IR (KBr) ν _max 3432, 2931, 1698, 1639, 1631, 1460, 1377, 1281, 1126, 1084, 1051, 795 cm⁻¹; ¹³C NMR (C₅D₅ N, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (C₅D₃ N, 400 MH_Z) spectroscopic data: see [Table 2]; positive ESIMS m/z [M – H]⁻ 485; HRESIMS m/z [M – H]⁻ 485.3263 (calcd. 485.3267 for C₃₀H₄₅O₅).

Schiglausin E (5): white powder (MeOH); m. p. 235–237 °C; [α]_D ^24.7 + 21.3 (c 0.12, MeOH); UV (MeOH) λ _max (log ε) 217 (4.67) nm; IR (KBr) ν _max 3419, 2941, 2871, 1720, 1693, 1436, 1378, 1279, 1204,1173, 1054, 981, 897, 862 cm⁻¹; ¹³C NMR (CDCl₃, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CDCl₃, 400 MH_Z) spectroscopic data: see [Table 2]; positive ESIMS m/z [M + Na]⁺ 523; HRESIMS m/z [M + Na]⁺ 523.3400 (calcd. 523.3399 for C₃₁H₄₈O₅Na).

Schiglausin F (6): white powder (MeOH); m. p. 115–117 °C; [α]_D ^15.4 + 14.9 (c 0.14, MeOH); UV (MeOH) λ _max (log ε) 217 (4.55) nm; IR (KBr) ν _max 3424, 2935, 2871, 1736, 1692, 1459, 1692, 1641, 1459, 1437, 1378, 1203, 1174, 1080, 1024, 901, 801, 723 cm⁻¹; ¹³C NMR (CDCl₃, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CDCl₃, 400 MH_Z) spectroscopic data: see [Table 3]; positive ESIMS m/z [M + Na]⁺ 523; HRESIMS m/z [M + Na]⁺ 523.3409 (calcd. 523.3399 for C₃₁H₄₈O₅Na).

Schiglausin G (7): colorless oil (MeOH); [α]_D ^15.2 + 11.5 (c 0.19, MeOH); UV (MeOH) λ _max (log ε) 218 (4.57) nm; IR (KBr) ν _max 3430, 2952, 1679, 1460, 1384, 1206, 1140, 897, 839, 803, 722, 527 cm⁻¹; ¹³C NMR (CDCl₃, 125 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CDCl₃, 500 MH_Z) spectroscopic data: see [Table 3]; positive ESIMS m/z [M + H]⁺ 499; HRESIMS m/z [M + H]⁺ 499.3419 (calcd. 499.3423 for C₃₁H₄₆O₅H).

Schiglausin H (8): colorless oil (MeOH); [α]_D ^24.8 + 73.7 (c 0.08, MeOH); UV (MeOH) λ _max (log ε) 205 (4.13) nm; IR (KBr) ν _max 3456, 2927, 1740, 1636, 1437, 1379, 1278, 1197, 1172, 1032, 1013, 894 cm⁻¹; ¹³C NMR (CD₃OD, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (CD₃OD, 400 MH_Z) spectroscopic data: see [Table 3]; positive ESIMS m/z [M + Na]⁺ 381; HRESIMS m/z [M + Na]⁺ 381.2405 (calcd. 381.2405 for C₂₃H₃₄O₃Na).

Schiglausin I (9): white powder (MeOH); m. p. 227–229 °C; [α]_D ^24.2 + 50.5 (c 0.12, MeOH); UV (MeOH) λ _max (log ε) 205 (3.75), 250 (3.26) nm; CD (CH₃OH, c 0.28) nm (Δε) 258 (+ 5.08); IR (KBr) ν _max 3458, 2978, 2947, 2926, 2868, 1721, 1624, 1378, 1244, 1128, 1053, 986, 854 cm⁻¹; ¹³C NMR (C₅D₅ N, 100 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (C₅D₅ N, 500 MH_Z) spectroscopic data: see [Table 3]; positive ESIMS m/z [M + Na]⁺ 491; HRESIMS m/z [M + Na]⁺ 491.3131 (calcd. 491.3137 for C₃₀H₄₄O₄Na).

Schiglausin J (10): white powder (MeOH); m. p. 226–228 °C; [α]_D ^16.9 − 10.14 (c 0.12, C₅H₅ N); UV (MeOH) λ _max (log ε) 218 (4.74) nm; IR (KBr) ν _max 3448, 2974, 2941, 2869, 1726, 1696, 1373, 1238, 1153, 1133, 1094, 1034, 1025, 979, 638 cm⁻¹; ¹³C NMR (C₅D₅ N, 125 MH_Z) spectroscopic data: see [Table 1]; ¹H-NMR (C₅D₅ N, 500 MH_Z) spectroscopic data: see [Table 3]; positive ESIMS m/z [M + Na]⁺ 537; HRESIMS m/z [M + Na]⁺ 537.3538 (calcd. 537.3555 for C₃₂H₅₀O₅Na).

X-ray crystal structure analysis

Colorless crystal of 1 was obtained in Me₂CO. Intensity data were collected at room temperature on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [10]. Molecular graphics were computed with PLATON. Crystallographic data of 1 have been deposited in the Cambridge Crystallographic Data Center (No. CCDC 821932). Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44 (0) (1223) 336 033); e-mail: 2Hdeposit@ccdc.cam.ac.uk]. Schiglausin A (1): C₃₀H₄₄O₅·C₃H₆O, M _w = 542.73, monoclinic, space group, P2₁, Z = 2, a = 7.8076 (2), b = 10.3864 (3), c = 18.9408 (5) Å, α = β = 90°, γ = 98.284 (1)°, V = 1519.94 (7) ų, µ (Cu Kα) = 0.635 mm⁻¹, ρ _calc = 1.186 g · cm⁻³; S = 1.044, final R factors: R ₁ = 0.0400 and wR ₂ = 0.1055 for 3913 observed from 11 864 independent and 3937 measured reflections [θ _max = 66.5, I > 2σ (I) criterion and 363 parameters]; maximum and minimum residues are 0.226 and − 0.219 e · Å⁻³, respectively. The Flack parameter is 0.04 (18).

Supporting information

1D and 2D NMR, UV, and IR spectra of 1 and 3, NMR spectroscopic data of compounds 2 and 4–10, X-ray crystallographic data of 1, and method of yeast two-hybrid system-based assay as well as detailed anti-FXR activities of compounds 1–14 (Table 1S) are available as Supporting Information.

Results and Discussion

Schiglausin A (1) was obtained as colorless crystals. Its molecular formula was determined to be C₃₀H₄₄O₅ by HRESIMS. Analysis of its NMR data indicated that compound 1 highly resembled schisanlactone H (12) [11]. The differences observed between them were the presence of two downfield-shifted signals for C-2 (δ _C 67.5) and C-4 (δ _C 88.6), and the absence of one methoxyl. The ¹H-¹H COSY correlations between H₂-1 with H-2 and the HMBC correlations from H₂-1 to C-2, C-3, C-5, C-9, and C-19 further established the existence of the oxygenated C-2 ([Fig. 2]). The HMBC correlations from H₃-27 to C-24, C-25, and C-26, from H-24 to C-22 and C-26, from H-22 to C-21, C-24, and C-26, indicated the presence of a six-membered α-methyl-α,β-unsaturated lactone ring. In addition, according to its molecular formula, there should be another ring formed in the structure of 1. To further determine its structure, 1 was crystallized in Me₂CO to afford a suitable crystal, and an X-ray diffraction was carried out. The small Flack parameter in combination with its relatively large standard deviation did not allow an unambiguous assignment of the absolute configuration of compound 1 on the basis of the X-ray data. In the CD spectrum, compound 1 showed a positive Cotton effect at 255 nm (Δε = + 2.31), similar to that of kadsuphilactone B [12], indicating an R configuration of C-22. Combining the observed ROESY correlations ([Fig. 3]), the X-ray diffraction ([Fig. 4]), and the CD spectrum, the absolute stereochemistry of the seven chiral centers, C-2, C-5, C-8, C-10, C-13, C-14, C-17, C-20, and C-22, were determined as S, R, S, S, R, S, R, S, and R, respectively. Thus, the structure of 1 was fully established as shown.

Schiglausin B (2) had the molecular formula C₃₀H₄₄O₆, as determined by HRESIMS. The comparison of the ¹³C NMR data of 2 with those of 1 indicated that they are analogous. The major difference was a hydroxyl group substituted at C-20 (δ _C 75.1 s) in 2. The HMBC correlations from H-23β and H-17 to C-20 and from H-21 to C-17, C-20, and C-22 confirmed the above deduction. In the CD spectrum, compound 2 showed a positive Cotton effect at 257 nm (Δε = + 2.97), similar to that of kadsuphilactone B [12], indicating an R configuration of C-22. The absence of correlation of H₃-21 with H₃-18 showed that the OH-20 was in β-orientation which was also confirmed by the ROESY correlation of H-16 with H₃-21 and H-22 with H-23. Therefore, the structure of 2 was established as shown.

Schiglausin C (3) was assigned to the molecular formula C₃₁H₄₈O₆ by HRESIMS. The ¹H NMR and ¹³C NMR data of 3 were quite similar to those of 12, the only difference was C-2 substituted by a hydroxyl group in 3. This deduction was confirmed by the HMBC correlations from H₂-1 to C-2 and from H-2 to C-3, and ¹H- ¹H COSY of H₂-1 with H-2. In the CD spectrum, compound 3 showed a positive Cotton effect at 254 nm (Δε = + 1.58), similar to that of kadsuphilactone B [12], indicating an R configuration of C-22. Accordingly, the structure of 3 was established as shown.

Schiglausin D (4) gave a molecular formula C₃₀H₄₅O₅ by HRESIMS. The comparison of the spectroscopic data of 4 with those of kadsuric acid (11) [13] showed that they are structurally similar. The difference found between 4 and 11 was one carbon with a signal at δ _C 70.8 (C-6) in 4 instead of a methylene (C-6) in 11. The HMBC correlations from H-6 to C-4, C-8, and C-10, from H-5 and H-8 to C-6, and ¹H-¹H COSY correlation of H-5 with H-6, suggested that a hydroxyl group was located at C-6. The ROESY correlation of H-6 with H-5 and no correlations observed of H-6 with H-8 and H-19 suggested that the hydroxyl was β-orientated. The obvious ROESY correlation of H-24 with H-26, together with a chemical shifts comparison of C-24 and C-26 with those of 11, determined the double bond between C-24 and C-25 to have a Z configuration. Consequently, the structure of 4 was established as shown.

The NMR spectra of compounds 5, 6, and 7 were quite similar to those of 4, which indicated all of them possessed the same skeleton as 4. The difference found between 4 and 5 was the presence of one methine at δ _C 66.3 (C-23), one methoxyl at δ _C 51.6, and the absence of one methine at δ _C 70.8 (C-6 in 4) in 5. The carbon signal at δ _C 66.3 was assigned to be C-23 on the basis of the HMBC correlation of H-22 with C-23, and the ¹H-¹H COSY correlation of H-24 with H-23, and a hydroxyl group should connect to C-23. The stereochemistry at C-23 of 5 was deduced on the basis of the observed correlation of H-23 with H-24 and the absence of a correlation between H-23 and H₃-21 in the ROESY spectra, as well as the comparison of its ¹³C NMR side-chain (C-20 – C-25) chemical shifts with those of ganoderic acids γ-θ [14], abiesatrine F–H [15], (23S*)-cycloarta-24-en-3β,23-diol, and (23R*)-cycloarta-24-en-3β,23-diol [16]. Therefore, the configuration at C-23 was assigned to be S*. The methoxyl was determined to be located at C-3 by the HMBC correlation of proton signal at δ _H 3.77 (MeO) with C-3.

The ¹³C NMR data of 6 were quite similar to those of 5. The obvious differences were the appearance of an oxygenated methylene at δ _C 66.7 and the absence of a methine at δ _C 66.7 in 6. These differences can be rationalized by a methyl oxygenated in 6 instead of a methylene oxygenated in 5. The oxygenated methylene (δ _C 66.7) was assigned to be C-29 by HMBC correlations of H₂-30 and H-5 with the carbon at δ _C 66.7, and from H₂-29 to C-4, C-5, and C-30. The NMR data of 7 were similar to those of 6, and the difference can be caused by the appearance of an α,β-unsaturated ketone and C-29 being deoxidized in 7. The α,β-unsaturated ketone formed between C-9, C-11, and C-12 was deduced by the downfield shift of C-13 from δ _C 43.9 in 6 to δ _C 57.9 in 7, and the downfield shift of C-9 from δ _C 142.0 in 6 to δ _C 163.0 in 7, together with HMBC correlations from H-17 and H₃-18 to C-12 ([Fig. 2]). Furthermore, in the ROESY spectra of 5, 6, and 7, the observed correlations of H-24 with H-26 determined that the double bond between C-24 and C-25 in the three compounds have a Z configuration. Thus, the structures of 5-7 were established as shown.

Schiglausin H (8) was assigned the molecular formula C₂₃H₃₄O₃ by its HRESIMS. Analysis of 1D NMR spectroscopic data showed that 8 was structurally similar to micranoic acid A [17]. The only difference found was the appearance of one methoxy group with a signal at δ _C 50.5 in 8, which was established to be located at C-3 on the basis of the HMBC correlation of δ _H 3.65 (OMe) with C-3 (δ _C 176.4) and H₂-1 and H₂-2 with C-3. Thus, the structure of 8 was established.

Schiglausin I (9) had a molecular formula of C₃₂H₅₀O₅, as determined by HRESIMS. The NMR spectra of 9 were similar to those of schisanlactone D [18], except that one hydroxyl group was located at C-20 (δ _C 75.1 s) in 9. In the CD spectrum, compound 9 showed a positive Cotton effect at 258 nm (Δε = + 5.08), similar to that of heteroclitalactone E [12], indicating an R configuration of C-22. The absence of correlation of H₃-21 with H₃-18 showed the OH-20 was in β-orientation, which was also confirmed by the ROESY correlation of H-16 with CH₃-21 and H-22 with H-23. The relative configuration of other chiral centers in 9 was established to be identical to that of schisanlactone D by analysis of its ROESY spectrum. Accordingly, the structure of 9 was established as shown.

Schiglausin J (10) was assigned the molecular formular C₃₂H₅₀O₅ by its HRESIMS. Its 1D NMR data were quite similar to those of 3β-hydroxy-lanost-9(11),24-dien-26-oic acid (14) [19]. The difference observed between the two compounds was the appearance of an acetoxyl group and the downfield-shifted signal for C-22 (δ _C 76.1). The HMBC correlation of H-22 with the acetoxyl signal at δ _C 170.1 suggested that the acetoxyl group was located at C-22. The ROESY correlations of H-22 with H-16α, H-20, and H-24, and H-20 with H-24 suggested that H-22 was in α-orientation which was also confirmed by comparing the ¹³C NMR side-chain (C-20 – C-23) chemical shifts with those of 22β-acetoxy-3α,15α-dihydroxylanosta-7,9(11),24-trien-26-oic acid and 22β-acetoxy-3β,15α-dihydroxylanosta-7,9(11),24-trien-26-oic acid [20], as well as 22(R)-and 22(S)-hydroxycholestanols [21]. The obvious ROESY correlation of H-24 with H-26 determined the double bond between C-24 and C-25 to have a Z configuration. As a result, the structure of 10 was established as shown.

The known compounds 11–14 were identified to be kadsuric acid (11) [13], schisanlactone H (12) [11], schisanlactone G (13) [22], and 3β-hydroxy-lanost-9(11),24-dien-26-oic acid (14) [19] by comparison of their spectral data with literature values.

The isolated compounds (purity >94 %) were evaluated for their FXR agonistic and antagonistic effects using the method described in the literature [23]. As a result, GS showed an IC₅₀ of 6.47 µM in our system; compounds 4, 5, 6, 7, 11, and 14 showed weak inhibitory activity against FXR with an inhibitory rate higher than 20 % at 25 µM, and other compounds were not bioactive in the current assay (see Table 1S in Supporting Information).

Acknowledgements

This project was supported financially by the NSFC (No. 20802082 and 30830115), Chinese Academy of Sciences (projects KSCX2-EW-Q-10 and KSCX1-YW-R-24), Major State Basic Research Development Program of China (No. 2009CB522303 and 2009CB940900), Yong Academic and Technical Leader Rising Foundation of Yunnan Province (2006PY01–47), Natural Science Foundation of Yunnan Province (2005XY04 and 2006B0042Q), and Fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (awarded to Dr. Hong-Lin Li).

Conflict of Interest

There were no conflicts of interest among all authors in this manuscript.

References

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  Search in Google Scholar
• 19 Liu J S, Huang M F. Studies on the constituents of Schisandra sphenanthera Rehd. et Wils. 4. The structures of anwuweizic acid and Dl-anwulignan, and the absolute-configurations of D-epigalbacin.  Acta Chim Sin. 1984;  42 264-270
  Search in Google Scholar
• 20 Lin L J, Shiao M S, Yeh S F. Seven new triterpenes from Ganoderma lucidum.  J Nat Prod. 1988;  51 918-924
  Search in Google Scholar
• 21 Letourneux Y, Khuonghuu Q, Gut M, Lukacs G. Identification of C-22 epimers in steroids by C-13 nuclear magnetic-resonance spectroscopy.  J Org Chem. 1975;  40 1674-1675
  Search in Google Scholar
• 22 Ren R, Luo G J, Li H M, Wu Z Y, Li H Z, Li R T. Schisanlactone G, a new 3,4-seco-lanostane triterpenoid from Schisandra sphenanthera.  Chin Chem Lett. 2009;  20 601-603
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• 23 Hyukjae C, Hoosang H, Jungwook C, Euno K, Jaehwan L, Sang-Jip N, Byoung C L, Boon J R, Heonjoong K. Tuberatolides, potent FXR antagonists from the Korean marine tunicate Botryllus tuberatus.  J Nat Prod. 2011;  74 90-94
  Search in Google Scholar

Prof. Han-Dong Sun
Dr. Wei-Lie Xiao

State Key Laboratory of Phytochemistry and Plant Resources in West China
Kunming Institute of Botany
Chinese Academy of Sciences

Lanhei Road 132

Kunming 650201

People's Republic of China

Phone: +86 87 15 22 32 51

Fax: +86 87 15 21 63 43

Email: hdsun@mail.kib.ac.cn

Email: xwl@mail.kib.ac.cn

References

• 1 Song W Z, Xiao P G. The medical plants and lignans of Schisandraceae in China.  Zhong Cao Yao. 1982;  13 40-43
  Search in Google Scholar
• 2 Xiao W L, Li R T, Huang S X, Pu J X, Sun H D. Triterpenoids from the Schisandraceae family.  Nat Prod Rep. 2008;  25 871-891
  Search in Google Scholar
• 3 Fan Y P, Duan L P, Dong X H, Dai Y, Li G P. A new lignan from fruit of Schisandra wilsoniana A. C. Smith and its anti-HIV activity.  Asian J Chem. 2009;  21 5488-5492
  Search in Google Scholar
• 4 Li X N, Pu J X, Du X, Yang L M, An H M, Lei C, He F, Luo X, Zheng Y T, Lu Y, Xiao W L, Sun H D. Lignans with anti-HIV activity from Schisandra propinqua var. sinensis.  J Nat Prod. 2009;  72 1133-1141
  Search in Google Scholar
• 5 Li Y K, Ma Y H, Peng Y F, Dai Y, Li G P. A new lignan from the fruit of Schisandra lancifolia and its anti-HIV activity.  Asian J Chem. 2009;  21 5794-5796
  Search in Google Scholar
• 6 Tanaka R, Tsujii H, Yamada T, Kajimoto T, Amano F, Hasegawa J, Hamashima Y, Node M, Katoh K, Takebe Y. Novel 3 alpha-methoxyserrat-14-en-21 beta-ol (PJ-1) and 3 beta-methoxyserrat-14-en-21 beta-ol (PJ-2)-curcumin, kojic acid, quercetin, and baicalein conjugates as HIV agents.  Bioorg Med Chem. 2009;  17 5238-5246
  Search in Google Scholar
• 7 Sun L J, Wang G H, Wu B, Wang J, Wang Q, Hu L P, Shao J Q, Wang Y T, Li J, Gu P M, Lu B. Effects of Schisandra on the function of the pituitary-adrenal cortex, gonadal axies and carbohydrate metabolism in rats undergoing experimental chronic psychological stress, navigation and strenuous exercise.  Natl J Androl. 2009;  15 126-129
  Search in Google Scholar
• 8 Lei F, Xiong W C. Dexamethasone combining traditional Chinese medicine Schisandra chinensis treatment intrahepatic cholestasis of pregnancy.  Sichuan Med J. 2004;  25 43-44
  Search in Google Scholar
• 9 Meng F Y, Sun J X, Li X, Yu H Y, Li S M, Ruan H L. Schiglautone A, a new tricyclic triterpenoid with a unique 6/7/9-fused skeleton from the stems of Schisandra glaucescens.  Org Lett. 2011;  13 1502-1505
  Search in Google Scholar
• 10 Sheldrick G M. SHELXS-97: Program for crystal structure solution. Göttingen: University of Göttingen; 1997
  Search in Google Scholar
• 11 Zhou S Y, Wang W G, Li H M, Zhang R B, Li H Z, Li R T. Schisanlactone H and sphenanthin A, new metabolites from Schisandra sphenanthera.  J Asian Nat Prod Res. 2009;  11 861-866
  Search in Google Scholar
• 12 Shen Y C, Lin Y C, Chiang M Y, Yeh S F, Cheng Y B, Liao C C. Kadsuphilactones A and B, two new triterpene dilactones from Kadsura philippinensis.  Org Lett. 2005;  7 3307-3310
  Search in Google Scholar
• 13 Yamada Y, Hsu C S, Iguchi K, Suzuki S, Hsu H Y, Chen Y P. Structure of kadsuric acid, anew seco-triterpenoid from Kadsura japonica Dunal.  Chem Lett. 1976;  1307-1310
  Search in Google Scholar
• 14 Min B S, Gao J J, Nakamura N, Hattori M. Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against Meth-A and LLC tumor cells.  Chem Pharm Bull. 2000;  48 1026-1033
  Search in Google Scholar
• 15 Yang X W, Li S M, Wu L, Li Y L, Feng L, Shen Y H, Tian J M, Tang J, Wang N, Liu Y H, Zhang W D. Abiesatrines A–J: anti-inflammatory and antitumor triterpenoids from Abies georgei Orr.  Org Biomol Chem. 2010;  8 2609-2616
  Search in Google Scholar
• 16 Lagoa J H, Brochinia C B, Roquea N F. Terpenoids from Guarea guidonia.  Phytochemistry. 2002;  60 333-338
  Search in Google Scholar
• 17 Li R T, Han Q B, Zhao A H, Sun H D. Micranoic acids A and B: two new octanortriterpenoids from Schisandra micrantha.  Chem Pharm Bull. 2003;  51 1174-1176
  Search in Google Scholar
• 18 Liu J S, Huang M F. On the structures of schisanlactone-C and schisanlactone-D, 2 new triterpene lactones from Schisandra sp.  Acta Chim Sin. 1984;  42 464-469
  Search in Google Scholar
• 19 Liu J S, Huang M F. Studies on the constituents of Schisandra sphenanthera Rehd. et Wils. 4. The structures of anwuweizic acid and Dl-anwulignan, and the absolute-configurations of D-epigalbacin.  Acta Chim Sin. 1984;  42 264-270
  Search in Google Scholar
• 20 Lin L J, Shiao M S, Yeh S F. Seven new triterpenes from Ganoderma lucidum.  J Nat Prod. 1988;  51 918-924
  Search in Google Scholar
• 21 Letourneux Y, Khuonghuu Q, Gut M, Lukacs G. Identification of C-22 epimers in steroids by C-13 nuclear magnetic-resonance spectroscopy.  J Org Chem. 1975;  40 1674-1675
  Search in Google Scholar
• 22 Ren R, Luo G J, Li H M, Wu Z Y, Li H Z, Li R T. Schisanlactone G, a new 3,4-seco-lanostane triterpenoid from Schisandra sphenanthera.  Chin Chem Lett. 2009;  20 601-603
  Search in Google Scholar
• 23 Hyukjae C, Hoosang H, Jungwook C, Euno K, Jaehwan L, Sang-Jip N, Byoung C L, Boon J R, Heonjoong K. Tuberatolides, potent FXR antagonists from the Korean marine tunicate Botryllus tuberatus.  J Nat Prod. 2011;  74 90-94
  Search in Google Scholar

Prof. Han-Dong Sun
Dr. Wei-Lie Xiao

State Key Laboratory of Phytochemistry and Plant Resources in West China
Kunming Institute of Botany
Chinese Academy of Sciences

Lanhei Road 132

Kunming 650201

People's Republic of China

Phone: +86 87 15 22 32 51

Fax: +86 87 15 21 63 43

Email: hdsun@mail.kib.ac.cn

Email: xwl@mail.kib.ac.cn

• Supplementary Material