Hydroxy Lignan Glucosides from Arctii Fructus - American Chemical

Sep 2, 2014 - KEYWORDS: Arctium lappa L., Arctii Fructus, lignan, isolation, health food, ... bang zi” in Chinese, is the dried mature fruit of Arct...
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Hepatoprotective Activity of Twelve Novel 7′-Hydroxy Lignan Glucosides from Arctii Fructus Ya-Nan Yang, Xiao-Ying Huang, Zi-Ming Feng, Jian-Shuang Jiang, and Pei-Cheng Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Twelve novel 7′-hydroxy lignan glucosides (1−12), including two benzofuran-type neolignans, two 8-O-4′ neolignans, two dibenzylbutyrolactone lignans, and six tetrahydrofuranoid lignans, together with six known lignan glucosides (13−18), were isolated from the fruit of Arctium lappa L. (Asteraceae), commonly known as Arctii Fructus. Their structures were elucidated using spectroscopy (1D and 2D NMR, MS, IR, ORD, and UV) and on the basis of chemical evidence. The absolute configurations of compounds 1−12 were confirmed using rotating frame nuclear overhauser effect spectroscopy (ROESY), the circular dichroic (CD) exciton chirality method, and Rh2(OCOCF3)4-induced CD spectrum analysis. All of the isolated compounds were tested for hepatoprotective effects against D-galactosamine-induced cytotoxicity in HL-7702 hepatic cells. Compounds 1, 2, 7−12, and 17 showed significantly stronger hepatoprotective activity than the positive control bicyclol at a concentration of 1 × 10−5 M. KEYWORDS: Arctium lappa L., Arctii Fructus, lignan, isolation, health food, hepatoprotective activity

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spectrometer (Jasco Inc., Easton, MD). High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed on an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany). Gas chromatography (GC) experiments were conducted on an Agilent 7890A instrument (Agilent Technologies, Waldbronn, Germany). 1H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR spectra were obtained using an INOVA 500 spectrometer (Varian, Inc., Palo Alto, CA). Column chromatography was performed using macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). Flash chromatography was conducted using Combiflash RF200 (Teledyne Isco Corp., Lincoln, NE). Preparative high-performance liquid chromatography (HPLC) was carried out on a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu Corp., Tokyo, Japan), using a YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm, YMC Corp, Kyoto, Japan). HPLC-diode array detection (DAD) analysis was performed on an Agilent 1200 series system with an Apollo C18 column (250 mm × 4.6 mm, 5 μm, Alltech Corp., KY). Plant Material. Arctii Fructus specimens were collected from Wuchang Town, Heilongjiang Province, China, in October 2011. The plant was identified by Professor Lin Ma (Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China). A voucher specimen (ID-S-2434) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China. Extraction and Isolation. Arctii Fructus (100 kg) compounds were extracted with 80% EtOH. After the solvent was evaporated under reduced pressure, the residue (4.2 kg) was suspended in water (10 L) and extracted with EtOAc (10 L) three times. The aqueous layer was evaporated under reduced pressure, and then chromatog-

INTRODUCTION Burdock (Arctium lappa L.) is a biennial herbaceous plant and a popular vegetable in China and Japan. Arctii Fructus, or “niu bang zi” in Chinese, is the dried mature fruit of Arctium lappa, which is cultivated in northeastern China. Arctii Fructus has been used in traditional Chinese medicine since antiquity, and its use has been recorded in every edition of the Chinese Pharmacopoeia. Tea made from Arctii Fructus is used to treat tonsillitis, pharyngolaryngitis, and constipation. Arctii Fructus has shown several biological activities, including antitumor,1 antidiabetic,2 anti-inflammatory,3 and anti-influenza effects.4 Phytochemical investigations have reported that Arctii Fructus contains several secondary metabolites, including lignans,5,6 caffeoylquinic acids,7 and fatty acids.8 Arctigenin, a lignan found in Arctii Fructus, inhibits proliferation of and promotes apoptosis in SMMC-7721 human hepatocellular carcinoma cells.9 Hence, the compound with hepatoprotective activity in Arctii Fructus was further studied in this article. In our investigation of the bioactive components from Arctii Fructus, we isolated 12 novel 7′-hydroxy lignan glucosides, together with six known lignan glucosides, and the structures of these molecules were established using spectroscopy (1D and 2D NMR, MS, IR, ORD, and UV) and chemical evidence. The absolute configurations of compounds 1−12 were confirmed using rotating frame nuclear overhauser effect spectroscopy (ROESY), the circular dichroic (CD) exciton chirality method, and Rh2(OCOCF3)4-induced CD spectrum analysis.

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MATERIALS AND METHODS

General Apparatus and Chemicals. Optical rotation was measured using a Jasco P-2000 polarimeter (Jasco Inc., Easton, MD). Infrared (IR) spectra were recorded on a Nicolet 5700 spectrometer (Thermo Scientific, FL) using an FT-IR microscope transmission method. CD spectra were measured on a Jasco J-815 CD © XXXX American Chemical Society

Received: April 17, 2014 Revised: August 26, 2014 Accepted: September 2, 2014

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dx.doi.org/10.1021/jf501859x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Structures of compounds 1−18.

Table 1. 1H NMR Data (500 MHz, DMSO-d6) for Compounds 1−6 position

3

4

2 5 6 7

6.96 7.06 6.84 5.49

d (2.0) d (8.5) dd (8.5, 2.0) d (6.5)

6.96 7.06 6.84 5.49

d (2.0) d (8.5) dd (8.5, 2.0) d (6.5)

7.57 d (2.0) 7.18 d (8.5) 7.75 dd (8.5, 2.0)

7.56 d (2.0) 7.18 d (8.5) 7.75 dd (8.5, 2.0)

8 9

3.42 3.73 3.63 6.83

m m m d (2.0)

3.43 3.72 3.61 6.82

m m m brs

5.58 t (5.0) 3.88 m

5.59 t (5.0) 3.88 m

6.81 4.57 1.75 1.68 3.48 3.44 4.88 3.24 3.28 3.15 3.26 3.65 3.42 3.75 3.78

d (2.0) m m m m m d (7.5) m m m m m m s s

6.82 4.57 1.75 1.68 3.48 3.44 4.88 3.24 3.28 3.15 3.26 3.65 3.42 3.75 3.78

brs m m m m m d (7.5) m m m m m m s s

6.92 6.68 6.68 4.53 1.71 1.64 3.46 3.39 5.08 3.28 3.39 3.17 3.28 3.67 3.46 3.81 3.74

6.92 6.69 6.69 4.53 1.71 1.64 3.46 3.39 5.08 3.28 3.39 3.17 3.28 3.67 3.46 3.81 3.73

2′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 3-CH3O 3′-CH3O

1

2

brs overlap overlap t (5.0) m m m m d (7.5) m m m m m m s s

raphy was performed on a macroporous adsorption resin (HP-20) column (10 L). After eluting with H2O (20 L), the adsorbed constituents were eluted with 15% ethanol (20 L), 30% ethanol (20 L), 50% ethanol (20 L), 70% ethanol (20 L), and 95% ethanol (10 L). The 30% ethanol part (86.4 g) was subjected to flash chromatography elution with H2O−MeOH (from 100:0 to 0:100) in gradient to yield 13 fractions (fractions A−M). Fraction D was further chromatographed over Sephadex LH-20 and eluted with MeOH−H2O (from 0:100 to 100:0) to give 12 fractions

5

brs overlap overlap t (5.0) m m m m d (7.5) m m m m m m s s

6

6.65 6.96 6.57 2.70 2.49 2.84

d (2.0) d (8.5) dd (8.5, 2.0) dd (13.5, 6.5) overlap dt (8.5, 6.0)

6.65 6.92 6.53 2.86 2.49 2.86

d (2.0) d (8.0) dd (8.0, 2.0) overlap dd (15.5, 7.5) overlap

6.78 6.72 6.66 4.34 2.46

d (2.0) d (8.5) dd (8.5, 2.0) t (5.0) m

6.81 6.72 6.69 4.55 2.49

d (2.0) d (8.0) dd (8.0, 2.0) t (5.0) overlap

4.01 4.13 4.83 3.24 3.26 3.15 3.28 3.66 3.45 3.71 3.73

t (8.5) t (8.5) d (7.5) m m m m brd (11.5) dt (11.5, 6.0) s s

3.94 m 4.83 3.24 3.26 3.15 3.28 3.66 3.45 3.70 3.73

d (7.5) m m m m brd (11.5) dt (11.5, 6.0) s s

(fractions D-1−12). Fractions D-5 and D-6 were obtained with the mobile phase of MeOH−H2O (0:100), and fraction D-8 was obtained with the mobile phase of MeOH−H2O (10:90). Fraction D-5 was purified using reversed-phase preparative HPLC with MeOH−H2O (25:75) as the mobile phase at a flow rate of 5 mL/min to give 17 (22 mg, 55 min). Fraction D-6 was purified using reversed-phase preparative HPLC with MeOH−H2O (25:75) as the mobile phase at a flow rate of 5 mL/min to give 7 (16 mg, 48 min) and 8 (19 mg, 44 min). Using the same method as that used for fraction D-6, 1 (24 mg, B

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Table 2. 13C NMR Data (125 MHz, DMSO-d6) for Compounds 1−12 position

1

2

3

4

5

6

7

8

9

10

11

12

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 3-CH3O 3′-CH3O

135.5 110.4 149.0 146.2 115.4 117.9 86.7 53.6 63.2 140.0 114.2 143.3 146.2 128.5 110.1 69.8 42.7 58.2 100.1 73.2 77.0 69.8 76.9 60.7 55.7 55.8

135.5 110.5 149.0 146.2 115.4 118.0 86.7 53.6 63.2 139.9 114.2 143.3 146.2 128.6 110.2 69.8 42.6 58.2 100.2 73.2 77.1 69.7 76.9 60.7 55.8 55.8

129.0 111.7 148.7 151.1 114.2 122.9 195.5 81.7 62.6 140.1 110.3 148.9 145.6 114.1 117.6 69.4 42.4 58.1 99.4 73.1 77.2 69.6 76.9 60.6 55.6 55.6

129.0 111.6 148.7 151.1 114.3 122.9 195.5 81.6 62.6 140.2 110.4 148.9 145.6 114.2 117.5 69.3 42.4 58.1 99.4 73.1 77.2 69.6 76.9 60.6 55.6 55.6

131.6 113.9 148.6 145.3 115.1 121.4 33.7 45.9 178.7 134.5 110.1 147.5 145.7 115.1 118.3 72.3 42.7 67.7 100.2 73.3 77.0 69.7 76.9 60.7 55.7 55.7

131.5 114.1 148.5 145.3 115.0 121.5 34.2 45.0 178.8 134.1 110.2 147.4 145.7 115.0 118.4 72.5 42.4 68.2 100.2 73.3 77.0 69.7 76.9 60.7 55.6 55.6

133.5 110.6 147.4 145.8 115.1 118.9 83.4 53.9 61.6 138.4 111.1 148.7 145.8 114.9 119.4 74.8 50.6 69.5 100.7 73.3 77.1 69.7 76.9 60.7 55.7 55.8

133.4 110.6 147.4 145.8 115.1 119.0 83.4 54.0 61.6 138.3 111.0 148.7 145.8 114.7 119.0 74.9 50.6 69.4 100.2 73.3 77.0 69.7 76.9 60.7 55.7 55.7

133.8 110.9 147.4 145.6 115.1 118.9 82.8 51.9 60.4 138.3 110.4 148.7 145.8 114.7 118.7 73.7 49.3 69.5 100.2 73.3 77.0 69.7 76.9 60.7 55.6 55.7

136.5 110.8 148.8 145.7 115.0 118.4 83.3 54.1 61.8 135.6 110.8 147.4 145.7 114.9 119.2 74.9 50.7 69.6 100.2 73.3 77.1 69.7 76.9 60.7 55.7 55.7

136.6 110.8 148.8 145.7 115.1 118.4 83.3 54.1 61.8 135.6 110.8 147.4 145.7 114.9 119.2 74.9 50.7 69.6 100.2 73.3 77.1 69.7 76.9 60.7 55.7 55.7

136.8 110.6 148.7 145.6 115.0 118.2 82.7 52.1 60.6 135.5 110.7 147.3 145.5 115.1 119.1 73.9 49.4 69.7 100.2 73.3 77.1 69.7 76.9 60.7 55.6 55.7

(7R,8S,7′S)-Dihydrodehydrodiconiferyl Alcohol-7′-hydroxy-4-Oβ-D-glucopyranoside (2). White amorphous powder; soluble in water or methanol; [α]20D −40.3° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 282 (−1.11), 243 (−1.79), 217 (+1.06) nm; IR νmax 3423, 2928, 1601, 1516, 1464, 1334, 1266, 1139, 1076, 897, 805 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1951 (calcd for C26H34O12Na, 561.1942). (8R,7′S)-4,9,7′,9′-Tetrahydroxy-3,3′-dimethoxyl-7-oxo-8−4′-oxyneolignan-4-O-β-D-glucopyranoside (3). White amorphous powder; soluble in water or methanol; [α]20D −31.4° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 331 (−0.95), 266 (+1.99), 231 (−1.53) nm; IR νmax 3342, 2933, 1676, 1593, 1512, 1421, 1267, 1137, 1076, 889, 801 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 577.1900 (calcd for C26H34O13Na, 577.1892). (8S,7′S)-4,9,7′,9′-Tetrahydroxy-3,3′-dimethoxyl-7-oxo-8−4′-oxyneolignan-4-O-β-D-glucopyranoside (4). White amorphous powder; soluble in water or methanol; [α]20D −44.8° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 346 (+0.29), 299 (−1.26), 259 (−2.54) nm; IR νmax 3331, 2926, 1677, 1593, 1512, 1420, 1267, 1137, 1076, 883, 800 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 577.1905 (calcd for C26H34O13Na, 577.1892). (8R,7′R,8′R)-4,4′,7′-Trihydroxy-3,3′-dimethoxyl-9-oxo Dibenzylbutyrolactone Lignan-4-O-β-D-glucopyranoside (5). White amorphous powder; soluble in water or methanol; [α]20D −26.7 (c 0.07, MeOH); CD (MeOH) λmax (Δε) 278 (−0.83), 229 (−7.37) nm; IR νmax 3386, 2916, 1757, 1601, 1514, 1453, 1423, 1385, 1269, 1227, 1157, 1076, 860, 821 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 559.1787 (calcd for C26H32O12Na, 559.1786). (8R,7′S,8′R)-4,4′,7′-Trihydroxy-3,3′-dimethoxyl-9-oxo Dibenzylbutyrolactone Lignan-4-O-β-D-glucopyranoside (6). White amorphous powder; soluble in water or methanol; [α]20D −33.9° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 307 (−1.09), 225 (−10.79) nm; IR νmax 3369, 2922, 1755, 1599, 1514, 1453, 1423, 1388, 1269, 1227,

42 min), 2 (27 mg, 46 min), and 3 (11 mg, 36 min) were isolated from fraction D-8. Fraction F was subjected to flash chromatography elution with H2O−MeOH (from 100:0 to 0:100) in gradient to yield 10 fractions (fractions F-1−10). Fraction F-3 was obtained with the mobile phase of MeOH−H2O (10:90), and was further purified using reversedphase preparative HPLC with MeOH−H2O (27:73) as the mobile phase at a flow rate of 5 mL/min to give 4 (33 mg, 31 min) and 10 (20 mg, 47 min). Fraction F-5 was obtained with the mobile phase of MeOH−H2O (20:80), and was subjected to chromatography over Sephadex LH-20 with H2O as the mobile phase to give 9 (15 mg) and 13 (27 mg). Fractions F-6 and F-7 were obtained with the mobile phase of MeOH−H2O (30:70). Fraction F-6 was chromatographed over Sephadex LH-20 with H2O elution to give 15 (31 mg). Fraction F-7 was purified using reversed-phase preparative HPLC with MeOH−H2O (30:70) as the mobile phase at a flow rate of 5 mL/ min to give 14 (19 mg, 55 min) and 16 (21 mg, 51 min). Fraction H was subjected to flash chromatography elution with H2O−MeOH in gradient to yield 12 fractions (fractions H-1−12). Fraction H-5 was obtained with the mobile phase of MeOH−H2O (20:80), and was further purified by reversed-phase preparative HPLC with MeOH−H2O (30:70) as the mobile phase at a flow rate of 5 mL/ min to give 5 (18 mg, 33 min) and 11 (10 mg, 39 min). Fraction H-6 was obtained with the mobile phase of MeOH−H2O (30:70), and was purified using reversed-phase preparative HPLC with MeOH−H2O (30:70) as the mobile phase at a flow rate of 5 mL/min to give 6 (13 mg, 35 min) and 12 (14 mg, 37 min). Fraction K was chromatographed over Sephadex LH-20 with H2O elution to give 18 (106 mg). The structures of compounds 1−18 are shown in Figure 1. Spectral Data. (7S,8R,7′S)-Dihydrodehydrodiconiferyl Alcohol-7′hydroxy-4-O-β-D-glucopyranoside (1). White amorphous powder; soluble in water or methanol; [α]20D −32.8° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 292 (+0.53), 245 (+0.81), 226 (−1.59) nm; IR νmax 3384, 2933, 1601, 1518, 1464, 1335, 1268, 1136, 1076, 859, 803 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1954 (calcd for C26H34O12Na, 561.1942). C

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Table 3. 1H NMR Data (500 MHz, DMSO-d6) for Compounds 7−12 position

7

8

2 5 6 7 8 9

6.87 6.72 6.72 4.46 2.14 3.48

brs overlap overlap d (8.0) m m

6.86 6.72 6.72 4.45 2.13 3.45

brs overlap overlap d (8.0) m m

2′ 5′ 6′ 7′ 8′ 9′

6.96 7.03 6.84 4.41 2.45 3.60 3.49 4.87 3.25 3.29 3.16 3.27 3.67 3.46 3.77 3.76

d (2.0) d (8.0) dd (8.0, 2.0) dd (8.5, 2.5) m m m d (7.5) m m m m m m s s

6.97 7.02 6.82 4.39 2.45 3.59 3.49 4.87 3.25 3.28 3.16 3.27 3.65 3.44 3.77 3.75

d (2.0) d (8.5) dd (8.5, 2.0) dd (8.5, 2.5) m m m d (7.5) m m m m m m s s

1″ 2″ 3″ 4″ 5″ 6″ 3-CH3O 3′-CH3O

9 6.85 6.71 6.71 4.51 1.73 3.14 3.07 6.87 6.98 6.72 4.37 2.38 4.07 3.72 4.84 3.26 3.28 3.15 3.27 3.66 3.44 3.72 3.75

10

brs overlap overlap d (7.0) m m m d (2.0) d (8.5) overlap dd (7.5, 4.5) m m m d (7.5) m m m m m m s s

1157, 1073, 857, 823 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 559.1798 (calcd for C26H32O12Na, 559.1786). (7R,8S,7′S,8′R)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β-D-glucopyranoside (7). White amorphous powder; soluble in water or methanol; [α]20D −85.1° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 277 (−1.84), 231 (−5.34) nm; IR νmax 3344, 2926, 1602, 1514, 1462, 1388, 1269, 1159, 1073, 858, 814 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1945 (calcd for C26H34O12Na, 561.1942). (7S,8R,7′R,8′S)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β-D-glucopyranoside (8). White amorphous powder; soluble in water or methanol; [α]20D +74.6° (c 0.06, MeOH); CD (MeOH) λmax (Δε) 285 (+0.45), 235 (+2.26) nm; IR νmax 3381, 2927, 1602, 1514, 1463, 1425, 1268, 1159, 1073, 859, 815 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1956 (calcd for C26H34O12Na, 561.1942). (7S,8R,7′S,8′S)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β-D-glucopyranoside (9). White amorphous powder; soluble in water or methanol; [α]20D +29.9° (c 0.07, MeOH); CD (MeOH) λmax (Δε) 281 (+0.88), 234 (+1.19) nm; IR νmax 3374, 2937, 1601, 1514, 1463, 1424, 1268, 1157, 1076, 859, 821 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1946 (calcd for C26H34O12Na, 561.1942). (7R,8S,7′S,8′R)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-D-glucopyranoside (10). White amorphous powder; soluble in water or methanol; [α]20D −84.2° (c 0.08, MeOH); CD (MeOH) λmax (Δε) 278 (−1.17), 229 (−5.75) nm; IR νmax 3372, 2888, 1601, 1514, 1463, 1427, 1268, 1159, 1076, 860, 818 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1948 (calcd for C26H34O12Na, 561.1942). (7S,8R,7′R,8′S)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-D-glucopyranoside (11). White amorphous powder; soluble in water or methanol; [α]20D +74.5° (c 0.06, MeOH); CD (MeOH) λmax (Δε) 291 (+1.14), 237 (+3.73) nm; IR νmax 3370, 2925, 1601, 1514, 1462, 1427, 1269, 1159, 1074, 860, 817 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125

11

6.92 7.03 6.83 4.51 2.14 3.47

d (2.0) d (8.5) dd (8.5, 2.0) d (8.5) m m

6.92 7.02 6.83 4.51 2.14 3.47

d (2.0) d (8.5) dd (8.5, 2.0) d (8.5) m m

6.90 6.72 6.72 4.33 2.42 3.60 3.50 4.88 3.25 3.29 3.16 3.28 3.68 3.45 3.76 3.75

brs overlap overlap dd (9.0, 2.5) m m m d (7.5) m m m m m m s s

6.90 6.72 6.72 4.34 2.42 3.60 3.50 4.88 3.24 3.29 3.15 3.26 3.68 3.44 3.76 3.75

brs overlap overlap d (8.5) m m m d (7.5) m m m m m m s s

12 6.91 7.03 6.83 4.55 1.73 3.14 3.07 6.80 6.67 6.61 4.30 2.36 4.07 3.74 4.87 3.25 3.29 3.15 3.27 3.67 3.44 3.72 3.75

d (2.0) d (8.0) dd (8.0, 2.0) d (8.5) m m m d (2.0) d (8.0) dd (8.0, 2.0) dd (8.5, 4.5) m m m d (7.5) m m m m m m s s

MHz) data, see Table 2; (+)-HRESIMS m/z 561.1954 (calcd for C26H34O12Na, 561.1942). (7S,8R,7′S,8′S)-4,9,4′,7′-Tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-D-glucopyranoside (12). White amorphous powder; soluble in water or methanol; [α]20D +55.2° (c 0.06, MeOH); CD (MeOH) λmax (Δε) 281 (+0.61), 231 (+1.92) nm; IR νmax 3372, 2933, 1601, 1514, 1463, 1426, 1269, 1157, 1076, 859, 820 cm−1; 1H NMR (DMSO-d6, 500 MHz) data, see Table 3; 13C NMR (DMSO-d6, 125 MHz) data, see Table 2; (+)-HRESIMS m/z 561.1952 (calcd for C26H34O12Na, 561.1942). Enzymatic Hydrolysis of 1−12. Compound 1 (6.0 mg) was dissolved in 0.1 M acetate buffer (3.0 mL). The reaction mixture was added to cellulase (9.0 mg, Sigma-Aldrich, St. Louis, MO) and left at 38 °C for 2 h. The solution was then transferred to a liquid−liquid extractor and extracted with CHCl3. The aqueous fraction was subjected to silica gel thin layer chromatography (TLC) [CHCl3− MeOH−H2O (7:3:1)] to show the presence of D-glucose (Rf = 0.16). The organic layer was evaporated in vacuo and purified using reversedphase preparative HPLC with MeOH−H2O (45:55) as the mobile phase to give 1a (3 mg), which was identified using NMR. Hydrolysis of 2−12 and sugar identification were performed according to the procedure described for 1. Hepatoprotective Effects on Cytotoxicity Induced by D-Galactosamine in HL-7702 Cells. Compounds 1−18 were tested for hepatoprotective effects using an MTT assay in HL-7702 cells. Each cell suspension containing 1 × 10−5 cells in 1 mL of Dulbecco’s modified Eagle’s medium containing fetal calf serum (10%), penicillin (100 units/mL), and streptomycin (100 μg/mL) was placed in a 96well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium containing bicyclol and the test samples was added, and the cells were cultured for 1 h. The cultured cells were exposed to 25 mM D-galactosamine for 24 h. The medium was then replaced with fresh medium containing 0.5 mg/mL MTT. After incubation of 4 h, the medium was removed, and DMSO was added to dissolve formazan crystals. The optical density (OD) of the formazan solution was measured using a microplate reader at 492 nm. Inhibition (%) was calculated using the following formula: inhibition (%) = [(ODsample − ODcontrol )/(ODnormal − ODcontrol )] × 100 D

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Figure 2. Key HMBC correlations of compounds 1, 3, 5, and 7.

Figure 3. Key ROESY correlations of compounds 1, 5, and 7.

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assigned to a lignan skeleton.11 In the heteronuclear multiplebond correlation (HMBC) spectroscopy spectrum (see Figure 2), the correlation peaks of H-7 resonated at δ 5.49 with C-1, C-2, C-6, C-8, C-9, C-4′, and C-5′, and H-7′ resonated at δ 4.57 with C-1′, C-2′, C-6′, C-8′, and C-9′, which confirmed the benzofuran-type lignan of 1. A detailed HMBC spectrum analysis revealed the positions of two methoxy groups located at C-3 and C-3′, respectively. Furthermore, the glucose unit was connected at C-4, according to the correlation from anomeric proton H-1″ resonated at δ 4.88 to C-4; 1 was hydrolyzed with cellulase to yield 1a and D-glucose, which was identified by TLC analysis. The β form of glucose was determined by the presence of an anomeric proton at δ 4.88 (1H, d, J = 7.5 Hz, H1″). On the basis of the above analysis, the planar structure of 1 was defined as dihydrodehydrodiconiferyl alcohol-7′-hydroxy-4O-β-D-glucopyranoside. The relative configuration of H-7 and H-8 in 1 was defined as trans from the characteristic correlation peak of H-7 with H-9 in the ROESY spectrum (see Figure 3). The positive Cotton effects at 245 and 292 nm in the CD spectrum revealed the 7S,8R configuration for 1.11 A positive Cotton effect at 346 nm in the Rh2(OCOCF3)4-induced CD spectrum indicated the 7′S configuration for 1a.12 Thus, compound 1 was defined as (7S,8R,7′S)-dihydrodehydrodiconiferyl alcohol-7′-hydroxy-4O-β-D-glucopyranoside. Compound 2 had the same molecular formula as 1, which was determined from HRESIMS at m/z 561.1951 [M + Na]+. Its NMR data (Tables 1 and 3) were almost identical to those of compound 1, which indicated that compound 2 had the same planar structure as 1. The absolute configurations for C-7, C-8, and C-7′ of 2 were identified by the same method as that used for 1. In the ROESY experiment, the correlation peak of H-7 with H-9 determined the trans configuration of H-7 and H8 in 2. The 7R,8S configuration for 2 was confirmed by the negative Cotton effects at 243 and 282 nm in the CD spectrum.

RESULTS AND DISCUSSION A previous study has shown strong inhibitory effects of two tetrahydrofuranoid lignans, taxiresinol and (7′R)-7′-hydroxytaxiresinol, on the elevation of serum levels of tumor necrosis factor-alpha (TNF-α) by D-galactosamine/LPS toxication.10 Furthermore, these two lignans protected hepatocytes from cytotoxicity induced by D-galactosamine/TNF-α in primary cultured mouse hepatocytes. Therefore, in the present study, all isolated compounds were tested for hepatoprotective effects on D-galactosamine-induced cytotoxicity in HL-7702 hepatic cells. Compounds 1, 2, 7−12, and 17 showed significantly stronger hepatoprotective activity than the positive control bicyclol at a concentration of 1 × 10−5 M. Analysis of structure−activity relationships revealed that the tetrahydrofuran ring in lignan glucosides might contribute to the hepatoprotective activity. Structure Elucidation. Compound 1 was obtained as a white powder, and its molecular formula was determined to be C26H34O12 on the basis of HRESIMS findings at m/z 561.1954 [M + Na]+. The 1H NMR spectrum of 1 (see Table 1) revealed an ABX system aromatic ring [δ 7.06 (1H, d, J = 8.5 Hz), 6.96 (1H, d, J = 2.0 Hz), 6.84 (1H, dd, J = 8.5, 2.0 Hz)] and a 1,3,4,5-tetra-substituted aromatic ring [δ 6.83 (1H, d, J = 2.0 Hz), 6.81 (1H, d, J = 2.0 Hz)] in the downfield region. 1H NMR spectrum analysis revealed two methoxy groups at δ 3.78 (3H, s) and 3.75 (3H, s) and a glucopyranosyl anomeric proton at δ 4.88 (1H, d, J = 7.5 Hz). Additionally, the 1H NMR spectrum revealed a methylene group at δ 1.75 (1H, m) and 1.68 (1H, m); two oxymethylene protons at δ 3.73 (1H, m), 3.63 (1H, m), 3.48 (1H, m), and 3.44 (1H, m); two oxymethine protons at δ 5.49 (1H, d, J = 6.5 Hz) and 4.57 (1H, m); and a methine proton at δ 3.42 (1H, m). The 13C NMR spectrum of 1 (see Table 2) showed 26 carbon signals, of which eight could be assigned to a glucose unit (δ 100.1, 77.0, 76.9, 73.2, 69.8, 60.7) and two methoxy groups (δ 55.8, 55.7), and the remaining 18 carbons could be E

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aromatic rings [δ 6.96 (1H, d, J = 8.5 Hz), 6.78 (1H, d, J = 2.0 Hz), 6.72 (1H, d, J = 8.5 Hz), 6.66 (1H, dd, J = 8.5, 2.0 Hz), 6.65 (1H, d, J = 2.0 Hz), and 6.57 (1H, dd, J = 8.5, 2.0 Hz)]. In addition, two methylene protons [δ 2.70 (1H, dd, J = 13.5, 6.5), 2.49 (1H, overlap), 4.13 (1H, t, J = 8.5), 4.01 (1H, t, J = 8.5)], three methane protons [δ 4.34 (1H, t, J = 5.0), 2.84 (1H, dt, J = 8.5, 6.0), 2.46 (1H, m)], two methoxy groups [δ 3.73(3H, s), 3.71 (3H, s)], and a glucopyranosyl anomeric proton [δ 4.83 (1H, d, J = 7.5 Hz)] were observed in the upfield region. The 13 C NMR spectrum of 5 revealed 26 carbon resonances (Table 2) corresponding to the glucose unit, two methoxy groups, and two C6−C3 units. These data indicated that 5 was a dibenzylbutyrolactone lignan with substitution of two methoxy groups and a glucose unit. In the HMBC spectrum (see Figure 2), correlation peaks from H-8 to C-1, C-7, C-9, C-7′, C-8′, and C-9′, from H-8′ to C-1′, C-7′, C-9′, C-7, C-8, and C-9, from OCH3-3/3′ to C-3/ 3′, and from H-1″ to C-4 confirmed the dibenzylbutyrolactone skeleton of 5, as well as the location of two methoxy groups and a glucose unit. The ROESY correlation (see Figure 3) from H-8 to H-7′ determined the trans configuration of H-8/8′. The 8R,8′R configuration was verified by the negative Cotton effect at 229 nm in the CD spectrum.13 5 was hydrolyzed with cellulase to yield 5a, which was treated with Rh2(OCOCF3)4 at room temperature. A negative Cotton effect at 340 nm in the Rh2(OCOCF3)4-induced CD spectrum indicated the 7′R configuration for 5a. Thus, compound 5 was defined as (7′R)-hydroxy matairesinol-4-O-β-D-glucopyranoside. Compound 6, an optical isomer of 5, displayed spectroscopic data features that were almost identical to those of 5. In the ROESY spectrum, the correlation peak of H-8/H-7′ was uncertain because of the overlap between H-8 and H-7a; therefore, the DMSO-d6 was replaced with CD3OD. 1D NMR data assignments were completed by comprehensive analysis of the 2D NMR data in CD3OD. The chemical shift of H-8 was separated with H-7a in the 1H NMR spectrum. The relative configuration of H-8/8′ was determined to be trans by the unambiguous correlation peak of H-8 with H-7′ in the ROESY spectrum. In combination with the negative Cotton effect at 225 nm in the CD spectrum, the absolute configuration of C-8 and C-8′ was established as 8R,8′R. Using the same method as that used for 5, the 7′S configuration was determined. Thus, 6 was defined as (7′S)-hydroxy matairesinol-4-O-β-D-glucopyranoside. Compound 7 was obtained as a white powder with a molecular formula of C26H34O12, on the basis of HRESIMS at m/z 561.1945 [M + Na]+ (calculated for C26H34O12Na, m/z 561.1942). In the 1H NMR spectrum (see Table 3), two sets of ABX system aromatic rings [δ 7.03 (1H, d, J = 8.0 Hz), 6.96 (1H, d, J = 2.0 Hz), 6.87 (1H, brs), 6.84 (1H, dd, J = 8.0, 2.0 Hz), 6.72 (1H, overlap), and 6.72 (1H, overlap)], together with two methine protons at δ 2.45 (1H, m), 2.14 (1H, m), two oxymethine protons at δ 4.46 (1H, d, J = 8.0 Hz), 4.41 (1H, dd, J = 8.5, 2.5 Hz), two oxymethylene protons at δ 3.60 (1H, m), 3.49 (1H, m), 3.48 (2H, m), two methoxy groups at δ 3.77 (3H, s), 3.76 (3H, s), and a glucopyranosyl anomeric proton at δ 4.87 (1H, d, J = 7.5 Hz), were observed. The 13C NMR spectrum (see Table 2) showed 26 carbon signals, consisting of two methoxy carbon signals, six carbon signals of a glucose unit, and 18 skeleton carbon signals of two C6−C3 units. These spectroscopic data were very similar to those of lanicepsides A, which was isolated from Saussurea laniceps.14 In the HMBC spectrum (see Figure 2), the correlation peaks of H-7 at δ 4.46

Hydrolysis of 2 with cellulase yielded 2a and D-glucose, which was identified by comparison with the D-glucose standard in TLC analysis. The absolute configuration for C-7′ in 2a was defined as S based on a positive Cotton effect at 350 nm in the Rh2(OCOCF3)4-induced CD spectrum. Therefore, compound 2 was defined as (7R,8S,7′S)-dihydrodehydrodiconiferyl alcohol-7′-hydroxy-4-O-β-D-glucopyranoside. Compound 3 exhibited the molecular formula C26H34O13, as determined using HRESIMS at m/z 577.1900 [M + Na]+. The 1 H NMR spectrum (see Table 1) of 3 showed six aromatic proton signals at δ 7.75 (1H, dd, J = 8.5, 2.0 Hz), 7.57 (1H, d, J = 2.0 Hz), 7.18 (1H, d, J = 8.5 Hz), 6.92 (1H, brs), 6.68 (1H, overlap), and 6.68 (1H, overlap), revealing the presence of two ABX system aromatic rings. In addition, the 1H NMR spectrum revealed two methoxy groups at δ 3.81 (3H, s) and 3.74 (3H, s) and a glucopyranosyl anomeric proton at δ 5.08 (1H, d, J = 7.5 Hz). In the 13C NMR spectrum (see Table 2), apart from two methoxy carbons and a glucose unit, the remaining 18 skeleton carbon signals suggested that the aglycone of 3 was also a lignan. In the HMBC spectrum (see Figure 2), the characteristic correlation of H-8 at δ 5.58 with C-4′ at δ 145.6 indicated that 3 was an 8-O-4′ neolignan. The correlation peaks of two methoxy groups at δ 3.81 and 3.74 with C-3 and C-3′ confirmed that the linkage position of these methoxy groups was at C-3 and C-3′, respectively. The correlation of H-1″ (δ 5.08) with C-4 (δ 151.1) indicated that the linkage point of the glucose unit was at C-4. On the basis of the above data, 3 was defined as 4,9,7′,9′-tetrahydroxy-3,3′-dimethoxyl-7-oxo-8−4′oxyneolignan-4-O-β-D-glucopyranoside. To designate the absolute configuration of C-8 and C-7′ in compound 3, CD exciton chirality and the Rh2(OCOCF3)4induced CD spectrum were assayed. Cellulase hydrolysis of 3 resulted in 3a and D-glucose. A negative exciton-split Cotton effect at 332 nm (Δε −0.05) and 261 nm (Δε +0.07) in 3a exhibited in the CD spectrum indicated that the two transition dipole moments of the chromophores (benzoyl moiety and benzene moiety) were oriented in an anticlockwise manner (see Supporting Information Figure S26). Thus, the absolute configuration of C-8 was assigned as 8R. Moreover, the positive Cotton effects at 328 and 374 nm in the Rh2(OCOCF3)4induced CD spectrum indicated the 7′S configuration for 3a. From the above analysis, 3 was defined as (8R,7′S)-4,9,7′,9′tetrahydroxy-3,3′-dimethoxyl-7-oxo-8−4′-oxyneolignan-4-O-βD-glucopyranoside (3). Compound 4, a white powder, had the same molecular formula (C26H34O13Na, m/z 577.1905) as 3. The UV, IR, and NMR spectroscopy data of 4 indicated that it was an 8-O-4′ system neolignan and an optical isomer of 3. Cellulase hydrolysis of 4 resulted in 4a and D-glucose. In the CD spectrum, a positive exciton-split Cotton effect at 332 nm (Δε +0.04) and 265 nm (Δε −0.06) caused by the exciton coupling of the benzoyl and benzene chromophores indicated the 8S configuration of 4a (see Supporting Information Figure S35). In the Rh2(OCOCF3)4-induced CD experiment, a positive Cotton effect at 367 nm indicated the 7′S configuration for 4a. Thus, compound 4 was defined as (8S,7′S)-4,9,7′,9′-tetrahydroxy-3,3′-dimethoxyl-7-oxo-8−4′-oxyneolignan-4-O-β-D-glucopyranoside (4). Compound 5 had the molecular formula C26H32O12 (m/z 559.1787 for C26H32O12Na in HRESIMS), and exhibited hydroxyl (3386 cm−1), carbonyl (1757 cm−1), and phenyl (1601 and 1514 cm−1) groups in the IR spectrum. The 1H NMR spectrum (see Table 1) showed two sets of ABX system F

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with C-1, C-2, C-6, C-8, C-9, C-8′, and C-9′, and of H-7′ at δ 4.41 with C-1′, C-2′, C-6′, C-8′, C-9′, and C-8 helped verify that 7 was a tetrahydrofuranoid lignan. The location of the glucose unit was determined to be at C-4′ from the correlation of H-1″ with C-4′ in the HMBC spectrum. All of the above data indicated the same planar structure for 7 as for lanicepsides A. The spectroscopic data of compounds 8 and 9 were in agreement with those of 7, which indicated that these two compounds were optical isomers of 7. The absolute configurations of these three compounds were determined by the ROESY spectrum, CD spectrum, and Rh2(OCOCF3)4induced CD assay. In the ROESY spectrum (see Figure 3), the correlation peaks of H-7/H-9, H-8/H-7′, and H-8′/H-9 helped confirm the trans configurations of H-7/H-8 and H-8/H-8′ in 7, 8, and 9. In the CD spectrum, a negative Cotton effect at 230 nm indicated the 8S configuration for 7.15 Moreover, positive Cotton effects at 235 and 234 nm indicated the 8R configuration for 8 and 9.16 From the above analysis, the absolute configurations of C-7, C-8, and C-8′ for these compounds were determined as 7R,8S,8′R (7), 7S,8R,8′S (8), and 7S,8R,8′S (9). To determine the absolute configuration at C-7′, the Rh2(OCOCF3)4-induced CD assay was performed; 7 was hydrolyzed with cellulase to yield 7a, which was treated with Rh2(OCOCF3)4 at room temperature. A positive Cotton effect at 371 nm in the Rh2(OCOCF3)4-induced CD spectrum indicated the 7′S configuration of 7a. The absolute configurations of C-7′ in 8 and 9 were confirmed using the same method as that used for 7. A negative Cotton effect at 350 nm for 8a and a positive Cotton effect at 350 nm for 9a indicated the 7′R and 7′S configurations, respectively. From the above analysis, compounds 7−9 were defined as (7R,8S,7′S,8′R)-4,9,4′,7′-tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β- D -glucopyranoside (7), (7S,8R,7′R,8′S)4,9,4′,7′-tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β-Dglucopyranoside (8), and (7S,8R,7′S,8′S)-4,9,4′,7′-tetrahydroy3,3′-dimethoxy-7,9′-epoxylignan-4′-O-β-D-glucopyranoside (9). The molecular formulas of compounds 10, 11, and 12 were determined to be C26H34O12 from HRESIMS ion m/z of 561.1948 [M + Na]+, 561.1954 [M + Na]+, and 561.1952 [M + Na]+, respectively. The UV, IR, and NMR spectroscopic data of 10−12 were almost identical to those of compounds 7−9. In comprehensive analysis of the ROESY and HMBC spectra of 10−12, the correlation peak of H-1″ with H-5 in the ROESY spectrum, and with C-4 in the HMBC spectrum, indicated that the glucose unit was located at C-4. The absolute configurations of these three compounds were established using the same methods as those used for 7−9. Compounds 10−12 were identified as (7R,8S,7′S,8′R)-4,9,4′,7′-tetrahydroy-3,3′-diethoxy-7,9′-epoxylignan-4-O-β- D -glucopyranoside (10), (7S,8R,7′R,8′S)-4,9,4′,7′-tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-D-glucopyranoside (11), and (7S,8R,7′S,8′S)4,9,4′,7′-tetrahydroy-3,3′-dimethoxy-7,9′-epoxylignan-4-O-β-Dglucopyranoside (12). On the basis of MS data, NMR data, and comparison with literature, known compounds (see Figure 1) were identified as (7R,8S)-7,9,9′-trihydroxy-3,3′-dimethoxy-8-O-4′-neolignan-4O-β-D-glucopyranoside (13),17 (7S,8R)-7,9,9′-trihydroxy-3,3′dimethoxy-8-O-4′-neolignan-4-O-β-D-glucopyranoside (14),17 (7R,8R)-7,9,9′-trihydroxy-3,3′-dimethoxy-8-O-4′-neolignan-4O-β-D-glucopyranoside (15),17 (7S,8S)-7,9,9′-trihydroxy-3,3′dimethoxy-8-O-4′-neolignan-4-O-β-D-glucopyranoside (16),17

(7R,7′R,8S,8′S)-(+)-neo-olivil-4-O-β-D-glucopyranoside (17),18 and arctiin (18).19 Cytotoxic Activity. In previous studies, arctigenin, the main component of Arctii Fructus, inhibited proliferation of and promoted apoptosis in SMMC-7721 human hepatocellular carcinoma cells.9 To clarify whether lignan glucosides in Arctii Fructus have cytotoxic activity, all of the isolated compounds were tested for cytotoxic activity using an MTT assay in HCT8, Bel-7402, BGC-823, A549, and A2780 cells. All of the tested compounds exhibited no significant cytotoxicity in cell model systems. Analysis of structures indicated that the glucose moiety in lignans may have reduced cytotoxic activity. Hepatoprotective Activity. Compounds 1−18 were tested for hepatoprotective activities against D-galactosamineinduced toxicity in HL-7702 cells. Compounds 1, 2, 7−12, and 17 showed stronger hepatoprotective activities than that shown by the positive control bicyclol at a concentration of 1 × 10−5 M. Among them, compounds 1 and 2 reduced D-galactosamineinduced damage in HL-7702 cells (cell survival rate of 51%) with 79% and 71% inhibition, respectively, while the positive control bicyclol gave a 61.0% inhibition. Meanwhile, compounds 7−12 and 17 had 61−70% inhibition. From the above experimental data, only benzofuran-type neolignans and tetrahydrofuranoid lignans showed hepatoprotective activity. Tetrahydrofuran rings in lignans might contribute to the hepatoprotective activity observed. Furthermore, compounds 1 and 2, which owned benzofuran rings, revealed stronger hepatoprotective activities than those of compounds 7−12 and 17, which owned tetrahydrofuran rings. In conclusion, we conducted a phytochemical investigation of Arctii Fructus, from which a series of 12 novel 7′-hydroxy lignan glucosides were isolated, along with six known lignan glucosides. To determine the absolute configurations for chiral carbons, the ROESY assay was combined with the CD exciton chirality method and Rh2(OCOCF3)4-induced CD spectrum analysis. Further investigation of the in vitro bioactivities of 1− 18 revealed that some compounds showed significantly stronger hepatoprotective activities than that shown by the positive control bicyclol. The structure−activity relationships revealed that the tetrahydrofuran ring in lignan glucosides might contribute to the hepatoprotective activity observed. These results provide additional phytochemical and bioactive information on Arctii Fructus, which is widely used in traditional Chinese medicine.

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ASSOCIATED CONTENT

S Supporting Information *

NMR, IR, MS, and CD spectra of compounds 1−12. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 10 63165231. Fax: 86-10-63017757. E-mail: [email protected]. Funding

This research was supported by the Nation Science and Technology Project of China (no. 2012ZX09301002-002). Notes

The authors declare no competing financial interest. G

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(18) Kikuchi, M.; Kikuchi, M. Studies on the constituents of Swertia japonica MAKINO II. On the structures of new glycosides. Chem. Pharm. Bull. 2005, 53, 48−51. (19) Rahman, M. M. A.; Dewick, P. M.; Jackson, D. E.; Lucas, J. A. Lignans of Forsythia Intermedia. Phytochemistry 1990, 29, 1971−1980.

ACKNOWLEDGMENTS We thank Ying-hong Wang for NMR measurements. REFERENCES

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