New norlignans and flavonoids of Dysosma versipellis

Phytochemistry Letters 16 (2016) 75–81

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New norlignans and flavonoids of Dysosma versipellis Yue Zhenga,1, Yang-Guo Xiea,1, Yu Zhanga , Tao Lic, Hui-Liang Lib , Shi-Kai Yanb , Hui-Zi Jina,* , Wei-Dong Zhanga,b,* a b c

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China Department of Phytochemistry, Second Military Medical University, Shanghai 200433, PR China Key Laboratory of Animal Parasitology, Ministry of Agriculture, Shanghai Veterinary Research Institute, Shanghai, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 January 2016 Received in revised form 20 January 2016 Accepted 7 March 2016 Available online xxx

Two new norlignans, dysosmanorlignans A and B (1–2); six new flavonoids, dysosmaflavones A–F (3–8); and 30 known compounds (9–38) were isolated from the roots of Dysosma versipellis. Their structures were determined by detailed analysis of MS and NMR spectroscopic data. Dysosmaflavones E and F both exhibited mild antibacterial activities against Streptococcus agalactiae,Pseudomonas aeruginosa, and Bacillus subtilis with MIC values ranging from 93.0 to 93.8 mg/mL. ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Dysosma versipellis Flavonoid Norlignan Antibacterial properties

1. Introduction

2. Results and discussion

Dysosma versipellis (Hance.) M. Cheng (Berberidaceae) is an endemic but endangered species in China. The dried roots of D. versipellis, called ”Bajiaolian” in Chinese, are used as folk medicine for the treatment of carbuncles and snakebite (Dong and Zeng, 1994; Jiang et al., 2011). Previous phytochemical investigations have shown that lignans, flavonoids and amino acids are major constituents of D. versipellis (Jiang and Chen, 1989; Shang et al., 2002; Ying et al., 1990; Gao et al., 2011). The lignans have received considerable focus due to their potent antitumor activities, but the norlignans and flavonoids were barely reported. To produce guidelines for reasonable use, we have conducted a full study of this plant. We herein describe the isolation of these new isolated compounds, the elucidation of their structure and the evaluation of their inhibitory activities against Riemerella anatipestifer, Bacillus subtilis, Enterococcus faecalis, Pseudomonas aeruginosa, Aeromonas hydrophila, Streptococcus agalactiae, and Streptococcus suis.

The CH2Cl2-soluble portion was subjected to CC over silica gel, Sephadex LH-20, and preparative HPLC to afford two new norlignans, dysosmanorlignans A and B (1–2), dysosmaflavones A–F (3–8), and 30 known compounds (9–38) (Fig 1). Compound 1 was obtained as a yellow oil and has a molecular formula of C23H24O8 by HRESIMS (m/z 451.1364 [M+Na] +, calc. for C23H24NaO8 451.1363). 1H NMR spectroscopic data showed three methoxyl groups at (dH 3.75, s, 6H) and (dH 3.71, s, 3H); four aromatic protons, H-20 (dH 6.70, s), H-60 (dH 6.70, s), H-2 (dH 6.82, s), and H-5 (dH 7.42, s); and a 2-hydroxyl propyl group, H-9 (dH 2.43, ddd, J = 13.5, 7.7, 0.7 Hz; dH 2.60, ddd, J = 13.5, 5.2, 0.8 Hz), H-11 (dH 1.18, d, J = 6.2 Hz), H-10 (dH 4.02, m). The 13C NMR spectrum revealed an aryltetralin lignan skeleton, which is similar to podophyllotoxin (see Table 1). The location of the carbonyl (C-7) was confirmed by the HMBC correlations of H-5, H-80 , and H-9 with C-7. The correlations of H-2, H-60 , and H-20 with C-70 indicated that C-70 is located between two aromatic rings. The olefinic bond was assigned between C-7 and C-70 due to the correlation of H-80 with C-7, C-1, and C-9 (Fig. 2). Previous ORD studies showed that 7aryltetralin lignans give Cotton values between 290 and 275 nm, that all 70 b-compounds give a negative Cotton effect, and that all 70 a-compounds give a positive one (Klyne et al., 1966; Swan and Klyne, 1965). The CD spectrum of 1 exhibited a positive Cotton effect at 285 nm, indicating that the C-70 was a(S) (Fig. 3). Thus, 1 was established to be (8S)-8-hydroxy-6-(2-hydroxypropyl)-8-

* Corresponding authors. E-mail addresses: [email protected] (H.-Z. Jin), [email protected] (W.-D. Zhang). 1 These authors contributed equally to this work and should be considered cofirst authors.

http://dx.doi.org/10.1016/j.phytol.2016.03.001 1874-3900/ ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

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Y. Zheng et al. / Phytochemistry Letters 16 (2016) 75–81

O

4

O

3

O

5 6 1 2

8

7 7

8 1'

HO

O

9

10

2'

6'

11

O

OH

OH

O HO

5'

3' 4'

O

O

O

O O

O

2

1 4'' 5'

5''

3''

2''

3'

1''

HO

8

7

O

OH

2

2' 1'

HO

OH

4'

6

4

5

OH

3

7 6

5' 6'

8

5

OH

O

O 4

2

3

OH

1' 2'

O

3'

O 3''

1'' 2''

O

O

OH

OH

4'

6'

4''

HO OH

O O

5''

OH

5

4

3

O

4'''

HO

3'''

2'''

5'''

3'

1''' 4''

O 5''

HO

3'' 2''

8

7

O

2

2' 1'

1''

6

5

OH

O

3

O

OH

OH

OH

OH

OH

4'

HO

HO

O

O

5' 6'

4

O

OH

O

16

OH

O

7

6

O OH

O

8

Fig. 1. Structures of compounds 1–8 isolated from D. versipellis.

Fig. 2. Selected 2D correlations of dysosmanorlignan A (1).

(3,4,5-trimethoxyphenyl) naphtho [2,3-d][1,3]dioxol-5(8H)-one and named dysosmanorlignan A. Compound 2 was obtained as a yellow oil, and its molecular formula was determined to be C23H24O8 on the basis of its HRESIMS (m/z 451.1364 [M + Na]+, calc. for C23H24NaO8 451.1363). The 1H and 13C NMR spectra of 2 were almost the same as for 1. However, their retention times on HPLC are different, suggesting differences in the absolute configurations. The CD spectrum of 2 exhibited a negative Cotton effect at 285 nm, indicating that C-70

was b(R) (Fig. 3). Thus, 2 was established as (8R)-8-hydroxy-6-(2hydroxypropyl)-8-(3,4,5-trimethoxyphenyl) naphtho[2,3-d][1,3] dioxol-5(8H)-one and named dysosmanorlignan B. Compound 3 was obtained as a yellowish oil, molecular formula (C21H20O7), ascertained via high resolution (HRESIMS) analysis, which indicated 12 of unsaturation. The IR spectrum indicated hydroxyl (3403 cm1), carbonyl (1652 cm1) and olefinic groups (1600 cm1). The 13C and DEPT NMR spectroscopic data of 3 revealed 21 carbon signals, including three methyls, one

Y. Zheng et al. / Phytochemistry Letters 16 (2016) 75–81 Table 1 1 H (500 MHz) and

13

77

C (125 MHz) NMR Data for Compounds 1–2 (d in ppm, J values in Hz).

Position

1

dC 1 2 3 4 5 6 7 8 9

144.7 107.0 152.0 147.9 104.0 124.6 184.3 132.7 38.9

10 11 10 20 30 40 50 60 70 80 OCH2O OCH3 OCH3

66.1 22.3 139.2 102.6 153.1 136.7 153.1 102.6 71.3 149.7 102.0 55.1 59.6

2

dH (J in Hz)

dC

6.82 s

7.42 s

2.43 ddd (13.5, 7.7, 0.7) 2.60 ddd (13.5, 5.2, 0.8) 4.02 m 1.18 d (6.2) 6.70 s

6.70 s 6.76 s 6.00 dt (16.4, 3.2) 3.75 s 3.71 s

144.7 107.0 152.1 148.0 104.0 124.7 184.2 132.5 38.9 66.1 22.0 139.2 102.6 153.1 136.8 153.1 102.6 71.3 149.8 102.0 55.1 59.7

dH (J in Hz) 6.79 s

7.42 s

2.50 d (6.38) 3.97 dt (12.6, 6.3, 6.3)

6.64 s

6.64 s 6.76 s 6.00 d (14.5) 3.74 s 3.72 s

Data were recorded in CD3OD.

Fig. 3. CD spectra of 1 and 2.

methylene, five methines, and twelve quaternary carbons. The 1H NMR spectroscopic data showed two pairs of olefinic protons at H8 (dH 6.21, d, J = 2.0 Hz), H-6 (dH 6.28, d, J = 2.0 Hz), and H-50 (dH 6.75, d, J = 8.2 Hz), H-60 (dH 6.81, d, J = 8.2 Hz). The NMR data also revealed a flavone skeleton. The presence of one 2-methyl-2butenyl group was based on the signals of one olefinic proton H-300 (dH 5.06, ddd, J = 6.9, 5.5, 1.0 Hz), two methyl groups H-400 (dH 1.47, d, J = 1.0 Hz,) and H-300 (dH 1.36, s), and one methylene group H-100 (dH 3.40, t, J = 7.0 Hz) (see Tables 2 and 3). These spectroscopic data indicated that 3 was a prenylated flavone derivative. The location of the 2-methyl-2-butenyl group was assigned to C-20 by the HMBC correlations of H-10 with C-10, C-20 and C-30 (Fig. 3). Thus, 3 was established as (E)-2-(3, 4-dihydroxy-2-(2-methylbut-2-en-1-yl) phenyl)-5,7-dihydroxy-3methoxy-4H-chromen-4-one and named dysosmaflavone A. Compound 4 was obtained as a yellowish oil with the molecular formula of C21H18O7, as deduced from high-resolution analysis. Careful comparison of the NMR spectroscopic data on 4 with the data on 3 indicated the presence of a similar flavone skeleton. The

difference between 4 and 3 was that the prenyl group was replaced by a 2,2-dimethyl-pyran ring, as deduced by a series of signals consisting of one pair of cis-coupled olefinic protons, H-100 (dH 6.27, d, J = 10.0 Hz) and H-200 (dH 5.77, d, J = 10.0 Hz), and two methyl groups, H-400 , 500 (dH 1.47, s) (see Tables 2 and 3). The HMBC spectrum also showed the correlations between H-100 and C-10, C-20 and C-30 , indicating that the 2,2-dimethylpyran ring was attached to the B ring by C-20 and C-30 . Thus, 4 was established as 5,7,80 trihydroxy-3-methoxy-20 ,20 -dimethyl-20 H,4H-[2,50 -bichromen]-4one and named dysosmaflavone B. Compound 5 was obtained as a yellowish oil and possessed the molecular formula C26H28O8, as revealed by high-resolution analysis, which indicated 13 of unsaturation. The flavone skeleton was deduced as in 3. The presence of one 3-methyl-2-butenyl group was based on the signals of one olefinic proton, H-2000 (dH 5.05, m); two methyl groups, H-5000 (dH 1.47, s) and H-4000 (dH 1.36, s); and one methylene group, H-1000 (dH 3.39, d, J = 6.7 Hz). The presence of one 2-hydroxy-3-methyl-3-butenyl group was based on the signals of one methylene proton, H-400 (dH 4.69, s) and (dH

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Y. Zheng et al. / Phytochemistry Letters 16 (2016) 75–81

4.74, s); one methyl proton, H-500 (dH 1.83, s); one methine proton, H-200 (dH 4.43, t, J = 6.9, 6.5 Hz); and one methylene proton, H-100 (dH 2.90, dd, J = 13.4, 6.9 Hz, dH 3.03, dd, J = 13.4, 6.5 Hz) (see Tables 2 and 3). The location of the 3-methyl-2-butenyl group was assigned to C-20 based on the HMBC correlations of H-1000 with C-10, C-20 and C-30 . The location of the 2-hydroxy-3-methyl-3-butenyl group was assigned to C-6 based on the HMBC correlations of H-100 with C-5, C-6 and C-7. Thus, 5 was established as 2-(3,4-dihydroxy2-(3-methylbut-2-en-1-yl) phenyl)-5,7-dihydroxy-6-(2-hydroxy3-methylbut-3-en-1-yl)-3-methoxy-4H-chromen-4-one and named dysosmaflavone C. Compound 6 was obtained as a yellowish oil with a molecular formula of C26H28O8, ascertained via high-resolution analysis, which indicated 13 of unsaturation. Careful comparison of the NMR spectroscopic data on 6 with the data on 5 indicated the flavone skeleton. The difference between the spectra of 6 and 5 was that the 2-hydroxy-3-methyl-3-butenyl group was replaced by a 3,4-dihydroxy-2,2-dimethyl-pyran ring, as deduced from a series of signals consisting of one methylene proton, H-100 (dH 2.65, dd, J = 17.0, 6.8 Hz, dH 2.96, dd, J = 17.0, 5.3 Hz); one methane proton, H200 (dH 3.83, dd, J = 6.8, 5.3 Hz); and two methyl groups, H-400 (dH 1.32, s) and H-500 (dH 1.36, s). The HMBC spectrum also showed the correlations of H-100 with C-5, C-6 and C-7, indicating that the ring was attached to the A ring by C-6 and C-7. Thus, 6 was established as 2-(3,4-dihydroxy-2-(3-methylbut-2-en-1-yl)phenyl)-5,7-dihydroxy-3-methoxy-8,8-dimethyl-7,8-dihydropyrano[3,2-g]chromen-4(6H)-one and named dysosmaflavone D. Compound 7 was obtained as a yellowish oil, with the molecular formula of C26H28O8, as deduced from high-resolution analysis. Careful comparison of the NMR spectra of 7 with 6 indicated a similar flavone skeleton. The difference between the spectra of 7 and 6 was that the 3-methyl-2-butenyl group was assigned to C-6 based on the HMBC correlations of H-100 with C-5, C-6 and C-7; however, the 3,4-dihydroxy-2,2-dimethyl-pyran ring was attached to the B ring by C-20 and C-30 , as deduced by the correlations of the methylene proton H-1000 with C-10, C-20 and C-30 (Fig. 3). Thus, 7 was established as 2-(3,8-dihydroxy-2,2-dimethylchroman-5-yl)-5,7-dihydroxy-3-methoxy-6-(3-methylbut-2en-1-yl)-4H-chromen-4-one and named dysosmaflavone E.

Table 3 13 C (125 MHz) NMR Data for Compounds 3–8 (d in ppm). Position

3

4

5

6

7

8

2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 1” 2” 3” 4” 5” 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 3-OCH3

160.3 139.1 178.7 161.7 98.4 164.5 93.4 157.4 104.8 121.5 128.1 143.3 146.9 111.7 121.2 25.3 130.6 122.7 24.2 16.4

161.9 139.3 178.4 161.8 98.5 164.6 93.4 157.5 104.8 117.9 120.8 140.2 147.7 122.1 114.8 119.8 131.6 75.8 26.1 26.1

59.5

59.5

160.1 139.0 178.7 159.1 108.6 163.1 93.0 155.6 104.5 121.6 128.0 143.3 147.1 111.7 121.2 28.1 74.8 147.1 109.7 16.2 25.3 122.6 130.6 16.4 24.3 59.5

158.7 138.9 178.7 159.5 104.8 160.6 94.4 155.2 103.5 121.5 128.1 143.3 146.9 111.7 121.5 24.6 78.5 67.9 20.0 24.2 25.4 122.7 130.6 16.4 24.2 59.5

158.4 139.3 178.5 158.3 111.5 162.4 92.5 155.3 104.6 120.1 121.1 140.9 147.9 112.5 121.9 20.8 121.9 130.7 16.4 24.5 29.0 68.7 77.0 19.4 24.1 59.8

159.4 139.2 178.6 158.4 111.5 162.3 92.6 155.2 104.6 122.2 126.0 144.1 147.1 112.8 121.6 20.8 121.9 130.7 24.5 16.5 34.4 76.3 147.8 109.7 16.4 59.7

Data were recorded in CD3OD at 125 MHz for

13

C NMR.

Compound 8 was obtained as a yellowish oil, molecular formula (C26H28O8), ascertained via high-resolution analysis, which indicated 13 of unsaturation. The flavone skeleton was deduced as in 5. The difference between the spectra of 8 and 7 was that the 4dihydroxy-2,2-dimethyl-pyran ring was replaced by a hydroxy-3methyl-3-butenyl group, and the group was assigned to C-20 based on the HMBC correlations of H-1000 with C-10, C-20 and C-30 . Thus, 8 was established as 2-(3,4-dihydroxy-2-(2-hydroxy-3-methylbut3-en-1-yl) phenyl)-5,7-dihydroxy-3-methoxy-6-(3-methylbut-2en-1-yl)-4H-chromen-4-one and named dysosmaflavone F (Fig. 4). Other known compounds were identified by the comparison of their NMR data to previous reports, including podophyllotoxin (9)

Table 2 1 H (500 MHz) NMR Data for Compounds 3–8 (d in ppm, J values in Hz). Position

3

4

6

6.28 d (1.9)

6.30 d (1.5) 6.21 d (1.5) 6.75 d (8.2) 6.81 d (8.2) 6.27 d (10.0) 5.77 d (10.0)

8 5

6.21 d (1.9) 0

6.75 d (8.2)

60

6.81

1”

3.40 t (7.0)

2” 3” 4” 5” 10 0 0 20 0 0 40 0 0 50 0 0 3–OCH3

d (8.2)

5.06 ddd (6.9, 5.5, 1.0) 1.47 d (1.0) 1.47 s 1.36 s 1.47 s

3.57 s

3.57 s

5

6

7

8

6.32 s

6.32 s

6.36 s

6.35 s

6.74 d (8.2)

6.74 d (8.2)

6.78 d (8.2)

6.80 d (8.2)

6.81 d (8.2)

6.81 d (8.2)

6.97 d (8.2)

6.87 d (8.2)

3.03 dd (13.4, 6.5), 2.90 dd (13.4, 6.9) 4.43 t (6.9, 6.5)

2.65 dd (17.0, 6.8), 2.96 dd (17.0, 5.3) 3.83 dd (6.8, 5.3)

3.34 m

3.34 m

5.26–5.20 m

5.23 t (7.1, 7.1)

4.69 s, 4.74 s 1.83 s 3.39 d (6.7)

1.32 s 1.36 s 3.39 m

5.05 m 1.36 s 1.47 s 3.56 s

5.06 m. 1.37 s 1.48 s 3.57 s

1.78 s 1.66 s 2.94 dd (17.1, 5.3), 2.69 dd (17.1, 7.3) 3.76 dd (7.3, 5.3) 1.32 s 1.40 s 3.58 s

1.66 s 1.59 s 2.71 dd (14.1, 3.6), 2.92 dd (14.1, 8.8) 4.37 dd (8.8, 3.6)

Data were recorded in CD3OD at 500 MHz for 1H; J in Hz within parentheses.

1.78 s 3.59 s

Y. Zheng et al. / Phytochemistry Letters 16 (2016) 75–81

79

Fig. 4. Selected 2D correlations of dysosmaflavones A and E (3 and 7).

(Zhao et al., 2008), epipodophyllotoxin (10) (Engelhardt et al., 2003), picropodophyllin (11) (Zhao et al., 2008), podophyllotoxone (12) (Jiang et al., 2011), picropodophyllone (13) (Pullockaran et al., 1989), deoxypodophyllotoxin (14) (Middel et al., 1995), 40 demethylpodophyllotoxin (15) (Zhan et al., 2013), 40 -demethylepipodophyllotoxin (16) (Xiao et al., 2014), podophyllotoxin glucoside (17) (Jiang et al., 2011), picropodophyllotoxin glucoside (18) (Broomhead and Dewick, 1990), 4-demethylpicropodophyllotoxin-7-O-b-glucopyranoside (19) (Zhao et al., 2001), 600 -acetylpodophyllotoxin-7-O-b-D-glucopyranoside (20) (Zhao et al., 2001), b-apopicropodophyllin (21) (Zhang et al., 2010), 40 -demethyldeoxy-podophyllotoxin 4-O-b-D-glucopyranoside (22) (Iida et al., 2010), NC370 (23) (Wichers et al., 1991), diphyllin (24) (Jiang et al., 2011), diphyllin 4-O-b-D-glucoside (25) (Jiang et al., 2011), 70 methoxylariciresinol (26) (Sun et al., 2012), dysosmarol (27) [2], busaliol (28) (Estévez-Braun et al., 1995), lantibetin (29) (Li et al., 2008), kaempferol (30) (Canzi et al., 2014), kaempferide (31) (Sasaki et al., 2014), p-hydroxybenzaldehyde (32) (Liu et al., 2005), 4-ethozybenzoic acid (33) (Chang et al., 2007), tyrosol (34) (Kwak et al., 2009), ethyl 3,4-dihydroxybenzoate (35) (Cheng et al., 2009), (6R,9R) 9-hydroxy-4-megastigmen-3-one (36) (Dabrosca et al., 2004), vomifoliol (37) (Zhang et al., 2000), and 5,7-di-t-butyl-1,4benzodioxin (38) (Kashima et al., 1987). We evaluated the antibacterial activities of compounds 18 along with two known flavonoids against Riemerella anatipestifer CH1, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 19433, Pseudomonas aeruginosa CI 1011, Aeromonas hydrophila CI 1004, Streptococcus agalactiae ATCC 13813 and Streptococcus suis ATCC 43765 (see SI). Ciprofloxacin was used as a positive control. Only dysosmaflavones E and F (7 and 8) exhibited mild antibacterial activities against S. agalactiae, P. aeruginosa and B. subtilis, with MIC values ranging from 93.0 to 93.8 mg/mL 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Jasco P-2000 Polarimeter. IR spectra were obtained on a Bruker FTIR Vector 22 spectrometer. UV spectra were obtained on a Shimadzu UV-2550 spectrometer. 1 H and 13C NMR spectra were recorded on Bruker Avance500 spectrometers. ESIMS spectra were acquired on an Agilent 1100 series mass and Autospec-Ultima ETOF apparatus, and HRESIMS spectra were measured on a Q-TOF micro-mass spectrometer (Waters, USA). A preparative column (Shimadzu PR C-ODSEV0233, C18, 5 mm, 300  10 mm, 500 mL/inj.) was used for preparative HPLC (Shimadzu LC-6AD). TLC analysis was performed on HSGF254 silica gel plates (10–40 mm, Yantai, China). Column chromatography (CC) was performed on silica gel (100– 200, 200–300 mesh, Yantai, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden).

3.2. Plant material Roots of D. versipellis were collected in Guizhou Province in China in October 2013 and identified by Prof. Han Ming Zhang, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University. An authentic specimen (No. 2013BJL) was deposited at the School of Pharmacy, Shanghai Jiao Tong University. 3.3. Extraction and isolation The roots of D. versipellis (10.0 kg) were chopped and extracted with 95% EtOH at room temperature under reflux for 3  12 h. The combined EtOH extracts were concentrated in vacuo to give the crude material (1684.1 g), which was then successfully portioned using CH2Cl2 and EtOAc. The CH2Cl2 fraction (773.8 g) was subjected to CC on silica gel and eluted with a CH2Cl2/MeOH gradient (100/1; 50/1; 20/1; 10/1; 5/1; 2:1; 1:1) to obtain nineteen fractions (Fr. 1–Fr. 19). Fr. 9 (2.7 g) was subjected to MIC and Sephadex LH-20 (MeOH) to yield three subfractions (Fr. 9.1–9.3). Compound 30 (31.0 mg) was obtained from Fr. 9.1 (637.7 mg) by preparative HPLC (ACN/H2O, 50:50). Compounds 1 (31.0 mg), 2 (10.3 mg), and 34 (22.5 mg) were obtained from Fr. 9.2 (755.0 mg) by preparative HPLC (ACN/H2O, 20:80). Compounds 3 (17.2 mg), 4 (10.1 mg), 5 (38.1 mg), 6 (1.5 mg), 7 (6.1 mg), 8 (7.5 mg), and 14 (33.6 mg) were obtained from Fr. 9.3 (781.8 mg) by preparative HPLC (MeOH/H2O, 50:50). Fr. 10 (9.9 g) was subjected to silica gel CC with an elution gradient (CH2Cl2/MeOH, 100:1–10:1) to give four subfractions (Fr. 10.1–10.4). Compounds 12 (54.8 mg), 13 (69.9 mg), and 29 (10.0 mg) were separated from Fr. 10.1 (900 mg) by preparative HPLC (MeOH/H2O, 45:55). Fr. 10.3 (2.6 g) was separated on a Sephadex LH-20 column (MeOH) to obtain three subfractions (Fr. 10.3.1–10.3.3). Compound 36 (4.0 mg) was isolated from Fr. 10.3.1 (205.5 mg) by preparative HPLC (ACN/H2O, 35:65). Compound 23 (4.0 mg) was isolated from Fr. 10.3.2 (608.2 mg) by preparative HPLC (MeOH/H2O, 55:45). Compounds 9 (150.0 mg) and 10 (301.0 mg) were obtained from Fr. 11 (20.8 mg) by silica gel CC (eluting with CH2Cl2/EA, 50:1) and purified by recrystallization. Fr. 15 (5.7 g) was separated over Sephadex LH-20 (MeOH), followed by preparative HPLC (MeOH/H2O, 70:30) to yield 16 (80.0 mg). Fr. 16 (3.8 g) was separated by CC (SiO2, PE/EA, 10:1–1:1) to obtain three subfractions (Fr. 16.1–16.3), of which Fr. 16.2 (980.0 mg) was subjected to preparative HPLC (MeOH/H2O, 70:30) to yield 15 (15.0 mg). Compounds 20 (8.8 mg), 22 (26.9 mg), and 27 (10.6 mg) were obtained from Fr. 18 (1.7 g) by preparative HPLC (MeOH/H2O, 60:40). Compound 17 (8.8 mg) was obtained from Fr. 19 (2.3 g) by preparative HPLC (ACN/MeOH/H2O, 25:5:70). The EtOAc extract (106.4 g) was subjected to silica gel CC with an elution gradient (CH2Cl2/MeOH, 100:1–1:1) to afford ten fractions (Fr. E.1–E.10). Compound 38 (18.9 mg) was obtained from Fr. E.1 (537.8 mg) by preparative HPLC (MeOH/H2O, 90:10). Fr. E.2 (3.1 g) was separated

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by CC (SiO2, CH2Cl2/MeOH, 50:1–1:1) to obtain three subfractions (Fr. E.2.1–E.2.3), of which Fr. E.2.2 (980.0 mg) was subjected to preparative HPLC (MeOH/H2O, 50:50) to yield compound 31 (16.7 mg). Fr. E.5 (3.1 g) was separated on a Sephadex LH-20 column (MeOH) to obtain three subfractions (Fr. E.5.1–E.5.3). Compound 21 (10.6 mg) was obtained from Fr. E.5.1 (500 mg) by preparative HPLC (MeOH/H2O, 55:45). Compound 37 (7.1 mg) was obtained from Fr. E.5.2 (960 mg) by preparative HPLC (MeOH/H2O, 45:55). Fr. E.6 (5.8 g) was separated by CC (SiO2, CH2Cl2/MeOH, 50:1–10:1) to obtain three subfractions (Fr. E.6.1–E.6.3), of which Fr. E.6.1 (670.0 mg) was subjected to preparative HPLC (MeOH/H2O, 55:45) to yield 11 (290.6 mg). Compounds 26 (8.9 mg) and 24 (4.8 mg) were obtained from Fr. E.7 (620 mg) by preparative HPLC (MeOH/H2O, 40:60). Fr. E.10 (15 g) was purified using silica gel CC (eluting with PE/EA, 10:1–2:1) and Sephadex LH-20 (MeOH) to give three subfractions (Fr. E.10.1–Fr. E.10.3). Fr. E.10.1 (600 mg) and Fr. E.10.3 was fractioned using Sephadex LH-20 (MeOH) and preparative HPLC. These separations resulted in compounds 28 (13.2 mg), 18 (185.2 mg), 19 (72.9 mg) and 25 (6.2 mg).

concentrations (MICs) were determined by serial dilution in 96well culture plates, performed according to the guidelines published by the Clinical and Laboratory Standards Institute. The tested substances were dissolved in DMSO. Ciprofloxacin (Sigma, Shanghai, China) was used as a positive control. Acknowledgment This work was supported by the Scientific Foundation of Shanghai Committee of Science and Technology (12401900501) and partially supported by the Global Research Network for Medicinal Plants (GRNMP) and King Saud University, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2016.03.001.

3.4. Structural elucidation of new products References Dysosmanorlignan A (1). Yellow amorphous powder, IR (KBr)  nmax 2961, 1592, 1125 cm1; ½a25 D + 80 (c 0.1, MeOH); UV (MeOH): lmax (log e) = 218 (2.45), 246 (2.13) nm; 1H and 13C NMR data, see

Table 1. HRESIMS (positive) 451.1364 [M+Na]+ (calc. for C23H24NaO8, 451.1363). Dysosmanorlignan B (2): Yellow amorphous powder, IR (KBr)  nmax 2961, 1592, 1125 cm1; ½a25 D 50 (c 0.1, MeOH); UV (MeOH): lmax (log e) = 210 (1.24), 246 (2.13) nm; 1H and 13C NMR data, see

Table 1. HRESIMS (positive) 451.1364 [M+Na]+ (calc. for C23H24NaO8, 451.1363). Dysosmaflavone A (3): Yellow oil; IR (KBr) nmax 3403, 1652, 1600 cm1; UV (MeOH): lmax (log e) = 213 (3.12) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 383.1154 [MH] (calc. for C21H19O7, 383.1136). Dysosmaflavone B (4): Yellow oil; IR (KBr) nmax 3403, 1652, 1600 cm1; UV (MeOH): lmax (log e) = 212 (3.05) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 383.1131 [M+Na]+ (calc. for C21H18O7, 383.1126). Dysosmaflavone C (5): Yellow oil; IR (KBr) nmax 3391, 1648, 1615 cm1; UV (MeOH): lmax (log e) = 219 (3.38) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 469.1870 [M+Na]+ (calc. for C26H29O8, 469.1857). Dysosmaflavone D (6): Yellow oil; IR (KBr) nmax 3422, 1651, 1605 cm1; UV (MeOH): lmax (log e) = 214 (3.30) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 469.1866 [M+Na]+ (calc. for C26H29O8, 469.1857). Dysosmaflavone E (7): Yellow oil; IR (KBr) nmax 3403, 1648, 1611 cm1; UV (MeOH): lmax (log e) = 218 (3.38) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 469.1874 [M+Na]+ (calc. for C26H29O8, 469.1857). Dysosmaflavone F (8): Yellow oil; IR (KBr) nmax 3395, 1648, 1610 cm1; UV (MeOH): lmax (log e) = 221 (3.39) nm; 1H and 13C NMR data, see Table 1; HRESIMS (positive) m/z 469.1868 [M+Na]+ (calc. for C26H29O8, 469.1857). 3.5. Anti-bacterial assay The standard bacterial strains Riemerella anatipestifer CH1, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 19433, Pseudomonas aeruginosa CI 1011, Aeromonas hydrophila CI 1004, Streptococcus agalactiae ATCC 13813, and Streptococcus suis ATCC 43765 (see Table 1 in SI) were obtained from the Shanghai Veterinary Research Institute. The minimum inhibitory

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