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Five new bioactive compounds from Chenopodium ambrosioides a

b

ac

a

Kun Song , Jian Zhang , Peng Zhang , Hong-Qing Wang , Chao a

a

a

a

Liu , Bao-Ming Li , Jie Kang & Ruo-Yun Chen a

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China b

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Department of Cell and Molecular Biology, Research Institute of Orthopedics & Traumatology, Foshan Hospital of TCM, Foshan 528000, China c

Department of Traditional Chinese Medicinal Chemistry, Beijing University of Chinese Medicine, Beijing 100102, China Published online: 22 May 2015.

To cite this article: Kun Song, Jian Zhang, Peng Zhang, Hong-Qing Wang, Chao Liu, Bao-Ming Li, Jie Kang & Ruo-Yun Chen (2015): Five new bioactive compounds from Chenopodium ambrosioides, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1042872 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042872

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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1042872

Five new bioactive compounds from Chenopodium ambrosioides Kun Songa, Jian Zhangb, Peng Zhanga,c, Hong-Qing Wanga, Chao Liua, Bao-Ming Lia, Jie Kanga* and Ruo-Yun Chena* a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; bDepartment of Cell and Molecular Biology, Research Institute of Orthopedics & Traumatology, Foshan Hospital of TCM, Foshan 528000, China; cDepartment of Traditional Chinese Medicinal Chemistry, Beijing University of Chinese Medicine, Beijing 100102, China

Journal of Asian Natural Products Research

(Received 1 April 2015; final version received 15 April 2015) Five new bioactive compounds, chenopodiumamines A – D (1 – 4) and chenopodiumoside A (5), were isolated from the ethanol extract of Chenopodium ambrosioides. The structures of these compounds were elucidated by various spectroscopic means (UV, IR, HR-ESI-MS, 1D and 2D NMR). Compounds 1– 3 had moderate antioxidant and anti-inflammatory activities. Keywords: Chenopodiaceae; Chenopodium ambrosioides; chenopodiumamines A– D; chenopodiumoside A; antioxidant activity; anti-inflammatory activity

1. Introduction Chenopodium ambrosioides, a species of Chenopodiaceae, is a plant which originally grows in tropical America and now distributes broadly throughout the temperate zone to the tropical areas around the world [1]. It has been used as a traditional herbal drug to treat various diseases including rheumatism pain, hookworms, ascariasis, dysmenorrheal, amenorrhea, eczema, and diseases induced by insect and snake bite [1]. It is also one of the major ingredients in Chinese herb compound named “Jing Huang Wei Kang soft capsule”, which is clinically used for the treatment of gastrosia and duodenal ulcer diseases. Previous investigations were mainly focused on the volatile components of the herb [2 –6], while studies targeted at the nonvolatile part were still a few [7,8]. Five new bioactive compounds were obtained after our ongoing effort to search for the bioactive nonvolatile compounds, and some of them had antioxidant and anti-

inflammatory activities. The structures were determined on the basis of extensive spectroscopic data analysis (Figure 1).

2. Results and discussion Compound 1 was obtained as white amorphous powder. The molecular formula C23H31NO5 of compound 1 was determined by HR-ESI-MS at m/z 424.2079 [M þ Na]þ. The IR spectrum showed the presence of hydroxyl (3353 cm21), carbonyl (1644 cm21), and aromatic ring (1514 and 1453 cm21) functional groups. The UV absorption maxima were observed at 279 and 225 nm. In the 1H NMR spectrum of compound 1, two groups of aromatic proton signals were observed and could be attributed to a 1,2,4-trisubstituted benzene moiety [dH: 6.90 (1H, br s), 6.79 (1H, br d, J ¼ 8.0 Hz), 6.77 (1H, d, J ¼ 8.0 Hz)] and a 1,4-disubstituted benzene moiety [dH 7.10 (2H, d, J ¼ 8.0 Hz), 6.74 (2H, d, J ¼ 8.0 Hz)], respectively. In addition,

*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis

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K. Song et al. 14

13 O

15

OH

O

3

H3CO

8

HO

N H

9

H3CO HO

1

11

6

9

8

12

1

11

HO

OH

OH O

3

1'

8' 8

1

11

9

N H

7'

4'

12

3

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4'

OCH3 9'

10

O –O

5

7 2

1N CH3 14

13

12

4 3

O O

7'

2 9

13

N H

12

1

H3CO

1'

8'

3

4'

7'

OH

OH O

13

1'

8'

1

OH

8

HO O 5'

4' 3'

1' 2'

OH OH 5

Figure 1. The structures of compounds 1 – 5.

three methines [dH 4.48 (d, J ¼ 8.5 Hz), 2.35 (dd, J ¼ 8.5, 6.0 Hz), 1.60 (m)], three methylenes [dH 3.38 (2H, m), 3.29 (2H, m), 2.73 (2H, m)], one aromatic methoxyl at dH 3.87 (3H, s) and three methyls at dH 1.10 (3H, t, J ¼ 7.0 Hz), 0.91 (3H, d, J ¼ 7.0 Hz), and 0.85 (3H, d, J ¼ 7.0 Hz) were also observed in 1H-NMR spectrum. According to the coupling constants, one of three methyls should be connected with a methylene and the other two should attach to a methine, respectively. The 13C NMR and HSQC spectra of compound 1 provided assignments of the protons and protonated carbons in the NMR spectrum, which exhibited 6 quaternary carbon signals, 10 tertiary carbons, 3 secondary carbons, 3 methyl carbons, and 1 methoxy group. In the HMBC spectrum, correlations of H-80 / C-9, 70 , 40 , and H-7/C-3, 5, 9 indicated the connection order of the main chain of compound 1 (Figure 2) [9]. The correlations

of H-8/C-11,12 and H-10/C-9 confirmed an isopropyl moiety was linked to C-8, and the correlations of H-13/C-7 and H-14/C-13 determined an ethoxyl was located at C-7 (Figure 2). In addition, the position of the methoxyl was established by an NOE experiment. Irradiation of the methoxyl signal at dH 3.87 enhanced the aromatic proton signal at dH 6.90 (br s, H-3), which demonstrated that the methoxyl was at C-2 (Figure 2). Therefore, the structure of 1 (chenopodiumamine A) was determined as shown in Figure 1. Compound 2 was obtained as white amorphous powder. The molecular formula C22H29NO6 of compound 2 was determined by HR-ESI-MS at m/z 426.1892 [M þ Na]þ. The IR spectrum showed the presence of hydroxyl (3357 cm21), carbonyl (1633 cm21), and aromatic ring (1517 and 1454 cm21) functional groups. The UV absorption

Journal of Asian Natural Products Research

O

O

H3CO

OH

OH

OH

O

H3CO N H

3

N H

OCH3

HO

HO 2

1

OH H3CO

OH

O

O O

N H

N

HO

CH3

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3

4

O OH O HO

O

OH OH 5

Figure 2. The key HMBC (

) and NOE (

maximum was observed at 280 and 228 nm. The 1H and 13C NMR spectroscopic data (Table 1) of compound 2 were similar to those of compound 1, except one more methoxyl substituted at C-20 , and a hydroxyl group replacing an ethoxy group at C-7. The HMBC correlations of H-3/C1, OH-1/C-1,2,6 implied that a methoxyl was located at C-2. In the same way, H-30 / C-10 , OH-10 /C-10 ,20 ,60 suggested that the other methoxyl was located at C-20 . Therefore, the structure of 2 (chenopodiumamine B) was determined and shown in Figure 1. Compound 3 was obtained as white amorphous powder and had the molecular formula C21H27NO5, as established by HR-ESI-MS at m/z 396.1782 [M þ Na]þ. The UV and IR spectroscopic features of 3 were similar to those of 1. The 1H and13C NMR spectroscopic data (Table 1) of

) correlations of compounds 1 – 5.

compound 3 were also similar to those of 1, except no signals of ethoxyl were observed in the NMR spectra of 3. Combined with the analysis of the data of HR-ESI-MS, it was demonstrated that a hydroxyl group was at C-7 in 3 instead of an ethoxy group at the same position in 1. In addition, the substituting position of the methoxyl determined by HMBC correlations of H-3/C-1,5, H-6/C-4,2, and H313/C-2 indicated that a methoxyl was located at C-2. Consequently, the structure of 3 (chenopodiumamine C) was elucidated as shown in Figure 1. The relative configurations of H-7 and H-8 of compounds 1– 3 have not been determined yet due to no corresponding NOE found. Compound 4 was obtained as white amorphous powder. The HR-ESI-MS ion at m/z 222.1486 [M þ H]þ indicated a

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K. Song et al.

Table 1.

1

H and 13C NMR spectral data of compounds 1 – 3. 1a

No. 1 2 3 4 5 6 7 8 9 10 11

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12 13 14 15 10 20 30 40 50 60 70 80 90 1OH 10 OH 7OH – NH

1

H

2b 13

1

C

3c 13

H

146.0 147.8 6.90 (br s) 110.2 6.84 (br s) 131.7 6.79 (br d, J ¼ 8.0 Hz) 120.1 6.68 (br d, J ¼ 8.0 Hz) 6.77 (d, J ¼ 8.0 Hz) 114.5 6.69 (d, J ¼ 8.0 Hz) 4.48 (d, J ¼ 8.5) 81.0 4.68 (t, J ¼ 7.0 Hz) 2.35 (dd, J ¼ 8.5, 59.9 2.17 (t, J ¼ 7.0 Hz) 6.0 Hz) 174.0 1.59– 1.61 (m) 27.3 1.64 – 1.66 (m) 0.85 (3H, d, 20.5 0.87 (3H, d, J ¼ 7.0 Hz) J ¼ 7.0 Hz) 0.91 (3H, d, 18.1 0.82 (3H, d, J ¼ 7.0 Hz) J ¼ 7.0 Hz) 3.29 (2H, m) 63.8 3.74 (3H, s) 1.10 (3H, t, 14.2 J ¼ 7.0 Hz) 3.87 (3H, s) 55.0 155.6 6.74 (d, J ¼ 8.0 Hz) 114.8 7.10 (d, J ¼ 8.0 Hz) 129.3 6.74 (d, J ¼ 1.5 Hz) 130.0 7.10 (d, J ¼ 8.0 Hz) 129.3 6.52 (dd, J ¼ 8.0,1.5 Hz) 6.74 (d, J ¼ 8.0 Hz) 114.8 6.66 (d, J ¼ 8.0 Hz) 2.72– 2.74 (2H, m) 34.5 2.49 – 2.51 (2H, m) 3.37– 3.39 (2H, m) 40.8 3.17 – 3.19 (2H, m) 3.74 (3H, s) 8.77 (s)

1

C

H

13

C

145.8 147.6 110.8 6.81 (d, J ¼ 1.8 Hz) 135.9 119.2 6.64 (dd, J ¼ 8.4, 1.8 Hz) 115.3 6.67 (d, J ¼ 8.4 Hz) 72.2 4.65 (t, J ¼ 6.0 Hz) 59.3 2.13 (t, J ¼ 6.0 Hz)

145.3 147.1 110.3 135.4 118.6

173.5 27.7 1.63– 1.65 (m) 21.7 0.78 (3H, d, J ¼ 7.2 Hz) 19.2 0.85 (3H, d, J ¼ 7.2 Hz) 55.0 3.73 (3H, s)

173.0 27.2 19.6

145.2 147.8 6.62 (d, J ¼ 8.4 Hz) 113.3 6.89 (d, J ¼ 8.4 Hz) 130.8 121.1 6.89 (d, J ¼ 8.4 Hz)

155.5 115.0 129.3 129.5 129.3

115.8 6.62 (d, J ¼ 8.4 Hz) 35.4 2.43– 2.45 (2H, m) 40.7 3.10– 3.12 (2H, m) 55.9 9.13 (s)

115.0 34.4 40.2

8.66 (s)

8.75 (s)

5.26 (d, J ¼ 7.0 Hz)

5.23 (d, J ¼ 6.0 Hz)

7.72 (t, J ¼ 6.0 Hz)

7.71 (t, J ¼ 5.4 Hz)

114.8 71.6 58.8

21.1 55.5

a

500 MHz for 1H and 125 MHz for 13C in CD3OD. 500 MHz for 1H and 125 MHz for 13C in DMSO-d6. c 600 MHz for 1H and 150 MHz for 13C in DMSO-d6. b

C13H19NO2 molecular formula. The IR spectrum showed the presence of a carbonyl group (1639 cm21) and pyridine ring (1611, 1458 cm21). The UV absorption maximum was at 205 and 273 nm. In the 1H NMR spectrum, two sets of isopropyl proton signals [dH 4.09 (m), 1.24 (6H, d, J ¼ 7.0 Hz), and 3.44 (overlapped),

1.35 (6H, d, J ¼ 6.5 Hz)], a methyl proton signal [dH 4.22 (3H, s)], and two aromatic proton signals [dH 8.76 (s), 7.73 (s)] were observed. The 13C NMR and HSQC spectra indicated the presence of a carbonyl group (dC 165.0), three aromatic quaternary signals (dC 164.9, 160.7, 139.8), two aromatic methine signals (dC

Journal of Asian Natural Products Research

Journal of Asian Natural Products Research 145.2, 121.8), five methyls (dC 44.5, 22.7, 22.7, 21.8, 21.8), and two aliphatic methine carbons (dC 30.0, 29.8). The HMBC correlations of H-14/C-2, 6 together with the chemical shifts suggested that the methyl group was connected with a nitrogen atom of the pyridine ring. Meanwhile, the correlations of H-2/C-7 indicated that the carbonyl group was located at C-3. Thus it was concluded that compound 4 had the skeleton of trigonelline [10]. The correlations of H-8/C-3,4,5 and H-11/C-5,6 indicated that two isopropyl groups were located at C-4 and C-6, respectively. Thus, the structure of compound 4 (chenopodiumamine D) was elucidated as shown in Figure 1. Compound 5 was obtained as white amorphous powder. The molecular formula C13H16O7 of compound 5 was determined by HR-ESI-MS at m/z 307.0794 [M þ Na]þ. The IR spectrum showed the presence of hydroxyl (3355 cm21), carbonyl (1684 cm21), and aromatic ring (1602 and 1561 cm21) functional groups. The UV absorption maximum was at 278 and 216 nm. In the 1 H NMR spectrum of compound 5, a group of aromatic proton signals were observed and attributed to a 1,4-disubstituted benzene moiety at dH 8.09 (2H, d, J ¼ 8.5 Hz), 7.13 (2H, d, J ¼ 8.5 Hz), and a characteristic AB spin system [dH 5.16 (1H, d, J ¼ 16.0 Hz) and 5.04 (1H, d, J ¼ 16.0 Hz)] showed the presence of an oxygenated methylene unit; the other six proton resonances from dH 4.18 to dH 5.77 indicated the existence of a pentose moiety. In addition, the presence of the anomeric proton at 5.77 (br s) indicated an almost planar conformation of the furanose ring, thus the relative configuration of H-10 was b [11]. The HSQC and 13C NMR spectra of compound 5 showed two secondary carbons [dC 75.5 (C-40 ), 65.4 (C-50 )], two tertiary carbons [dC 110.6 (C10 ), 77.9 (C-20 )], and one quaternary carbon [dC 80.7 (C-30 )] signals of pentose moiety, suggesting the presence of an

5

apiose moiety according to the literature [11,12]. In the HMBC spectrum, correlations of H-3/C-7,1 showed the carbonyl group connected to C-4. The correlations of H-8/C-7,10 revealed the apiose moiety connected with C-8 of the aglycone. Therefore, the structure of 5 (chenopodiumoside A) was determined as shown in Figure 1. The antioxidant and anti-inflammatory activities of 1–3 were tested, and the results showed that 1 – 3 exhibited moderate antioxidant activities. The inhibitory rates of the three compounds (1–3) and positive control (Vit E) against malondialdehyde (MDA) were 61.1%, 60.9%, 59.1%, and 98% at a concentration of 1024 M, respectively. Moreover, compounds 1–3 showed significant inhibitory effects against LPSinduced TNF-a or IL-6 gene expressions by 64%, 47%, and 66%, or 84%, 65%, and 87%, respectively, at the concentration of 50 mg/ml (Figure 3). However, they failed to show significant inhibitory effects at 5 mg/ml (Figure 3), and only showed a slight tendency. The results suggested that compounds 1–3 had moderate anti-inflammatory activities against LPS-induced TNF-a and IL-6 gene expression. Anti-oxidative and anti-inflammatory activities are provided to decrease oxidative stresses in cells, so that inflammation diseases, such as rheumatoid arthritis and gastric duodenal ulcers, are prevented or treated [13,14]. Increased intake of antioxidants may decrease freeradical damage to joint linings, which diminish swelling and pain [14]. Numerous studies have implicated that TNF-a and IL6 are important inflammatory cytokines that modulate the generation and amplification of the immune response involved in inflammatory diseases [15]. Compounds 1 –3 showed moderate antioxidant and anti-inflammatory activities, which partly revealed the reason that C. ambrosioides were used to treat gastrosia, duodenal ulcer diseases, or rheumatism pain in China for a long time [1].

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Figure 3. Inhibitory effects of compounds 1 – 3 (5 or 50 mg/ml) in the gene expression of proinflammatory cytokine TNF-a and IL-6 induced by LPS in the macrophages (means ^ SD, n ¼ 3) (*P , 0.05).

3.

Experimental

3.1. General experimental procedures The optical rotations were measured by Jasco P2000 polarimeter (JASCO, Tokyo, Japan). IR spectra were carried out on a Nicolet 5700 spectrophotometer with KBr disks (Thermo, Waltham, MA, USA). UV spectra were determined by a Jasco V650 spectrophotometer (JASCO, Tokyo, Japan). 1H NMR, 13C NMR, NOE, and 2D NMR spectra were recorded with an INOVA (500 and 600 M) spectrometer (Varian, Palo Alto, CA, USA) using TMS as internal standard, and values are given in ppm. HR-ESI-MS was performed on an Agilent 1100 LC/MSD Trap-SL mass spectrometer (Agilent, Santa Clara, CA, USA). Sephadex LH-20 (Pharmacia, Uppsala, Sweden), silica gel (Qingdao Marine Chemical Factory, 200– 300 mesh, Qingdao, China), and RP-C18 (Merck, 40– 60 mm, Darmstadt, Germany) were used for column chromatography, and silica gel GF-254 (Qingdao Marine Chemical Factory, Qingdao, China) was used for TLC. 3.2. Plant material The herb of C. ambrosioides was provided by the medicinal material base of Tian Shi

Li Company in Shanxi Province in 2011 and was authenticated by Associate Professor Lin Ma, Institute of Material Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. A voucher specimen (No. 99054) has been deposited at the Herbarium of Institute of Material Medica, Chinese Academy of Medical Sciences & Peking Union Medical College.

3.3. Extraction and isolation The air-dried powder of C. ambrosioides (31.6 kg) was extracted with 95% EtOH for three times under reflux. After evaporation of the solvents under reduced pressure, the residue (2076 g) was suspended in H 2O, and extracted with petroleum ether, chloroform, EtOAc, and n-butyl alcohol successively. The chloroform fraction (185 g) was chromatographed over a silica gel column and eluted with a petroleum ether– EtOAc gradient eluent (v/v 85:15 to 0:100). Fraction 4 (20 g) was then chromatographed over a silica gel column, eluted with CH2Cl2 –CH3OH (v/v 100:1, 80:1, 60:1, 40:1, 20:1, 0:1) to give 20 subfractions. Sub-fraction 7 (1.5 g) was

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further subjected to a C-18 column, eluted with an isocratic eluent (MeOH – H2O, ¼ 40:60) to yield compounds 1 (15 mg) and 3 (6 mg). Sub-fraction 14 (1.8 g) was purified by Sephadex LH-20, eluted with MeOH to yield compound 2 (6 mg). The n-butylalcohol fraction (240 g) was chromatographed over a D101 macroporous resin column and was eluted by ethanol – H2O (v/v 0:100, 30:70, 50:50, 70:30, 95:5). Sub-fraction 2 (30 g) was chromatographed over a C-18 column (50 mm, 500 g), eluted with an isocratic eluent (MeOH –H2O, 3:7) to give compounds 4 (8 mg) and 5 (7 mg). 3.3.1. Chenopodiumamine A (1) White amorphous powder; ½a20 D 2 9.2 (c 0.12, MeOH); UV (MeOH) lmax (nm) (log 1) 225 (4.89), 279 (4.35); IR (KBr) nmax 3353, 3014, 1644, 1514, 1453 cm21; 1 H (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) spectral data (see Table 1); HRESIMS: m/z 424.2079 [M þ Na] þ (calcd for C23H31NO5Na, 424.2094). 3.3.2.

Chenopodiumamine B (2)

White amorphous powder; ½a20 D 2 7.2 (c 0.10, MeOH); UV (MeOH) lmax (nm) (log 1) 228 (4.03), 280 (3.65). IR (KBr) nmax 3357, 1633, 1517, 1454 cm21; 1H (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) spectral data see Table 1); HR-ESI-MS: m/z 426.1892 [M þ Na]þ (calcd for C22H29NO6Na, 426.1887). 3.3.3.

Chenopodiumamine C (3)

White amorphous powder; ½a20 D 2 6.7 (c 0.11, MeOH); UV (MeOH) lmax (nm) (log 1) 224 (3.82), 278 (3.33); IR (KBr) nmax 3337, 1613, 1515, 1452 cm21; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectral data see Table 1); HR-ESI-MS: m/z 396.1782

7

[M þ Na]þ (calcd for C21H27NO5Na, 396.1781). 3.3.4. Chenopodiumamine D (4) White amorphous powder; ½a20 D þ 4.4 (c 0.21, MeOH); UV (MeOH) lmax (nm) (log 1) 205 (4.37), 273 (3.40); IR (KBr) nmax 1639, 1611, 1458 cm21; 1H NMR (DMSO-d6, 500 MHz): d 8.76 (s, H-2), 7.73 (s, H-5), 4.22 (3H, s, H-14), 4.08– 4.10 (m, H-8), 3.44 (overlapped, H-11), 1.35 (6H, d, J ¼ 6.5 Hz, H-12, 13), 1.24 (6H, d, J ¼ 7.0 Hz, H-9, 10); 13C NMR (DMSO-d6, 125 MHz): d 165.0 (C-7), 164.9 (C-4), 160.7 (C-6), 145.2 (C-2), 139.8 (C-3), 121.8 (C-5), 44.5 (C-14), 30.0 (C-8), 29.8 (C-11), 22.7 (C-9, 10), 21.8 (C12, 13); HR-ESI-MS: m/z 222.1486 [M þ H] þ (calcd for C13H 20NO 2, 222.1489). 3.3.5.

Chenopodiumoside A (5)

White amorphous powder; ½a20 D 2 60.6 (c 0.15, MeOH); UV (MeOH) lmax (nm) (log 1) 216 (4.02), 278(4.04); IR (KBr) nmax 3355, 3278, 1684, 1602, 1561, 1246 cm 21; 1H NMR (pyridine-d5, 500 MHz): d 8.09 (2H, d, J ¼ 8.5 Hz, H3, 5), 7.13 (2H, d, J ¼ 8.5 Hz, H-2, 6), 5.77 (br s, H-10 ), 5.16 (d, J ¼ 16.0 Hz, H-8a), 5.04 (d, J ¼ 16.0 Hz, H-8b), 4.87 (s, H-20 ), 4.56 (d, J ¼ 9.5 Hz, H-40 a), 4.35 (d, J ¼ 9.5 Hz, H-40 b), 4.18 (2H, s, H-50 ); 13 C NMR (pyridine-d5, 125 MHz): d 194.3 (C-7), 164.1 (C-1), 131.1 (C-3, 5), 127.3 (C-4), 116.3 (C-2, 6), 110.6 (C-10 ), 80.7 (C-30 ), 77.9 (C-20 ), 75.5 (C-40 ), 70.5 (C-8), 65.4 (C-50 ); HR-ESI-MS: m/z 307.0794 [M þ Na] þ (calcd for C13H 16O 7Na, 307.0788). 3.4. Absolute configurations of the sugars The absolute configuration of apiose was determined according to a reported procedure [16]. Compound 5 (2.0 mg) was hydrolyzed by 1 M HCl (1 ml) at 1008C for

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K. Song et al.

2 h, then it was diluted with H2O and extracted with EtOAc (3 £ 5 ml). After the H2O layer was dried under vacuum, the residue was dissolved in pyridine (0.5 ml) containing L -cysteine methyl ester hydrochloride (2 mg) and heated at 608C for 2 h. o-Tolylisothiocyanate (2 ml) was then added and the mixture was heated at 608C for 2 h. The reaction mixture was directly analyzed by an Agilent 1260 HPLC with a DAD detector at 254 nm. The Apollo C-18 HPLC column (5 mm, 250 mm £ 4.6 mm, Alltima, Washington, DC, USA) was used at 358C, and eluted with a gradient of CH3CN – H2O (25:75) (flow rate of 0.8 ml/ min). The reaction condition for D -apiose was the same as described above and its retention time (33.05 min) was used for comparison with that of reaction mixtures. A peak at 33.15 min of the sugar derivatives from 5 coincided with the derivatives of D -apiose (33.05 min). 3.5. Antioxidant bioassay The antioxidant assay was carried out as reported previously [17]. In brief, 1.5 mg of microsomal protein in 1 ml of 0.1 M phosphate buffered saline (pH 7.4) was incubated with 0.2 mM cysteine and test samples at 378C for 15 min. Lipid peroxidation was initiated by addition of 0.05 mM FeSO4. After 15 min of incubation at 378C, 1 ml of 20% trichloroacetic acid was added to stop the reaction. To determine the content of MDA formation, the mixture was centrifuged at 3000 rpm for 10 min, and 1 ml of supernatant was incubated with 1 ml of 0.67% thiobarbituric acid at 1008C for 10 min. After cooling down, the absorbance was measured at 532 nm. The percentage of inhibition of the MDA formation was used to indicate the potency of the test samples and positive control.

3.6. Anti-inflammatory bioassay RAW 264.7 macrophage cell line was purchased from the cell bank of the

Chinese Academy of Science. As described previously [18], cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin (100 units/ml), streptomycin (100 mg/ml), and glutamine (4 mM). Cells were seeded in 12-well cell culture plates at appropriate density of cells per well. At 80% confluence, cells were pretreated in the presence of various concentrations of the indicated compounds for 2 h before lipopolysaccharide (LPS) stimulation. Then the cells were treated with or without 100 mg/ml LPS (Invitrogen, San Diego, CA, USA) for 18 h followed by collection of RNA for realtime polymerase chain reaction (PCR). Total RNA from cultured cells was extracted by using TRIzol (or TRI reagent) (MRC Inc., Cincinnati, OH, USA), according to a method described previously [19], followed by DNase digestion and column cleanup with Qiagen (Valencia, CA, USA) mini-columns. Reverse transcription was performed with the iScript cDNA synthesis kit from BioRad (Hercules, CA, USA). Real-time PCR was performed with SYBR Green and the 7500 Fast Sequence Detection System (Applied Bio systems, Foster City, CA, USA). TNF-a and IL-6 gene expression data were normalized to the housekeeping gene GAPDH. All primers for real-time PCR analysis were designed with Primer Express software 2.0.0 (Applied Bio systems) (Table S1).

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This work was financially supported by grants from National Science and Technology Major Projects for “Major New Drugs

Journal of Asian Natural Products Research innovation and Development” [grant number 2012ZX09103201-043].

Journal of Asian Natural Products Research

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Five new bioactive compounds from Chenopodium ambrosioides.

Five new bioactive compounds, chenopodiumamines A-D (1-4) and chenopodiumoside A (5), were isolated from the ethanol extract of Chenopodium ambrosioid...
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