SUPPORTING INFORMATION for. Identification of key structural characteristics of Schisandra chinensis lignans

Transcription

1 SUPPORTING INFORMATION for Identification of key structural characteristics of Schisandra chinensis lignans involved in P-Glycoprotein inhibition Jiří Slanina #, Gabriela Páchniková #, Martina Čarnecká, Ludmila Porubová Koubíková,, Lenka Adámková, Otakar Humpa, Karel Šmejkal, Iva Slaninová * Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, Building A16, 625 Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Building A6, 625 Brno, Czech Republic Central European Institute of Technology, Masaryk University, Kamenice 5, Building A4, 625 Brno, Czech Republic Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1-3, Brno, Czech Republic Table of contents: 1. Plant material, isolation and characterization of compounds... S2 S5 2. Circular dichroism spectra of compounds... S6 S H and 13 C NMR spectra of 1-5 and S9 S17 S1

2 1. Plant material, isolation and characterization of compounds Plant material. The seeds of Schisandra chinensis were purchased from Herbaton Ltd. (Klčov, Slovakia), originated from (Vladivostok, Russia). The seeds were identified by Ing. Pavel Musil (head of Centre of Medicinal Plant of Masaryk University) and voucher specimens was deposited at Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno. The seeds were airdried and then powdered. Isolation of lignans. The lignans were isolated from 349 g of Schisandra chinensis seeds according to the modified method of Ikeya (Ref. 2). The seeds were extracted with petroleum ether in a Soxhlet apparatus for 2 days. The extract was evaporated in vacuo to give an oily residue (112 g), which was treated with methanol 2 times. The methanol portions were combined (47 g) and further fractioned by column chromatography on silica gel (19 g) with petroleum ether, benzene, diethyl ether and ethanol to give 12 fractions: a (5 ml of petroleum ether, 16.8 g), b (5 ml of petroleum ether, 4. g), c (1 ml of petroleum ether, 2.5 g), d (2 ml of benzene, 4.7 g), e (25 ml of benzene, 2.8 g), f (4 ml of benzene, 2.3 g), g (5 ml of benzene, 2.4 g), h (15 ml of benzene, 2. g), i (25 ml of diethyl ether, 7.3 g), j (5 ml of diethyl ether, 1.3 g), k (1 ml of diethyl ether, g), l (5 ml of ethanol, g). The fractions f - h were combined (6.7 g) and rechromatographed on silica gel column (1 g) with benzene containing an increasing amount of diethyl ether. Fractions were analysed by HPLC and combined if they contained the same compounds. The fractions eluted with benzene containing from 2 % to 1 % diethyl ether gave a residue (2.99 g), which was crystallized from methanol to form 1 (338 mg). A part of mother liquors after crystallization of 1 was purified gradually by semi-preparative reversed phase column (2 mg load, Separon SGX C18, 7 µm, 25 mm 8 mm, Tessek Prague, Czech Rep.) with a mixture methanol water 63:37 (v/v) to give 5 (77 mg) and 8 (4 mg). The fractions, originated from portion f h, which were eluted with a mixture containing from 15 % to 2 % diethyl ether in benzene, were combined with fr. i and this mixture (9.6 g) was fractionated over silica gel column (1 g) with a mobile phase composed from petroleum ether and ethyl acetate. The fraction eluted with a mixture containing 45% of ethyl acetate and 55% of petroleum ether afforded a residue (2.41 g). Repeated chromatography of this residue (silica gel, 1 g, from 1 % to 3 % diethyl ether in benzene) gave a fraction (1.82 g). A part of this eluate was purified gradually by semipreparative reversed phase column (2 mg load, Separon SGX C18, 7 µm, 25 mm 8 mm, Tessek Prague, Czech Rep.) with a mixture methanol water 63:37 (v/v) to give 11 (212 mg). The fraction e (2.8 g) was fractionated on silica gel column (1 g) with benzene containing an increasing amount of diethyl ether. Benzene fractions (8 mg) were repeatedly chromatographed on Separon SGX C18 column (7 µm, 25 mm x 8 mm) to yield 3 (13 mg). The fractions eluted with benzene containing from % to 2 % diethyl ether gave a residue (447 mg), which was purified gradually by semi-preparative reversed phase column (1 mg load, Separon SGX C18, 7 µm, 25 mm 8 mm) with a mixture methanol water 8:2 (v/v) to give (+)-2 (143 mg). The fraction d (4.7 g) was fractionated on silica gel column (1 g) with petroleum ether containing an increasing amount of benzene. The fractions eluted with from 75 % to 1 % benzene afforded a residue (2.4 g), which was crystallized from methanol to give 1 (16 mg). A part of mother liquors after removal of crystalline 1 was purified gradually by semi-preparative reversed phase column (1 mg load, Separon SGX C18, 7 µm, 25 mm 8 mm) with a mixture methanol water 77:23 (v/v) to give (±)-4 (62 mg) and 1 (58 mg). The collected fractions b and c (6.5 g) were subjected to silica gel column (1 g) and eluted with a discontinuous gradient of benzene and diethyl ether. The fractions eluted with benzene diethyl ether 9:1 (v/v) and crystallized from methanol yielded 9 (28 mg). (-)-gomisin N (1): colourless prisms (MeOH); mp C; [α] 2 D - 65 (c 6, CHCl 3 ); 1 H NMR (CDCl 3, 3 MHz) δ 6.56 (1H, s, H-4), 6.48 (1H, s, H-11), 5.95 (2H, s, OCH 2 O), 3.9 (3H, s, 2- OCH 3 ), 3.89 (3H, s, 3-OCH 3 ), 3.82 (3H, s, 14-OCH 3 ), 3.55 (3H, s, 1-OCH 3 ), 2.59 (1H, dd, J = 13.6, 7.2 Hz, H-6a), 2.5 (1H, dd, J = 13.6, 2.3 Hz, H-6b), 2.24 (1H, dd, J = 13.3, 9.5 Hz, H-9a), 2.3 (1H, d, J = 13.3 Hz, H-9b), 1.9 (1H, m, H-7), 1.78 (1H, m, H-8), 8 (3H, d, J=7.2 Hz, 8-CH 3 ), 4 (3H, d, J=7.1 Hz, 7-CH 3 ); 13 C NMR (CDCl 3, 75 MHz): δ (C, C-1), (C, C-3), (C, C-12), (C, C-14), 14 (C, C-2), (C, C-1), (C, C-13), (C, C-5), (C, C-16), S2

3 121.4 (C, C-15), 11 (CH, C-4), 12.9 (CH, C-11), 1 (CH 2, OCH 2 O), 6 (CH 3, 2-OCH 3 ), 6 (CH 3, 1-OCH 3 ), 59.6 (CH 3, 14-OCH 3 ), 55.9 (CH 3, 3-OCH 3 ), 4 (CH, C-8), 39.1 (CH 2, C-6), 35.5 (CH 2, C-9), 33.5 (CH, C-7), 21.5 (CH 3, 8-CH 3 ), 12.8 (CH 3, 7-CH 3 ); ESIMS m/z 41 [M+H] + (1); HRESIMS m/z (calcd for C 23 H 29 O 6, ). The CD spectrum of 1 is given in Fig. S1 The structure of 1 was also verified using X-ray crystallographic analysis, published previously in Ref. 3. The data are identical with that of (-)-gomisin N (1) (Ref. 23, 29 and 39). (+)-deoxyschizandrin ((+)-2): colourless prisms (MeOH); [α] 2 D 95 (c 2, CHCl 3 ); 1 H NMR (CDCl 3, 3 MHz) δ 6.55 (1H, s, H-11), 6.54 (1H, s, H-4), 3.9 (3H, s, 3-OCH 3 ), (3H, s, 12- OCH 3 ), 3.89 (3H, s, 13-OCH 3 ), 3.88 (3H, s, 2-OCH 3 ), (3H, s, 1-OCH 3 ), 3.59 (3H, s, 14- OCH 3 ), 2.59 (1H, dd, J = 13.6, 7.1 Hz, H-9a), 2.5 (1H, dd, J = 13.6, 2.1 Hz, H-9b), 2.29 (1H, dd, J = 13.2, 9.5 Hz, H-6a), 2.6 (1H, dd, J = 13.2, 1.3 Hz, H-6b), 1.92 (1H, m, H-8), 1.82 (1H, m, H-7), 1 (3H, d, J = 7.1 Hz, 7-CH 3 ), 5 (3H, J = 7.1 Hz, 8-CH 3 ); 13 C NMR (CDCl 3,75 MHz) δ (C, C-3), (C, C-12), (C, C-14), (C, C-1), 14 (C, C-13), (C, C-2), (C, C-5), (C, C-1), (C, C-15), (C, C-16), 11 (CH, C-11), 17.2 (CH, C-4), 6 (CH 3, 13-OCH 3 ), 6 (CH 3, 2-OCH 3 ), 69 (CH 3, 14-OCH 3 ), 69 (CH 3, 1-OCH 3 ), 55.9 (CH 3, 12- OCH 3 ), 55.9 (CH 3, 3-OCH 3 ), 47 (CH, C-7), 39.1 (CH 2, C-9), 35.6 (CH 2, C-6), 33.7 (CH, C-8), 21.7 (CH 3, 7-CH 3 ), 12.7 (CH 3, 8-CH 3 ); ESIMS m/z 417 [M+H] + (1); HRESIMS m/z (calcd for C 24 H 33 O 6, ). The CD spectrum of (+)-2 is given in Fig. S2. The data are identical with that of (+)-deoxyschizandrin (Ref. 23, 29 and 39). (-)-deoxyschizandrin ((-)-2) was prepared by methylation of (-)-8 with methyl iodide. Methylation of (-)-8 (6.7 mg) with methyl iodide ( ml) and K 2 CO 3 (9.5 mg) in acetone (1 ml) afforded (-)-2 (4.7 mg), which had the same retention time, co-elute with (+)-2 on reversed phase HPLC (Chromolith Performance RP 18e, Merck, 1 cm 4.6 mm, acetonitrile water 5:5 (v/v)) and showed the same fragmentation pattern by ESIMS. ESIMS m/z 417 [M+H] + (1). The CD spectrum of (-)-2 is given in Fig. S2. tigloylgomisin P (3): colourless prisms (MeOH); mp 123 C; 1 H NMR (CDCl 3, 5 MHz) δ 6.91 (1H, qd, J=7.1 Hz, J=1.5 Hz, O 2 C-C(CH 3 )=CH(CH 3 )), 6.88 (1H, s, H-4), 6.5 (1H, s, H-11), 5.98, 5.97 (each 1H, d, J=5. Hz, OCH 2 O), 5.54 (1H, s, H-6), 3.91 (3H, s, 2-OCH 3 ), 3.89 (3H, s, 3-OCH 3 ), 3.83 (3H, s, 14-OCH 3 ), 3.62 (3H, s, 1-OCH 3 ), 2.19 (1H, d, J=13.8 Hz, H-9a), 2.9 (1H, s, 7-OH), 2.8 (1H, dd, J=1, 13.8 Hz, H-9b), 1.9 (1H, dd, J=9.3, 7.1 Hz, H-8), (3H, m, O 2 C- C(CH 3 )=CH(CH 3 )), 1.81 (3H, dd, J=7., 1.1 Hz, O 2 C-C(CH 3 )=CH(CH 3 )), 1.12 (3H, s, 7-CH 3 ), 1.1 (3H, d, J=7. Hz, 8-CH 3 ); 13 C NMR (CDCl 3, 125 MHz) δ (C, O 2 C-C(CH 3 )=CH(CH 3 )), (C, C-3), (C, C-1), (C, C-12), (C, C-14), (C, C-2), (CH, O 2 C- C(CH 3 )=CH(CH 3 )), (C, C-1), (C, C-13), 133. (C, C-5), (C, O 2 C- C(CH 3 )=CH(CH 3 )), (C, C-15), (C, C-16), 16.2 (CH, C-4), 13. (CH, C-11), 1 (CH 2, OCH 2 O), 77.6 (CH, C-6), 75.2 (C, C-7), 6 (CH 3, 2-OCH 3 ), 6 (CH 3, 1-OCH 3 ), 6. (CH 3, 14-OCH 3 ), 55.9 (CH 3, 3-OCH 3 ), 46.6 (CH, C-8), 36.7 (CH 2, C-9), 18.8 (CH 3, C7-CH 3 ), 17.5 (CH 3, C8-CH 3 ), 14.4 (CH 3, O 2 C-C(CH 3 )=CH(CH 3 )), 12.2 (CH 3, O 2 C-C(CH 3 )=CH(CH 3 )); ESIMS m/z 532 [M+NH 4 ] + (21), 415 [M-tiglic acid+h] + (1); HRESIMS m/z (calcd for C 23 H 27 O 7 [M - tiglic acid + H] +, CD spectrum of 3 is given in Fig. S3. The data are identical with that of tigloylgomisin P (Ref. 24). (±)-γ-schizandrin ((±)-4): colourless prisms (MeOH); mp 125 C; [α] 2 D (c.99, CHCl 3 ); 1 H NMR (CDCl 3, 3 MHz) δ 6.55 (1H, s, H-11), 6.49 (1H, s, H-4), 5.97, 5.95 (each 1H, d, J=4.4 Hz, OCH 2 O), 3.9 (3H, s, OCH 3 ), 3.89 (3H, s, OCH 3 ), 3.83 (3H, s, OCH 3 ), 3.53 (3H, s, OCH 3 ), 2.56 (1H, dd, J = 13.5, 7.4 Hz, H-6a), 2.47 (1H, dd, J = 13.5, 1.9 Hz, H-6b), 2.25 (1H, J = 13.2, 9.5 Hz, H-9a), 2.4 (1H, br. d, J = 13.2 Hz, H-9b), 1.91 (1H, m, H-7), 1.8 (1H, m, H-8), (3H, d, J = 7.2 Hz, 8-CH 3 ), 5 (3H, d, J = 7.1 Hz, 7-CH 3 ); 13 C NMR (CDCl 3, 75 MHz) δ (C, C-12), (C, C-14), (C, C-3), (C, C-1), (C, C-13), (C, C-1), (C, C-2), (C, C-5), (C, C- 15), (C, C-16), 17.4 (CH, C-11), 15.9 (CH, C-4), 1 (CH 2, OCH 2 O), 6 (CH 3, 13-OCH 3 ), 6 (CH 3, 14-OCH 3 ), 59.6 (CH 3, 1-OCH 3 ), 55.9 (CH 3, 12-OCH 3 ), 4 (CH, C-8), 38.9 (CH 2, C-6), 35.4 (CH 2, C-9), 33.8 (CH, C-7), 21.9 (CH 3, 8-CH 3 ), 12.4 (CH 3, 7-CH 3 ); ESIMS m/z 41 [M+H] + (1). HRESIMS m/z (calcd for C 23 H 29 O 6, CD spectrum of 3 is given in Fig. S4. The data are identical with that of (±)-γ-schizandrin (Ref. 25 and 28). S3

4 (+)-γ-schizandrin ((+)-4) and (-)-γ-schizandrin ((-)-4) were obtained by separation 18 mg of (±)-4 on a short semi-preparative Chiralcel OD column (5 1 mm i.d., Daicel, sample injection 1-2 mg) with a mobile phase containing 25 % (v/v) of propane-2-ol in n-hexane. Fractions containing lignans were collected, evaporated and dissolved in 2 ml of methanol, and passed through a silica gel solid-phase extraction cartridge (Strata Silica, 1 mg) to remove a chiral selector, which can be potentially desorbed from the chiral column during separation. After methanol evaporation, 7.8 mg of (+)-4 and 5.2 mg of (-)-4 were prepared. ESI mass spectra of both (+)-4 and (-)-4 were identical with that of (±)- 4. CD spectra of both (+)-4 and (-)-4 are shown in Fig. S4. angeloylgomisin H (5): amorphous solids; 1 H NMR (CDCl 3, 5 MHz) δ 6.7 (1H, s, H-4), 6.57 (1H, s, H-11), 5.89 (1H, m, O 2 C-C(CH 3 )=CH(CH 3 )), 3.92 (3H, s, OCH 3 ), 3.88 (3H, s, OCH 3 ), 3.85 (3H, s, OCH 3 ), 3.85 (3H, s, OCH 3 ), 3.56 (3H, s, OCH 3 ), 2.76 (1H, d, J=13.8 Hz, H-6a), 2.72 (1H, dd, J=14.3, 2. Hz, H-9a), 2.42 (1H, dd, J=14.3, 7.7 Hz, H-9b), 2.35 (1H, d, J=13.8 Hz, H-6b), 1.89 (1H, qd, J=7.2 Hz, J=7.7 Hz, H-8), 1.77 (6H, m, 2 angeloyl CH 3 ), 1.71 (1H, s, 7-OH), 1.26 (3H, s, 7-CH 3 ), 6 (3H, d, J=7.2 Hz, 7-CH 3 ); 13 C NMR (CDCl 3, 125 MHz) δ (C, O 2 C-C(CH 3 )=CH(CH 3 )), (C, C-1), (C, C-3), (C, C-12), (C, C-14), 14 (C, C-2), (C, C-13), 138.2, (O 2 C-C(CH 3 )=CH(CH 3 ) and O 2 C-C(CH 3 )=CH(CH 3 )), 133.8, (each C, C-5 and C-1), (C, C-16), (C, C-15), (CH, C-11), 11 (CH, C-4), 72. (C, C-7), 6, 6, 6 (each CH 3, 3 OCH 3 ), 56.1 (2 CH 3, 2 OCH 3 ), 41.9 (CH, C-8), 4 (CH 2, C-6), 34.3 (CH 2, C-9), 29.8 (CH 3, 7-CH 3 ), 2 (CH 3, O 2 C-C(CH 3 )=CH(CH 3 )), 15.9 (CH 3, 8-CH 3 ), 15.3 (CH 3, O 2 C- C(CH 3 )=CH(CH 3 )); ESIMS m/z 51 [M+H] + (55), 41 [M-angelic acid+h] + (1), 483 [M-H 2 O+H] + (94); HRESIMS m/z (calcd for C 23 H 29 O 6 [M-angelic acid+h] +, The CD spectrum of 5 is given in Fig. S3. The data are identical with that of angeloylgomisin H (Ref. 19). Schisantherin C (6) was isolated from fruit of S. chinensis (Ref. 18). (-)-schisantherin C (6): amorphous solids; [α] 2 D -1 (c 2, CHCl 3 ); 1 H NMR (CDCl 3, 3 MHz) δ 6.76 (1H, s, H-4), 6.54 (1H, s, H-11), 5.99 (1H, q, J=1.4 Hz, O 2 C-C(CH 3 )=CH(CH 3 )), 5.92, 5.87 (each 1H, d, J=1.5 Hz, OCH 2 O), 5.66 (1H, br. s, H-6), 5.57 (1H, d, J=1.5 Hz, OH), 3.91 (3H, s, OCH 3 ), 3.9 (3H, s, OCH 3 ), 3.7 (3H, s, OCH 3 ), 3.58 (3H, s, OCH 3 ), 2.31 (1H, dd, J=13.9, Hz, H-9a), 2.17 (1H, dd, J=13.9, 1 Hz, H-9b), 1.97 (1H, m, H-8), 1.68 (3H, d, J= Hz, O 2 C-C(CH 3 )=CH(CH 3 )), 1.58 (3H, br. s, O 2 C-C(CH 3 )=CH(CH 3 )), 1.32 (3H, s, 7-CH 3 ), 1.15 (3H, d, J=7. Hz, 8-CH 3 ); 13 C NMR (CDCl 3, 75 MHz) δ 166. (C, O 2 C-C(CH 3 )=CH(CH 3 )), (C, C-3), (C, C-1), (C, C- 13), (C, C-2), (C, C-14), 139. (C, C-5), 138. (CH, O 2 C-C(CH 3 )=CH(CH 3 )), (C, C-12), (C, C-1), (C, O 2 C-C(CH 3 )=CH(CH 3 )), 122. (C, C-15), 122. (C, C-16), 11 (CH, C-4), 12.8 (CH, C-11), 1 (CH 2, OCH 2 O), 84.5 (CH, C-6), 72.6 (C, C-7), 61.1 (CH 3, 3- OCH 3 ), 6 (CH 3, 1-OCH 3 ), 59.3 (CH 3, 14-OCH 3 ), 56.2 (CH 3, 2-OCH 3 ), 42.9 (CH, C-8), 36.9 (CH 2, C-9), 28.6 (CH 3, C7-CH 3 ), 19.2 (CH 3, C8-CH 3 ), 14.3 (CH 3, O 2 C-C(CH 3 )=CH(CH 3 )), 11.9 (CH 3, O 2 C- C(CH 3 )=CH(CH 3 )). HRESIMS m/z (calcd for C 28 H 38 NO 9 [M+NH 4 ] +, ). The CD spectrum of 6 is given in Fig. S3. The data are identical with that of (-)-schisantherin C (Ref. 27). Gomisin G (7) was isolated from fruit of S. chinensis (Ref. 18). (-)-gomisin G (7): amorphous solids; [α] 2 D -12 (c, CHCl 3 ); 1 H NMR (CDCl 3, 3 MHz) δ 7.45 (1H, m, Ph-H), 7.38 (2H, m, Ph-H), 7.27 (2H, m, Ph-H), 6.74 (1H, s, H-4), 6.68 (1H, s, H-11), 6.8, 6.1 (each 1H, br. s, OCH 2 O), 5.85 (1H, br. s, H-6), 3.98 (3H, s, 12-OCH 3 ), 3.81 (3H, s, 13-OCH 3 ), 3.33 (3H, s, 1-OCH 3 ), 3.18 (3H, s, 14-OCH 3 ), 2.45 (1H, dd, J=13.5, 1. Hz, H-9a), 2.19 (1H, d, J=13.5 Hz, H-9b), 2.1 (1H, m, H-8), 1.33 (3H, s, 7-CH 3 ), 1.12 (3H, d, J=7.1 Hz, 8-CH 3 ); 13 C NMR (CDCl 3 ) δ (C, O-CO-Ph), C, C-12), (C, C-14), 149. (C, C-3), 14 (C, C-1), 14. (C, C-13), (C, C-2), 13 (C, C-1), 13 (C, C-5), 129.8, 129.8, 129.8, 129.6, 129.6, (each CH, Phe), (C, C-15), (C, C-16), 17.8 (CH, C-11), 16. (CH, C-4), 1 (CH 2, OCH 2 O), 85.4 (CH, C-6), 72.8 (C, C-7), 6 (CH 3, 1-OCH 3 ), 6 (CH 3, 13-OCH 3 ), 6 (CH 3, 14- OCH 3 ), 56.4 (CH 3, 12-OCH 3 ), 42.8 (CH, C-8), 37.1 (CH 2, C-9), 28.7 (CH 3, C7-CH 3 ), 19.2 (CH 3, C8- CH 3 ). HRESIMS m/z (calcd for C 3 H 36 NO 9 [M+NH 4 ] +, ). The CD spectrum of 7 is given in Fig. S3. The data are identical with that of (-)-gomisin G (Ref. 2). gomisin J (8): amorphous solids; [α] 2 D - 91 (c.96, CHCl 3 ); 1 H NMR (CDCl 3, 5 MHz) δ 6.638, (each 1H, s, H-4 and H-11), 5.71, 5.67 (each 1H, s, Ar-OH), (3H, s, OCH 3 ), (3H, s, S4

5 OCH 3 ), 3.53 (6H, s, 2 OCH 3 ), 2.56 (1H, dd, J=13.6, 7.5 Hz, H-6a), 2.47 (1H, dd, J=13.6, 1.9 Hz, H- 6b), 2.25 (1H, dd, J=13.2, J=9.5 Hz, H-9a), 2.4 (1H, d, J=13.2 Hz, H-9b), (1H, m, H-7), (1H, m, H-8), 9 (3H, d, J=7.2 Hz, 8-CH 3 ), 5 (3H, d, J=7.1 Hz, 7-CH 3 ); 13 C NMR (CDCl 3, 125 MHz) δ 15, 15 (each C, C-1 and C-14), 148.7, (each C, C-3 and C-12), 14 (C, C-1), 137.7, (each C, C-2 and C-13), (C, C-5), (C, C-16), (C, C-15), (CH, C-4), 11 (CH, C-11), 6 (2 CH 3, 2-OCH 3 and 13-OCH 3 ), 6. (2 CH 3, 1-OCH 3 and 14-OCH 3 ), 4 (CH, C-8), 38.9 (CH 2, C-6), 35.3 (CH 2, C-9), 33.8 (CH, C-7), 21.7 (CH 3, 8-CH 3 ), 12.6 (CH 3, 7-CH 3 ). ESIMS m/z 389 [M+H] + (1). HRESIMS m/z (calcd for C 22 H 29 O 6, ). The data are identical with that of gomisin J (Ref. 22 and 39). (+)-gomisin J ((+)-8) and (-)-gomisin J ((-)-8) were obtained by repeated separation 2 mg of 8 on a short semi-preparative Chiralcel OD column (5 1 mm i.d., Daicel, sample injection 1 mg) with a mobile phase n-hexane - propane-2-ol 99:1 (v/v). Fractions containing lignans were collected, evaporated and dissolved in 2 ml of methanol, and passed through a silica gel solid-phase extraction cartridge (Strata Silica, 1 mg) to remove a chiral selector, which can be potentially desorbed from the chiral column during separation. After methanol evaporation, 2. mg of (+)-8 and 15.7 mg of (-)-8 were isolated. ESI mass spectra of both (+)-8 and (-)-8 were identical with that of 8. CD spectra of (+)-8 and (-)-8 are shown in Fig. S5. (-)-wuweizisu C (9): colourless prisms (MeOH); mp C; [α] 2 D - 46 (c 8, CHCl 3 ); 1 H NMR (CDCl 3,7 MHz) δ 6.493, (each 1H, s, H-4 and H-11), 5.97, 5.954, 5.946, 5.94 (each 1H, d, J = 1.5 Hz, 2 OCH 2 O), 3.85, 3.83 (each 3H, s, 2 OCH 3 ), 2.54 (1H, dd, J = 13.6, J = 7.2 Hz, H- 6a), 2.44 (1H, dd, J = 13.7, 1.6 Hz, H-6b), 2.24 (1H, dd, J = 9.5, 13.5 Hz, H-9a), 2. (1H, d, J = 13.3 Hz, H-6b), (1H, m, H-7), (1H, m, H-8), 6 (3H, d, J = 7.2 Hz, 8-CH 3 ), 3 (3H, d, J = 7.2 Hz, 7-CH 3 ); 13 C NMR (CDCl 3, 175 MHz) δ 148.6, (each C, C-3 and C-12), 141.1, 14 (each C, C-1 and C-14), (C, C-1), 134.6, (each C, C-2 and C-13), (C, C-5), 122., 12 (C, C-15 and C-16), 16.1 (CH, C-4), 13.1 (CH, C-11), 1 (CH 2, 2 OCH 2 O), 59.6 (CH 3, 2 OCH 3 ), 4 (CH, C-8), 38.7 (CH 2, C-6), 35.2 (CH 2, C-9), 33.5 (CH, C-7), 21.7 (CH 3, 8-CH 3 ), 12.5 (CH 3, 7-CH 3 ); ESIMS m/z 385 [M+H] +. HRESIMS m/z (calcd for C 22 H 25 O 6 [M+H] +, The CD spectrum of 9 is given in Fig. S1. The data are identical with that of (-)- wuweizisu C (Ref. 26 and 28). (+)-gomisin A (1): colourless needles (MeOH); mp C, [α] 2 D + 57 (c 3, CHCl 3 ); 1 H NMR (CDCl 3, 7 MHz) δ 6.63 (1H, s, H-4), 6.49 (1H, s, H-11), 5.98, 5.97 (each 1H, d, J = 1.3 Hz, OCH 2 O), 3.91 (6H, s, 2 OCH 3 ), 3.85 (3H, s, OCH 3 ), 3.52 (3H, s, OCH 3 ), 2.7 (1H, d, J = 13.5 Hz, H-6a), 2.58 (1H, dd, J = 14.2, 2.1 Hz, H-9a), 2.35 (1H, J = 13.5 Hz, H-6b), 2.33 (1H, dd, J = 14., 7.3 Hz, H-9b), 1.9 (1H, br. s, 7-OH), 1.87 (1H, m, H-8), 1.26 (3H, s, 7-CH 3 ), 2 (3H, d, J = 7.3 Hz, 8- CH 3 ); 13 C NMR (CDCl 3, 175 MHz) δ (C, C-3), (C, C-1), (C, C-12), 141.1, 14 (each C, C-2 and C-14), (C, C-13), (C, C-1), 132. (C, C-5), (C, C-16), (C, C-15), 11 (CH, C-4), 15.9 (CH, C-11), 1 (CH 2, OCH 2 O), 71.6 (C, C-7), 6 (CH 3, 2-OCH 3 ), 6, 59.7 (each CH 3, 1-OCH 3 and 14-OCH 3 ), 55.9 (CH 3, 3-OCH 3 ), 42. (CH, C-8), 4 (CH 2, C-6), 33.6 (CH 2, C-9), 3 (CH 3, 7-CH 3 ), 15.8 (CH 3, 8-CH 3 ); ESIMS m/z 399 [M-H 2 O+H] + (1). HRESIMS m/z (calcd for C 23 H 27 O 6 [M-H 2 O+H] +, ). The CD spectrum of 1 is given in Fig. S1. The data are identical with that of (+)-gomisin A (Ref. 2, 28 and 39). (+)-schizandrin (11): colourless prisms (MeOH); mp C; [α] 2 D + 77 (c 4, CHCl 3 ); 1 H NMR (CDCl 3, 7 MHz) δ 6.62 (1H, s, H-4), 6.55 (1H, s, H-11), 3.92, 3.9, 3.895, 3.888, 3.6, 3.59 (each 3H, s, 6 OCH 3 ), 2.67 (1H, d, J = 13.8 Hz, H-6a), 2.64 (1H, dd, J = 14., 1.2 Hz, H-9a), 2.39 (1H, J = 13.8 Hz, H-6b), 2.37 (1H, m, H-9b), 1.91 (1H, br. s, 7-OH), 1.87 (1H, m, H-8), 1.27 (3H, s, 7- CH 3 ), 2 (3H, d, J = 7.3 Hz, 8-CH 3 ); 13 C NMR (CDCl 3, 175 MHz) δ (C, C-3), 152. (C, C-12), 151.8, (each C, C-1 and C-4), 14, 14 (each C, C-2 and C-13), (C, C-1), (C, C-5), (C, C-16), (C, C-15), 11, 19.9 (each CH, C-4 and C-11), 71.8 (C, C-7), 6 (2 CH 3, 2-OCH 3 and 13-OCH 3 ), 65, 6 (each CH 3, 1-OCH 3 and 14-OCH 3 ), 55.9, 55.8 (each CH 3, 3-OCH 3 and 12-OCH 3 ), 41.8 (CH, C-8), 4 (CH 2, C-6), 34.1 (CH 2, C-9), 29.8 (CH 3, 7-CH 3 ), 15.9 (CH 3, 8-CH 3 ); ESIMS m/z 433 [M+H] + (6), 415 [M-H 2 O+H] + (1); HRESIMS m/z (calcd for C 24 H 31 O 6 [M-H 2 O+H] +, The CD spectrum of 11 is given in Fig. S1 (Ref. 2, 28 and 39). The data are identical with that of (+)-schizandrin (Ref. 2, 28 and 39). S5

6 2. Circular dichroism spectra of compounds Figure S1. Circular dichroism (CD) spectra of (-)-gomisin N (1), (-)-wuweizisu C (9), (+)-gomisin A (1) and (+)-schizandrin (11) were recorded in methanol. Figure S2. Circular dichroism (CD) spectra of (+)-deoxyschizandrin ((+)-2) and (-)-deoxyschizandrin ((-)-2) were recorded in methanol. S6

7 Figure S3. Circular dichroism (CD) spectra of tigloylgomisin P (3), angeloylgomisin H (5), (-)- schisantherin C (6) and (-)-gomisin G (7) were recorded in methanol. Figure S4. Circular dichroism (CD) spectra of (+)-γ-schizandrin ((+)-4) and (+)-γ-schizandrin ((+)-4) were recorded in methanol. S7

8 Figure S5. Circular dichroism (CD) spectra of (+)-gomisin J ((+)-8) and (-)-gomisin J ((-)-8) were recorded in methanol. S8

9 H and 13 C NMR spectra of compounds JS_1-1H-NMR.1r.esp Figure S6. 1 H NMR spectrum of gomisin N (1) in CDCl 3 (3 MHz). JS_1-13C-NMR.1r.esp Figure S7. 13 C NMR spectrum of gomisin N (1) in CDCl 3 (75 MHz). S9

10 JS_ r.esp Figure S8. 1 H NMR spectrum of (+)-deoxyschizandrin, (+)-2, in CDCl 3 (3 MHz). deoxyschizandrin-13c-nmr.1r.esp Figure S9. 13 C NMR spectrum of (+)-deoxyschizandrin, (+)-2, in CDCl 3 (75 MHz). S1

11 JS r.esp Figure S1. 1 H NMR spectrum of tigloylgomisin P (3) in CDCl 3 (5 MHz)..35 JS-22-tigloylgomisin P r.esp Figure S C NMR spectrum of tigloylgomisin P (3) in CDCl 3 (125 MHz). S11

12 JS_2-gama-schizandrin-1H-NMR.1r.esp Figure S12. 1 H NMR spectrum of (±)-γ-schizandrin, (±)-4, in CDCl 3 (3 MHz). JS_2-gama-schizandrin-13C-NMR.1r.esp Figure S C NMR spectrum of (±)-γ-schizandrin, (±)-4, in CDCl 3 (75 MHz). S12

13 JS-21-angeloylgomisin-H.51.1r.esp Figure S14. 1 H NMR spectrum of angeloylgomisin H (5) in CDCl 3 (5 MHz). 3 Js-21-angeloylgomisin-13C-NMR.1r.esp Figure S C NMR spectrum of angeloylgomisin H (5) in CDCl 3 (125 MHz). S13

14 JS-12-gomisin J.1H-NMR.1r.esp Figure S16. 1 H NMR spectrum of gomisin J (8) in CDCl 3 (5 MHz). 6 JS-12-gomisin J-13C-NMR.1r.esp Figure S C NMR spectrum of gomisin J (8) in CDCl 3 (125 MHz). S14

15 wuweizisu C-1H-NMR.1r.esp Figure S18. 1 H NMR spectrum of wuweizisu C (9) in CDCl 3 (7 MHz). wuweizisu C-13C-NMR.1r.esp Figure S C NMR spectrum of wuweizisu C (9) in CDCl 3 (175 MHz). S15

16 gomisin-a-1h-nmr.1r.esp Figure S2. 1 H NMR spectrum of gomisin A (1) in CDCl 3 (7 MHz). gomisin-a-13c-nmr.1r.esp Figure S C NMR spectrum of gomisin A (1) in CDCl 3 (175 MHz). S16

17 schizandrin-1h-nmra-i.1r.esp Figure S22. 1 H NMR spectrum of schizandrin (11) in CDCl 3 (7 MHz). schizandrin-13c-nmr.1r.esp Figure S C NMR spectrum of schizandrin (11) in CDCl 3 (175 MHz). S17