New Butyrolactone Type Lignans from Arctii ... - ACS Publications

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New Butyrolactone Type Lignans from Arctii Fructus and Their Antiinflammatory Activities 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

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.jafc.5b02838

S Supporting Information *

ABSTRACT: Arctiidilactone (1), a novel rare butyrolactone lignan with a 6-carboxyl-2-pyrone moiety, and 11 new butyrolactone lignans (2−12) were isolated from the fruits of Arctium lappa L., together with 5 known compounds (13−17). Their structures were elucidated by interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ORD, and HRESIMS) and comparison to literature data. The absolute configurations of compounds 1−12 were determined by a combination of rotating-frame nuclear Overhauser effect spectroscopy (ROESY), circular dichroism (CD) spectroscopy, and Rh2(OCOCF3)4induced CD spectroscopy. All of the compounds were tested for their anti-inflammatory properties in terms of suppressing the production of NO in lipopolysaccharide-induced BV2 cells. Compounds 1, 6, 8, and 10 exhibited stronger anti-inflammatory effects than the positive control curcumin, particularly 1, which exhibited 75.51, 70.72, and 61.17% inhibition at 10, 1, and 0.1 μM, respectively. KEYWORDS: Arctium lappa L., Arctii Fructus, lignan, butyrolactone lignan, anti-inflammatory activity

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INTRODUCTION

Arctii Fructus is the dried mature fruits of Arctium lappa L., which is a vegetable for health care in China and Japan. It has traditionally been used as a health tea to treat tonsillitis, pharyngolaryngitis, and constipation in China. Modern pharmacological studies on Arctii Fructus have revealed a broad range of biological activities, such as anti-influenza,1 cytotoxic,2,3 antidiabetic,4 anti-inflammatory,5 and gastroprotective activities.6 Phytochemical studies on Arctii Fructus have already yielded many types of chemicals, including lignans,7 phenols,8 and fatty acids.9 In our previous study on the H2Osoluble fraction of an EtOH extract of Arctii Fructus, we reported 12 novel 7′-hydroxy lignan glucosides and their hepatoprotective activity.10 In our further investigation of the bioactive compounds from Arctii Fructus, we isolated a novel rare butyrolactone lignan with a 6-carboxyl-2-pyrone moiety, named arctiidilactone (1); a new apolignan glucoside without an aromatic ring, named arctiiapolignan A (2); a new sesquineolignan with a dihydrobenzofuran unit and a butyrolactone unit, named arctiisesquineolignan A (3); three new dibenzylbutyrolactone lignans (4−6); and six new dibenzylbutyrolactone lignans with an additional hydroxyl at C-7/7′ (7−12), together with five known compounds (13−17). Their structures and absolute configurations were characterized on the basis of spectroscopic data, including CD and Rh2(OCOCF3)4-induced CD spectra. In consideration of our previous report for the bioactivity of lignan glucosides,10 the hepatoprotective activities of 1−17 were tested through in vitro assays. Furthermore, tea made from Arctii Fructus is traditionally used to treat tonsillitis, so the anti-inflammatory and cytotoxic activities of compounds 1− 17 are described in this paper. © XXXX American Chemical Society

MATERIALS AND METHODS

General Apparatus and Chemicals. The optical rotations were measured on a Jasco P-2000 polarimeter (Jasco Corp., Tokyo, Japan). The UV spectra were recorded on a Jasco V650 spectrophotometer (Jasco Corp.). The IR spectra were measured on a Nicolet 5700 spectrometer (Thermo Scientific, Waltham, MA, USA). The CD spectra were recorded on a Jasco J-815 CD spectrometer (Jasco Corp.). HRESIMS was recorded on an Agilent 1100 series LC-MSD ion trap mass spectrometer (Agilent Technologies, Waldbronn, Germany). 1D NMR and 2D NMR spectra were performed on a Bruker Avance 500 MHz NMR spectrometer (Bruker-Biospin, Billerica, MA, USA). Column chromatography was carried out with Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) and macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo, Japan). Flash chromatography was set up on Combiflash RF200 (Teledyne Isco Corp., Lincoln, NE, USA). Preparative high-performance liquid chromatography (P-HPLC) was performed on a Shimadzu LC-6AD instrument with an SPD-20A detector (Shimadzu Corp., Tokyo, Japan), using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm; YMC Corp., Kyoto, Japan). High-performance liquid chromatography diode array detection (HPLC-DAD) analysis was set up on an Agilent 1260 series system (Agilent Technologies) with an Apollo C18 column (250 × 4.6 mm, 5 μm; Alltech Corp., Lexington, KY, USA). Plant Material. Arctii Fructus was collected in October 2011 in Wuchang town, Heilongjiang province, China. The plant material was identified as the fruits of A. lappa L. 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) has been put in the Herbarium of the Department of Chemistry of Natural Products, Institute of Materia Media, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Received: June 8, 2015 Revised: August 4, 2015 Accepted: August 27, 2015

A

DOI: 10.1021/acs.jafc.5b02838 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry


Figure 1. Structures of compounds 1−17. Extraction and Isolation. Air-dried mature fruits (100 kg) of A. lappa L. were extracted with 80% EtOH (3 × 600 L) under reflux three times. The solvent was evaporated under reduced pressure. Then, the residue (4.2 kg) was suspended in H2O (10 L) and extracted with EtOAc (3 × 10 L). The aqueous layer (400 g) was subjected to an HP-20 macroporous adsorbent resin (4000 g) column and eluted with H2O (20 L), 15% EtOH (20 L), 30% EtOH (20 L), 50% EtOH (20 L), and 95% EtOH (10 L), which yielded five corresponding fractions (fractions A−E). Fraction C (86.4 g) was submitted to flash chromatography with a C18 column (55 × 8 cm, 20 μm), eluting with a 10-step gradient of MeOH/H2O (2 L for one step, from 0:100 to 100:0) to provide 13 fractions (fractions C1−C13). Fraction C11 (301 mg) was separated on a Sephadex LH-20 (100 g) column, eluting with H2O (1.2 L) to yield four fractions (fractions C11-1−C11-4). Then fraction C11-1 (25 mg) was further purified by reverse-phase preparative HPLC, eluting with MeOH/H2O (25:75) at a flow rate of 5 mL/min to afford 1 (5 mg, 48 min). Fraction C12 (812 mg) was separated on a Sephadex LH-20 column (H2O, 1.5 L) and then further purified by reversephase preparative HPLC, eluting with MeOH/H2O (40:60) at a flow rate of 5 mL/min to afford 2 (27 mg, 45 min), 12 (5 mg, 51 min), and 13 (33 mg, 32 min). Fraction C13 (1.695 g) was subjected to a Sephadex LH-20 column and eluted with H2O (2 L) to give eight fractions (fractions C13-1−C13-8). Purification of fraction C13-3 (97 mg) with MeOH/H2O (30:70) on reverse-phase preparative HPLC at a flow rate of 5 mL/min afforded 6 (10 mg, 37 min) and 7 (13 mg, 49 min). Fraction C13-5 (255 mg) was applied successively to chromatography on Sephadex LH-20 (H2O, 1.2 L) to yield six fractions (fractions C13-5-1−C13-5-6). Compound 14 (100 mg, 57 min) was obtained from fraction C13-5-3 (188 mg) on reverse-phase preparative HPLC using MeOH/H2O (27:78) at a flow rate of 5 mL/ min. Fraction C13-8 (127 mg) was applied successively to chromatography on Sephadex LH-20 (H2O, 1 L) to yield five fractions (fractions C13-8-1−C13-8-5). Fraction C13-8-3 (157 mg) was performed on reverse-phase preparative HPLC with MeOH/H2O (30:70) at a flow rate of 5 mL/min to afford 9 (80 mg, 47 min) and 11 (10 mg, 57 min). Fraction D (210 g) was submitted to flash chromatography with a C18 column (55 × 8 cm, 20 μm), eluting with a 10-step gradient of MeOH/H2O (4 L for one-step, from 0:100 to 100:0) to yield 40 fractions (fractions D1−D40). Fraction D27 (1.4 g) was separated on a Sephadex LH-20 column and eluted with H2O (1.5 L) to give six fractions (fractions D27-1−D27-6). Fraction D27-3 (316 mg) was subjected to reverse-phase preparative HPLC, eluting with MeOH/ H2O (35:65) as the mobile phase at a flow rate of 5 mL/min, to afford 4 (7 mg, 23 min), 5 (6 mg, 31 min), 10 (5 mg, 44 min), and 17 (24 mg, 59 min). Fraction D13 (2.3 g) was separated into four fractions (fractions D13-1−D13-4) on Sephadex LH-20 eluted with H2O (2 L). Fraction D13-1 (42 mg) gave 3 (10 mg, 35 min) after purification over a reverse-phase preparative HPLC column with MeOH/H2O (40:60) at a flow rate of 5 mL/min. Fraction D13-2 (116 mg), with Sephadex LH-20 (H2O, 1 L) and reverse-phase preparative HPLC with MeOH/ H2O (45:55) at a flow rate of 5 mL/min, yielded 15 (5 mg, 25 min) and 16 (82 mg, 42 min). Fraction D13-4 (29 mg) was purified via

reverse-phase preparative HPLC with MeOH/H2O (35:65) as the mobile phase at a flow rate of 5 mL/min to afford 8 (4 mg, 27 min). The structures of compounds 1−17 are shown in Figure 1. Spectral Data. Arctiidilactone (1): white amorphous powder; [α]D25 −28.9 (c 0.26, MeOH); CD (MeOH) λmax (Δε) 236 (−1.21), 279 (−0.34) nm; UV (MeOH) λmax (log ε) 230 (3.51), 285 (3.29), 305 (3.22) nm; IR (KBr) νmax 3364, 2922, 2851, 1765, 1712, 1640, 1515, 1465, 1391, 1263, 1238, 1157, 1077, 1024 cm−1; (+)-HRESIMS m/z 389.1235 [M + H]+ (calcd for C20H21O8, 389.1236). For 1H and 13C NMR spectroscopic data, see Tables 1 and 2. Arctiiapolignan A (2): white amorphous powder; [α]D25 +6.97 (c 0.18, MeOH); CD (MeOH) λmax (Δε) 227 (−4.39) nm; UV (MeOH) λmax (log ε) 230 (3.10), 280 (2.70) nm; IR (KBr) νmax 3403, 2912, 1764, 1591, 1515, 1464, 1420, 1263, 1238, 1159, 1078, 1029 cm −1 ; (+)-HRESIMS m/z 451.1584 [M + Na]+ (calcd for C20H28O10Na, 451.1580). For 1H and 13C NMR spectroscopic data, see Tables 1 and 2. D Arctiisesquineolignan A (3): white amorphous powder; [α]25 +18.54 (c 0.15, MeOH); CD (MeOH) λmax (Δε) 230 (−3.55), 248 (+1.2), 275 (−0.58), 295 (+0.39) nm; UV (MeOH) λmax (log ε) 228 (3.87), 279 (3.42) nm; IR (KBr) νmax 3413, 2921, 1760, 1598, 1514, 1454, 1421, 1266, 1226, 1141, 1075, 1030 cm−1; (+)-HRESIMS m/z 883.2981 [M + Na]+ (calcd for C42H52O19Na, 883.3000). For 1H and 13 C NMR spectroscopic data, see Tables 1 and 2. Arctigenin-4-O-α-D-galactopyranosyl-(1→6)-O-β-D-glucopyranoside (4): white amorphous powder; [α]D25 +6.3 (c 0.07, MeOH); CD (MeOH) λmax (Δε) 230 (−3.02), 272 (−0.34) nm; UV (MeOH) λmax (log ε) 230 (3.12), 278 (2.69) nm; IR (KBr) νmax 3382, 2926, 1761, 1593, 1515, 1454, 1421, 1265, 1235, 1155, 1081, 1029 cm−1; (+)-HRESIMS m/z 719.2529 [M + Na]+ (calcd for C33H44O16Na, 719.2527). For 1H and 13C NMR spectroscopic data, see Tables 1 and 2. Arctigenin-4-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (5): white amorphous powder; [α]D25 −52.6 (c 0.12, MeOH); CD (MeOH) λmax (Δε) 231 (−6.82), 274 (−0.78) nm; UV (MeOH) λmax (log ε) 229 (3.58), 278 (3.19) nm; IR (KBr) νmax 3418, 2929, 1760, 1592, 1515, 1454, 1421, 1265, 1235, 1158, 1068, 1025 cm−1; (+)-HRESIMS m/z 689.2430 [M + Na]+ (calcd for C32H42O15Na, 689.2421). For 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 5′-Propanediolmatairesinoside (6): white amorphous powder; [α]D25 −53.4 (c 0.11, MeOH); CD (MeOH) λmax (Δε) 229 (−6.49), 278 (−1.19) nm; UV (MeOH) λmax (log ε) 230 (3.48), 280 (3.10) nm; IR (KBr) νmax 3426, 2928, 1758, 1597, 1513, 1461, 1422, 1267, 1228, 1157, 1072, 1030 cm−1; (+)-HRESIMS m/z 617.2207 [M + Na]+ (calcd for C29H38O13Na, 617.2210). For 1H and 13C NMR spectroscopic data, see Tables 1 and 2. (7′R,8R,8′R)-Rafanotrachelogenin-4-O-β-D-glucopyranoside (7): D −13.6 (c 0.12, MeOH); CD white amorphous powder; [α]25 (MeOH) λmax (Δε) 230 (−2.00), 267 (−0.22) nm; UV (MeOH) λmax (log ε) 230 (3.41), 277 (3.08) nm; IR (KBr) νmax 3423, 2931, 1759, 1594, 1514, 1463, 1421, 1266, 1232, 1156, 1075, 1027 cm−1; (+)-HRESIMS m/z 573.1954 [M + Na]+ (calcd for C27H34O12Na, 573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. B


Article

Journal of Agricultural and Food Chemistry Table 1. 1H NMR Data for 1 in Acetic Acid-d4 and for 2−6 in DMSO-d6 (500 MHz) no. 2 3 5 6 7 8 9

1 6.41, s 7.01, s

2.83, m 2.69, m 2.89, brdd (8.0, 5.0)


1′ 2′ 5′ 6′ 7′ 8′ 9′

6.73, 6.82, 6.68, 2.80, 2.71, 2.63, 4.46, 4.08,

d (1.5) d (8.0) dd (8.0, 1.5) m m m dd (8.0, 9.0) dd (8.0, 9.5)

2

5

6

6.77, d (2.0)

6.77, d (2.0)

6.79, d (1.5)

6.84, 6.73, 2.82, 2.65, 3.03,

d (8.0) d (8.0) dd (13.5, 6.5) dd (13.5, 8.5) m

6.98, d (8.5) 6.68, dd (8.5, 1.5) 2.82, m

7.04, 6.70, 2.85, 2.75, 2.69,

d (8.5) dd (8.5, 2.0) dd (13.5, 5.5) dd (13.5, 7.0) m

6.98, 6.70, 2.83, 2.76, 2.69,

d (8.5) dd (8.5, 2.0) dd (13.5, 5.5) dd (13.5, 7.0) m

6.97, d (8.5) 6.64, dd (8.5, 1.5) 2.81, m

4.22, 3.89, 4.04, 3.33, 2.60,

dd dd dd dd m

6.62, 6.81, 6.57, 2.47,

d (1.5) d (8.0) dd (8.0, 1.5) overlap

6.62, 6.81, 6.58, 2.47,

d (2.0) d (8.0) dd (8.0, 2.0) m

6.48, brs

2.47, 4.10, 3.88, 4.75, 3.24,

overlap dd (8.5, 8.5) dd (8.5, 8.5) d (7.5) m

2.50, 4.09, 3.88, 4.79, 3.22,

overlap dd (8.5, 8.5) dd (8.5, 8.5) d (7.5) m

2.74, m

6.56, brs 6.60, 2.56, 2.46, 2.44, 4.11, 3.87,

3″

3.01, m

4″ 5″ 6″

3.04, m 3.10, m 3.63 dd (11.5, 5.5) 3.43, m

7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 1″″ 2″″ 3″″ 4″″ 5″″ 6″″ 3.74, s 3.70, s

2.72, m

(8.5, 8.5) (8.5, 8.5) (10.5, 3.0) (10.5, 3.0)

3.95, d (7.5) 2.93, m

3.81, s 3.81, s

4

6.78, d (1.5)

1″ 2″

3-CH3O 4-CH3O 3′-CH3O 4′-CH3O 3″-CH3O

3

6.81, brs

brs m m overlap dd (8.5, 8.5) dd (8.5, 8.5)

6.94, d (2.0)

7.04, d (8.5) 6.83, dd (8.5, 2.0) 5.46, 3.42, 3.68, 3.59, 4.87, 3.22, 3.27, 3.14,

d (4.5) m m m d (7.5) m m m

3.25, 3.64, 3.42, 4.82, 3.22, 3.27, 3.14, 3.25, 3.64, 3.43, 3.70,

m overlap overlap d (7.5) m m m m overlap m s

3.73, s

3.25, m

3.23, m

3.14, 3.48, 3.62, 3.54,

m m overlap overlap

3.08, 3.43, 3.82, 3.43,

4.64, 3.59, 3.60, 3.64,

d (3.0) m m m

4.80, d (2.0) 3.72, m

3.57, m 3.39, m

6.47, 2.52, 2.42, 2.42, 4.07, 3.84, 3.14, 3.64, 3.57, 3.64, 3.57,

brs m overlap overlap dd (8.5, 8.5) dd (8.5, 8.5) m m m m m

4.82, 3.23, 3.24, 3.14,

d (7.5) m m m

m m dd (9.5, 4.5) m

3.85, d (9.5) 3.57, d (9.5) 3.33, overlap

3.24, m 3.64, overlap 3.43, m

3.69, s

3.70, s

3.71, s

3.68, s 3.70, s

3.69, s 3.68, s

3.71, s

3.72, s

573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. (7S,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside (9): white amorphous powder; [α]D25 −48.5 (c 0.21, MeOH); CD (MeOH) λmax (Δε) 230 (−55.97), 278 (−5.38) nm; UV (MeOH) λmax (log ε) 230 (3.48), 278 (3.04) nm; IR (KBr) νmax 3390, 2936, 1757, 1595, 1515, 1454, 1419,

(7′S,8R,8′R)-Rafanotrachelogenin-4-O-β-D-glucopyranoside (8): D white amorphous powder; [α]25 −19.2 (c 0.23, MeOH); CD (MeOH) λmax (Δε) 232 (−4.42), 283 (+1.15) nm; UV (MeOH) λmax (log ε): 228 (3.04), 278 (2.68) nm; IR (KBr) νmax 3423, 2931, 1759, 1514, 1463, 1421, 1266, 1232, 1156, 1075, 1027 cm−1; (+)-HRESIMS m/z 573.1956 [M + Na]+ (calcd for C27H34O12Na, C


Article

Journal of Agricultural and Food Chemistry Table 2. 13C NMR Data for 1 in Acetic acid-d4 and for 2−12 in DMSO-d6 (125 MHz)


no. 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 1″″ 2″″ 3″″ 4″″ 5″″ 6″″ 3-CH3O 4-CH3O 3′-CH3O 4′-CH3O 3″-CH3O

1 161.5 118.7 155.8 112.5 148.2 161.9 34.4 44.0 179.2 130.5 111.5 149.0 147.8 112.2 120.8 37.9 41.7 72.0

2

3

4

5

6

7

8

9

10

11

12

131.7 112.9 149.2 147.8 112.3 121.9 37.3 38.0 71.5

132.3 114.3 149.1 145.8 115.6 121.7 33.9 46.0 178.9

132.5 114.2 149.2 145.7 116.4 121.8 34.3 46.0 178.9

132.4 114.2 149.1 145.7 115.8 121.9 34.2 46.0 178.9

132.3 114.3 149.0 145.7 115.5 121.8 33.8 45.9 179.0

132.0 114.3 149.0 145.7 115.5 121.8 34.2 43.2 179.1

131.9 114.2 148.9 145.7 115.3 121.8 34.8 42.8 179.3

137.1 110.3 149.0 145.9 115.3 117.9 70.5 52.8 178.1

134.2 110.1 147.8 145.8 115.4 118.1 70.4 52.8 178.0

136.6 111.2 148.8 146.1 115.1 118.7 71.0 52.5 176.6

136.6 111.2 148.8 146.0 115.1 118.7 71.8 52.5 176.6

66.9 46.0 177.7

132.2 113.1 144.0 146.5 129.5 117.3 37.5 41.4 71.2 135.8 110.9 149.4 146.6 115.7 118.4 87.2 53.8 63.5 100.7 73.7 77.5 70.1 77.3 61.1 100.5 73.7 77.4 70.1 77.3 61.1 56.2

131.5 112.8 149.1 147.8 112.3 120.9 37.4 41.2 71.2 101.1 73.7 77.5 70.5 75.4 66.8

131.5 112.8 149.1 147.8 112.4 120.8 37.4 41.2 71.2 100.8 73.7 77.3 70.4 76.0 68.1

128.8 110.4 147.7 143.1 128.5 121.1 37.5 41.2 71.1 44.3 62.2 62.2

136.4 110.1 149.0 148.4 112.0 118.3 72.4 46.3 68.0 100.6 73.7 77.5 70.1 77.3 61.1

135.9 110.0 149.0 148.3 111.8 118.4 72.8 45.4 68.9 100.6 73.7 77.5 70.2 77.4 61.2

131.4 112.6 149.2 147.6 112.2 120.7 38.9 36.1 72.1 100.7 73.8 77.5 70.2 77.4 61.2

131.6 112.6 149.0 147.6 112.1 120.6 38.8 35.8 71.8

131.8 113.0 149.1 147.8 112.7 121.0 38.1 39.5 71.8 100.6 73.7 77.5 70.1 77.4 61.1

130.1 113.3 148.0 145.4 115.9 121.3 38.2 39.5 71.0 100.6 73.7 77.4 70.1 77.4 61.0

99.1 70.0 69.4 68.8 71.3 61.0

109.8 76.4 79.2 73.8 63.7

100.7 73.7 77.4 70.1 77.3 61.1

55.9

56.1

56.2

55.9

55.8

55.8

55.9

55.9

56.0

56.1

55.8 56.1

55.9 55.9

56.1

56.1 56.0

55.9 56.0

56.1 55.9

55.7 56.0

55.9 56.1

56.1

104.3 73.9 77.3 70.4 77.1 61.4

55.0 54.9

55.9 56.0

56.1

1264, 1234, 1141, 1077, 1028 cm−1; (+)-HRESIMS m/z 573.1949 [M + Na]+ (calcd for C27H34O12Na, 573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. (7S,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan (10): white amorphous powder; [α]D25 −44.3 (c 0.11, MeOH); CD (MeOH) λmax (Δε) 234 (−8.20), 285 (−0.37) nm; UV (MeOH) λmax (log ε) 231 (2.98), 279 (2.97) nm; IR (KBr) νmax 3387, 2921, 1760, 1595, 1516, 1464, 1423, 1265, 1237, 1154, 1026 cm −1; (+)-HRESIMS m/z 411.1416 [M + Na] + (calcd for C21H24O7Na, 411.1420). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. (7R,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside (11): white amorphous powder; [α]D25 −30.4 (c 0.13, MeOH); CD (MeOH) λmax (Δε) 227 (−3.11), 276 (−1.00) nm; UV (MeOH) λmax (log ε) 229 (3.51), 279 (3.07) nm; IR (KBr) νmax 3410, 2920, 1758, 1594, 1514, 1464, 1420,

1265, 1235, 1156, 1076, 1028 cm−1; (+)-HRESIMS m/z 573.1950 [M + Na]+ (calcd for C27H34O12Na, 573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. (7R,8S,8′R)-4,7,4′-Trihydroxy-3,3′-dimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside (12): white amorphous powder; [α]D25 −37.5 (c 0.35, MeOH); CD (MeOH) λmax (Δε) 227 (−0.89), 275 (−0.36) nm; UV (MeOH) λmax (log ε) 230 (3.27), 280 (2.90) nm; IR (KBr) νmax 3392, 2917, 1755, 1601, 1514, 1454, 1426, 1270, 1226, 1156, 1075, 1031 cm−1; (+)-HRESIMS m/z 559.1795 [M + Na]+ (calcd for C26H32O12Na, 559.1791). For 1H and 13C NMR spectroscopic data, see Tables 2 and 3. Enzymatic Hydrolysis of 2−9, 11, and 12. Compound 2 (3.0 mg) was dissolved in 0.1 M sodium citrate buffer (5.0 mL), and then cellulase (3.0 mg, Sigma-Aldrich, St. Louis, MO, USA) was added. The reaction mixture was maintained at 40 °C for 5 h. The solution was then transferred to a liquid−liquid extractor and extracted with EtOAc D


Article

Journal of Agricultural and Food Chemistry Table 3. 1H NMR Data for 7−12 in DMSO-d6 (500 MHz) no. 2 5 6 7 8 2′ 5′ 6′ 7′


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

7

8

6.65, 6.95, 6.56, 2.70, 2.53, 2.83, 6.78, 6.88, 6.74, 4.36,

d (1.5) d (8.0) dd (8.0, 1.5) dd (13.5, 6.5) dd (13.5, 5.5) m d (1.5) d (8.0) dd (8.0, 1.5) brs

6.60, 6.89, 6.48, 2.82, 2.63, 2.82, 6.82, 6.86, 6.75, 4.60,

2.47, 4.14, 4.00, 4.83, 3.23, 3.26, 3.14, 3.24, 3.65, 3.43, 3.69,

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

2.50, overlap 4.00, d (7.5)

3.72, s 3.71, s

4.82, 3.23, 3.28, 3.14, 3.23, 3.65, 3.44, 3.67,

d (2.0) d (8.0) dd (8.0, 2.0) overlap dd (15.5, 7.5) overlap d (2.0) d (8.0) dd (8.0, 2.0) d (5.0)

d (7.5) m m m m overlap m s

3.70, s 3.73, s

9

10

11

12

6.97, 7.03, 6.86, 5.10,

d (1.5) d (8.5) dd (8.5, 1.5) t (4.0)

6.90, 6.73, 6.79, 5.08,

d (1.5) d (8.5) dd (8.5, 1.5) brs

6.90, 7.00, 6.78, 4.83,

d (1.5) d (8.5) dd (8.5, 1.5) t (4.0)

6.89, 7.01, 6.77, 4.81,

d (1.5) d (8.0) dd (8.0, 1.5) t (3.5)

2.66, 6.45, 6.75, 6.39, 2.32, 2.05, 2.78, 4.15, 3.89, 4.87, 3.24, 3.28, 3.16, 3.28, 3.65, 3.46, 3.65,

dd (6.5, 2.5) d (2.0) d (8.5) dd (8.5, 2.0) dd (13.5, 9.0) dd (13.5, 6.0) m t (8.5) dd (8.5, 3.0) d (7.5) m m m m overlap m s

2.61, 6.42, 6.71, 6.38, 2.28, 1.98, 2.78, 4.12, 3.87,

dd (7.0, 3.0) d (1.5) d (8.5) dd (8.5, 1.5) dd (13.5, 9.5) dd (13.5, 5.5) m t (8.5) dd (8.5, 6.5)

3.67, s

2.75, 6.73, 6.84, 6.66, 2.79, 2.61, 2.61, 3.95, 3.84, 4.87, 3.23, 3.27, 3.15, 3.27, 3.64, 3.43, 3.71,

dd (7.5, 4.0) d (2.0) d (8.0) dd (8.0, 2.0) dd (11.5, 4.5) m m t (8.5) t (8.5) d (7.5) m m m m dd (11.5, 4.5) m s

2.74, 6.69, 6.66, 6.54, 2.74, 2.60, 2.60, 3.95, 3.82, 4.85, 3.23, 3.27, 3.15, 3.27, 3.64, 3.43, 3.71,

m d (1.5) d (8.0) dd (8.0, 1.5) m m m t (8.5) dd (8.5, 6.5) d (7.5) m m m m dd (11.5, 4.5) m s

3.66, s 3.72, s

3.71, s 3.70, s

3.72, s 3.68, s

3.72, s

Figure 2. Key HMBC correlations in compounds 1−3 and 7. Anti-inflammatory Effects of Compounds. Compounds 1−17 were tested for anti-inflammatory activity on BV2 cells.12,13 After the BV2 cells were pre-incubated for 24 h at 37 °C under a 5% CO2 atmosphere in a 96-well plate, the cells were treated with various concentrations of the test compounds (1 × 10−5, 1 × 10−6, and 1 × 10−7 M), followed by stimulation with LPS for 24 h. The production of NO was determined by measuring the concentration of nitrite in the culture supernatant. NaNO2 was used to generate a standard curve. The absorbances at 540 nm were measured. Curcumin was utilized as a positive control.

(5.0 mL). The organic layer was evaporated under reduced pressure and purified using reversed-phase preparative HPLC with MeOH/ H2O (45:55) as the mobile phase to afford 2a (1.1 mg, 43 min). The aqueous layer was evaporated and dried under reduced pressure to yield a monosaccharide residue. This residue was purified by preparative silica gel thin-layer chromatography (PTLC) [CHCl3/ MeOH/H2O (7:3:1)] to afford glucose (Rf = 0.2) with positive optical rotation ([α]D25 +36.5). Hydrolysis of 3−9, 11, and 12 and sugar identification were performed according to the procedure described for 2. The D-galactose (Rf = 0.16, [α]D25 + 62.8) and D-apiose (Rf = 0.5, [α]D25 +8.7) were confirmed by TLC analysis [CHCl3/MeOH/H2O (7:3:1)] in comparison with standard samples. Rh2(OCOCF3)4-Induced CD Experiments of Compounds 7a and 8a. Compound 7a (0.2 mg) was dissolved in dried chloroform (1 mL). The CD spectrum of 7a in the 450−300 nm region was measured. Then, Rh2(OCOCF3)4 (2 mg) was added in the solution, and the mixture was stirred at room temperature for 10 min. The CD spectrum of the reaction mixture in the 450−300 nm region was also measured. On the basis of the bulkiness rule for secondary alcohols,11 a negative Cotton effect at 340 nm (the E band) in the Rh2(OCOCF3)4-induced CD spectrum (Figure S49, Supporting Information) indicated the 7′R configuration of 7a. Similarly, the 7′S configuration of 8a was confirmed.

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RESULTS AND DISCUSSION Structure Elucidation of the New Compounds. Compound 1, a white amorphous powder, had the molecular formula C20H20O8 (m/z 389.1235 [M + H]+ in HRESIMS), which indicated 11 degrees of unsaturation. The IR spectrum exhibited absorption bands attributable to hydroxyl groups (3364 cm−1), carbonyl groups (1765, 1712, and 1640 cm−1), and aromatic rings (1515 and 1465 cm−1). In the 1H NMR spectrum (Table 1), ABX-system aromatic proton signals [δH 6.73 (1H, d, J = 1.5 Hz, H-2), 6.82 (1H, d, J = 8.0 Hz, H-5), and 6.68 (1H, dd, J = 8.0, 1.5 Hz, H-6)] and two proton signals that displayed as a broad singlet [δH 6.41 (1H, s, H-3) and 7.01 E


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C42H52O19 on the basis of the positive HRESIMS ion (m/z 883.2981 [M + Na]+). The 1H NMR spectroscopy data (Table 1) of 3 revealed two sets of ABX-system aromatic rings [δH 6.78 (1H, d, J = 1.5 Hz, H-2), 6.98 (1H, d, J = 8.5 Hz, H-5), 6.68 (1H, dd, J = 8.5, 1.5 Hz, H-6), 6.94 (1H, d, J = 2.0 Hz, H2″), 7.04 (1H, d, J = 8.5 Hz, H-5″), 6.83 (1H, dd, J = 8.5, 2.0 Hz, H-6″)] and a 1,3,4,5-tetrasubstituted aromatic ring [δH 6.56 (1H, brs, H-2′), 6.60 (1H, brs, H-6′)]. Additionally, two oxymethylene protons [δH 4.11 (1H, dd, J = 8.5, 8.5 Hz, H9′a), 3.87 (1H, dd, J = 8.5, 8.5 Hz, H-9′b), 3.68 (1H, m, H9″a), 3.59 (1H, m, H-9″b)], an oxymethine proton [δH 5.46 (1H, d, J = 4.5 Hz, H-7″)], two methylene protons [δH 2.82 (2H, m, H-7), 2.56 (1H, m, H-7′a), 2.46 (1H, m, H-7′b)], three methine protons [δH 2.74 (1H, m, H-8), 2.44 (1H, overlap, H-8′), 3.42 (1H, m, H-8″)], three methoxyl protons [δH 3.70 (3H, s, 3-OCH3), 3.73 (3H, s, 3′-OCH3), 3.72 (3H, s, 3″-OCH3)], and two glucopyranosyl anomeric protons [δH 4.87 (1H, d, J = 7.5 Hz, H-1‴), 4.82 (1H, d, J = 7.5 Hz, H1″″)] were observed in the upfield region. The 13C NMR spectrum (Table 2) of 3 displayed 42 carbon signals, 12 of which were assigned to two glucose units; the remaining 30 carbons were assigned to three C6−C3 units and three methoxy groups. These data revealed that 3 was a sesquineolignan.17 In the HMBC spectrum (Figure 2), the characteristic correlations from H-7 (δH 2.82) to C-1, C-2, C-9, and C-8′, from H-7′ (δH 2.56, 2.46) to C-1′, C-2′, C-9′, and C8, and from H-7″ (δH 5.46) to C-4′, C-5′, C-1″, and C-9″ suggested the presence of a dihydrobenzofuran unit and a benzylbutyrolactone unit in compound 3. The HMBC correlation peaks of three methoxy groups at δH 3.70, 3.73, and 3.72 with C-3, C-3′, and C-3″ confirmed the linkage positions of these methoxy groups. Two glucose units were determined to be located at C-4 and C-4″ on the basis of the correlations of H-1‴ with C-4 and H-1″″ with C-4″ in the HMBC spectrum. The chemical shifts of H-9′ (δH 4.11 and 3.87) indicated the trans configuration of H-8/H-8′. Moreover, the correlation peaks of H-7″ with H-9″ and of H-8″ with H-2″ and H-6″ in the ROESY spectrum (Figure 3) indicated the

(1H, s, H-5)] were observed in the downfield region. In addition, two oxygenated methylene protons at δH 4.46 (1H, dd, J = 8.0, 9.0 Hz, H-9′a) and 4.08 (1H, dd, J = 8.0, 9.5 Hz, H9′b), four methylene protons at δH 2.83 (1H, m, H-8a), 2.69 (1H, m, H-8b), 2.80 (1H, m, H-7′a), and 2.71 (1H, m, H-7′b), two methine protons at δH 2.89 (1H, brdd, J = 8.0, 5.0 Hz, H9) and 2.63 (1H, m, H-8′), and two methoxy groups at δH 3.81 (3H, s) and 3.81 (3H, s) were also observed. The 13C NMR spectrum (Table 2) of 1 displayed 20 carbon signals, including two C6−C3 units and two methoxy groups. On the basis of the aforementioned data, compound 1 was determined as a butyrolactone lignan similar to arctigenin, except that NMR resonances for the 6-carboxyl-2-pyrone moiety in 1 were observed in place of the resonances for the 4-hydroxy-3methoxyphenyl unit in arctigenin. This interpretation was verified by the HMBC and 13C NMR spectra. The correlations from H-8′ to C-8, C-9, C-10, C-1′, C-7′, and C-9′ and from H9 to C-4, C-8, C-10, C-7′, C-8′, and C-9′ indicated the presence of a benzylbutyrolactone moiety in 1. Detailed analysis of the 13 C NMR spectrum, together with the remaining five degrees of unsaturation, implied the existence of a 6-carboxyl-2-pyrone moiety; this moiety was verified by the HMBC correlations (Figure 2) from H-3 to C-2 and C-5 and from H-5 to C-6 and C-7. The connection between C-8 and C-4 was also determined by the key correlations from H-8 to C-3, C-4, and C-5. The previous study indicated that the relative configuration of H-9 and H-8′ could be determined by the ΔδH9′a−H9′b values (ΔδH9′a−H9′b ≥ 0.2 for trans, ΔδH9′a−H9′b ≈ 0 for cis).14,15 Therefore, the trans configuration of H-9 and H-8′ was established by the H-9′a (δH 4.46) and H-9′b (δH 4.08) signals in the 1H NMR spectrum. This result, combined with the negative Cotton effect at 230 nm in the CD spectrum, suggested that the absolute configurations of C-9 and C-8′ in 1 were 9R and 8′R, respectively.16 Consequently, 1 was identified as in Figure 1 and named arctiidilactone. Compound 2 was obtained as a white amorphous powder; its molecular formula was confirmed to be C20H28O10 on the basis of the positive HRESIMS ion observed at m/z 451.1584 [M + Na]+. The 1H NMR spectrum (Table 1) of 2 showed three aromatic proton signals at δH 6.81 (1H, brs, H-2), 6.84 (1H, d, J = 8.0 Hz, H-5), and 6.73 (1H, d, J = 8.0 Hz, H-6), revealing the presence of an ABX-system aromatic ring. Additionally, two methoxy groups at δH 3.74 (3H, s) and 3.70 (3H, s) and a glucopyranosyl anomeric proton at δH 3.95 (1H, d, J = 7.5 Hz, H-1″) were also observed in the upfield region. In the 13C NMR spectrum (Table 2), signals associated with a C3−C6− C3 unit, two methoxy groups, and a glucopyranosyl group were observed. All of the aforementioned spectroscopic data indicated that 2 was a butyrolactone-type lignan, but without an aromatic ring. The HMBC correlations (Figure 2) from H-7 to C-1, C-2, C-6, C-8, C-9, and C-2′ and from H-1′ to C-8, C2′, and C-3′ confirmed the benzylbutyrolactone skeleton of 2. The linkage point of the glucose was identified by the correlation between the anomeric proton H-1″ of glucose and C-1′ in the HMBC experiment. The 8R,2′S configurations of 2 were deduced from the chemical shifts of H-9 (δH 4.22, 3.89) in the 1H NMR spectrum (Table 1) and from the negative Cotton effect at 227 nm in the CD spectrum. Thus, 2 was determined to be arctiiapolignan A. The IR spectrum of compound 3 showed absorptions for a hydroxyl group (3413 cm−1), methylene (2921 cm−1), benzene ring (1598, 1514, and 1454 cm−1), and carbonyl group (1760 cm−1). The molecular formula of 3 was determined to be

Figure 3. Key ROESY correlations in compounds 1, 3, and 7.

trans configuration of H-7″/H-8″. In the CD spectrum, the negative Cotton effect at 230 revealed the 8R,8′R configurations of the benzylbutyrolactone unit,16 and the negative Cotton effect at 290 nm revealed the 7″R,8″S configurations of the dihydrobenzofuran unit.18 Therefore, compound 3 was confirmed to be arctiisesquineolignan A. The spectroscopic data for compound 4 were similar to those of arctiin, except for an additional galactose unit.19 The downfield shift of C-6″ (δC 66.8) of glucose in 4 suggested that the linkage point of the galactose was at C-6″ of glucose, which was further confirmed by the correlations between the anomeric proton H-1‴ of galactose and C-6″ of glucose in the HMBC experiment. The trans configuration of H-8 and H8′ was established on the basis of the H-9′a (δH 4.10) and H9′b (δH 3.88) signals in the 1H NMR spectrum (Table 1). The F


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8R,8′R. Cellulase hydrolysis of 7 resulted in 7a (the aglycone) and D-glucose. The 7′R configuration was supported by a negative Cotton effect at 340 nm in the Rh2(OCOCF3)4induced CD spectrum of 7a (Figure S49, Supporting Information).11 Consequently, 7 was identified as Figure 1 and named (7′R,8R,8′R)-rafanotrachelogenin-4-O-β-D-glucopyranoside. Compound 8 was obtained as a white amorphous powder. Its molecular formula was determined to be C27H34O12 on the basis of the positive HRESIMS ion observed at m/z 573.1956 [M + Na]+. A comparison of the IR, UV, and NMR data (Table 2) of 8 with those of 7 revealed that these two compounds possessed the same planar structure. As previously described, the ROESY correlation of H-7′/H-8 indicated the trans configuration of H-8/H-8′ in 8. In the CD spectrum, a negative Cotton effect at 227 nm helped confirm the 8R,8′R configurations for 8. The 7′S configuration was determined by a positive Cotton effect at 340 nm in the Rh2(OCOCF3)4induced CD spectrum of 8a (the aglycone) (Figure S57, Supporting Information), which was obtained by cellulase hydrolysis of 8. Thus, compound 8 was deduced to be (7′S,8R,8′R)-rafanotrachelogenin-4-O-β-D-glucopyranoside. Compound 9 had the same molecular formula as 8: C27H34O12. A comparison of the NMR spectra of 9 and 8 revealed that the differences between these two compounds were the linkage point of the alcoholic hydroxyl group. In the HMBC spectrum, the correlation peaks from H-7 (δH 5.10) to C-1, C-2, C-6, C-8, C-9, and C-8′ and from H-7′ (δH 2.32, 2.05) to C-1′, C-2′, C-6′, C-8′, C-9′, and C-8, together with the chemical shifts of H-7/7′ and C-7/7′, indicated that the hydroxyl was located at C-7. Thus, the planar structure of 9 was elucidated as 4,7-dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside. The relative configuration of H-8 and H-8′ in 9 was defined as trans on the basis of the characteristic correlation peak of H-8′ with H-7 in the ROESY spectrum. The 8S,8′R configuration for 9 was confirmed by the negative Cotton effects at 243 and 282 nm in the CD spectrum. Cellulase hydrolysis of 9 resulted in 9a (the aglycone), which was purified by preparative HPLC. The 7,8erythro configuration of 9a was deduced on the basis of the coupling constant J7,8 (4.0 Hz) in the 1H NMR experiment (Figure S66, Supporting Information).22 Thus, 9 was elucidated to be (7S,8S,8′R)-4,7-dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside. A comparison of the molecular formula (C21H24O7) and NMR data of 10 with those of 9 (Tables 2 and 3) indicated the absence of glucose unit resonances in 10. Using the same methods as described for 9 (Figures S74−S76, Supporting Information), we elucidated the 7S,8S,8′R configurations of 10. Thus, compound 10 was determined to be (7S,8S,8′R)-4,7dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone lignan. Compound 11 had the same planar structure as 9, as indicated by a comparison of their NMR spectra and molecular formulas. The absolute configurations of C-8 and C-8′ were established as 8S,8′R by the correlation of H-7/8′ in the ROESY spectrum and the negative Cotton effects at 243 and 282 nm in the CD spectrum. However, in the 1H NMR spectrum of 11a (the aglycone) (Figure S84, Supporting Information), which was obtained by hydrolysis of 11, the coupling constant of H-7 (J7,8 = 8.5 Hz) suggested that the relative configuration between C-7 and C-8 was threo instead of the erythro configuration in 9.21 Thus, the 7R configuration was

absolute configurations of C-8 and C-8′ in 4 were defined as 8R,8′R on the basis of the negative Cotton effect at 230 nm observed in the CD spectrum. Thus, 4 was identified as Figure 1 and named arctigenin-4-O-α-D-galactopyranosyl-(1→6)-O-βD-glucopyranoside. Compound 5 was demonstrated to have the molecular formula C32H42O15 on the basis of HRESIMS with m/z 689.2430 [M + Na]+. The IR spectrum exhibited signals for a hydroxyl group (3418 cm−1), a methylene (2929 cm−1), aromatic rings (1592, 1515, and 1454 cm−1), and a carbonyl group (1760 cm−1). Detailed analysis of the 1D NMR spectra (Table 1) suggested that the structure of 5 was similar to that of 4. The difference between these two compounds was the presence of an apiofuranose group20 at C″-6 in 5 instead of a galactose group at C″-6 in 4, which was supported by the HMBC correlation between the apiofuranose anomeric proton H-1‴ (δH 4.80) and C-6″ (δC 68.1) in 5. The absolute configurations of C-8 and C-8′ in 5 were also confirmed by the same method used to confirm those in 4. The chemical shifts of H-9′a (δH 4.09) and H-9′b (δH 3.88) verified the trans configuration of H-8/8′. This result, combined with the negative Cotton effect at 230 nm in the CD spectrum, indicated the 8R,8′R configurations for 5. Thus, compound 5 was determined to be arctigenin-4-O-β-D-apiofuranosyl-(1→6)O-β-D-glucopyranoside. Compound 6 had the molecular formula C29H38O13, and its IR spectrum exhibited absorption bands that were attributed to a hydroxyl (3426 cm−1), a methylene (2928 cm−1), a carbonyl group (1758 cm−1), and an aromatic ring (1597, 1513, and 1461 cm−1). Comprehensive analysis of the NMR spectra indicated that compound 6 was a dibenzylbutyrolactone lignan glucoside with an additional C3 unit. In the HMBC spectrum, the correlation peaks from H-2″ (δH 3.57, 3.64) and H-3″ (δH 3.57, 3.64) to C-1″ (δC 44.3) and from H-1″ (δH 3.14) to C-2″ (δC 62.2) and C-3″ (δC 62.2), together with their chemical shifts, demonstrated the propanediol structure of the C3 unit. In addition, the HMBC correlations from H-1″ (δH 3.14) to C4′ (δC 143.1), C-5′ (δC 128.5), and C-6′ (δC 121.1) indicated that C-1″ of the propanediol moiety was connected to C-5′ of the dibenzylbutyrolactone lignan by a C−C bond. The absolute configurations of C-8 and C-8′ were determined by the procedure described for 4 and 5. A negative Cotton effect at 229 nm observed in the CD spectrum, in combination with the chemical shifts of H-9′ at δH 4.07 and 3.84, verified the 8R,8′R configurations for 6. On the basis of the aforementioned information, compound 6 was confirmed to be 5′-propanediolmatairesinoside. Compound 7 was obtained as a white amorphous powder with a molecular formula of C27H34O12 on the basis of an ion peak at m/z 573.1954 [M + Na]+ in the HRESIMS spectrum. The spectroscopic data (Tables 2 and 3) for 7 were almost identical to the data for rafanotrachelogenin-4-O-β-D-glucopyranoside,21 which was isolated from Trachelospermum lucidum. However, the absolute configurations of rafanotrachelogenin-4O-β-D-glucopyranoside had not yet been identified. To determine the absolute configuration of C-8, C-7′, and C-8′ in compound 7, the ROESY spectrum, CD exciton chirality, and Rh2(OCOCF3)4-induced CD spectrum were assayed. The relative configuration of H-8 and H-8′ in 7 was defined as trans on the basis of the characteristic correlation peaks of H-7′ with H-8 in the ROESY spectrum (Figure 3). In combination with the negative Cotton effect at 230 nm in the CD spectrum, the absolute configuration of C-8 and C-8′ was confirmed as G


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Scheme 1. Plausible Biogenetic Pathway of 1

butyrolactone unit (3); three new dibenzylbutyrolactone lignans (4−6); six new dibenzylbutyrolactone lignans with an additional hydroxyl at C-7/7′ (7−12); and five known compounds (13−17). A literature search revealed that no butyrolactone lignan with a 6-carboxyl-2-pyrone moiety has been previously reported. A plausible biogenetic pathway of 1 is proposed in Scheme 1. The 6-carboxyl-2-pyrone might be formed from a 3,4-dihydroxy phenyl moiety through oxidative cleavage, oxidation, and esterification.

determined and the structure of 11 was characterized as Figure 1 and named (7R,8S,8′R)-4,7-dihydroxy-3,3′,4′-trimethoxyl-9oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside. The molecular formula of compound 12 was determined to be C26H32O12 on the basis of the positive HRESIMS ion observed at m/z 559.1795 [M + Na]+. The UV, IR, and NMR spectra of 12 were almost identical to the corresponding spectra of 11; the difference between the compounds was that 12 contained a 4′-OH group in place of the 4′-OCH3 in 11. The 7R,8S,8′R configuration of 12 was also confirmed by the ROESY correlation and CD spectrum and by the coupling constant of H-7 (J7,8 = 8.0 Hz) in 12a (the aglycone) (Figure S93, Supporting Information). On the basis of these results, 12 was determined to be (7R,8S,8′R)-4,7,4′-trihydroxy-3,3′dimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside. The known compounds matairesinol-4,4′-di-O-β-D-glucopyranoside (13),23 arctiin (14),24 styraxlignolide E (15),25 styraxlignolide D (16),25 and arctigenin-4-O-β-D-gentiobioside (17)26 were identified by comparison of these physical and spectral data with literature values. In addition, their absolute configurations were established from NMR and CD data. Cytotoxic Activity. All of the isolated compounds were tested for cytotoxic activity using an MTT assay in HCT-8, Bel7402, BGC-823, A549, and A2780 cells.27 Compounds 1−17 exhibited no significant cytotoxicity in the cell model systems at a concentration of 10 μM. Hepatoprotective Activity. Given our previous bioactivity screening results for the lignan glucosides, the hepatoprotective activities10 of 1−17 were tested through in vitro assays. Unfortunately, all of the tested compounds exhibited weak hepatoprotective activities at a concentration of 10 μM. Anti-inflammatory Activity. Compounds 1−17 were tested for anti-inflammatory activity by suppressing the production of NO in lipopolysaccharide-induced BV2 cells. From the data obtained, compounds 1, 6, 8, and 10 exhibited stronger anti-inflammatory effects than the positive control curcumin at a concentration of 10 μM. Among them, compound 1 demonstrated 75.51, 70.72, and 61.17% inhibition at 10, 1, and 0.1 μM, respectively, whereas the positive control curcumin showed 53.02, 36.74, and 0% inhibition at 10, 1, and 0.1 μM. Furthermore, compound 6 exhibited 71.59, 38.64, and 2.27% inhibition at 10, 1, and 0.1 μM, respectively. Compound 8 exhibited 87.77, 53.24, and 11.51% inhibition at 10, 1, and 0.1 μM, respectively. Compound 10 exhibited 63.31, 40.29, and 37.41% inhibition at 10, 1, and 0.1 μM, respectively. In the continued search for bioactive natural products from Arctii Fructus, 17 butyrolactone type lignans (1−17) were isolated, including a novel rare butyrolactone lignan with a 6carboxyl-2-pyrone moiety, named arctiidilactone (1); a new apolignan glucoside without an aromatic ring (2); a new sesquineolignan with a dihydrobenzofuran unit and a

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02838. NMR, IR, MS, and CD spectra of compounds 1−12 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(P.-C.Z.) Phone: +86-10-63165231. E-mail: pczhang@imm. ac.cn. Fax: 86-10-63017757. Author Contributions ∥

Y.-N.Y. and X.-Y.H. contributed equally to this work.

Funding

This research was supported by the National Science and Technology Project of China (No. 2012ZX09301002-002) and the Fundamental Research Funds for the Central Institutes (No. 2014TD03). Notes

The authors declare no competing financial interest.

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

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New Butyrolactone Type Lignans from Arctii ... - ACS Publications

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