Fitoterapia 97 (2014) 15–22
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Chemical constituents from the linseed meal Li Song, Xian-Fen Wang, Yan Wu, Wen-Yi He, Chun-Suo Yao ⁎, Jian-Gong Shi ⁎ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 22 March 2014 Accepted in revised form 13 May 2014 Available online 24 May 2014 Keywords: Linum usitatissimum L Linseed meal Linaceae Linustam
a b s t r a c t One megastigmane derivative 1, one methyl jasmonate glycoside derivative 2, and two C-28 steroids with 3β,5β-cis-dihydroxyl conformation 3 and 4, together with eight known compounds 5–12 were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L). Structures of 1–4 were elucidated by spectroscopic methods including NMR, HRESIMS, and Mo2(OAc)4-induced CD. The absolute configuration of 1 and 3 was determined by observing their induced circular dichroism after addition of Mo2(OAc)4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. The structure of 4a was corrected as 4 by an extensive analysis of its 1D and 2D NMR, in combination with the Mo2(OAc)4-induced CD in DMSO. The effect of all the isolates on nitric oxide (NO) generation by stimulated macrophages was evaluated, and none of them showed active. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Flaxseed (Linum usitatissimum L), a herbaceous plant belonging to Linaceae family, is an important industrial crop grown worldwide for its fiber and oilseed [1]. Flaxseed contains 35% of its mass as oil, of which 55% is α-linolenic acid (ω-3 fatty acid) and 15–18% is linoleic acid [2]. Concentrations of lignans up to 3% (w/w) have been reported in flaxseed, making flax one of the richest edible sources of lignans. In contrast to most plants in which free lignans are present, the lignans in flaxseeds are incorporated into an oligomeric structure. Flaxseed lignans showed multi-faced bioactivities, and attracted a lot of interests for the researchers all over the world. Besides lignans, flavonoids, cyclic peptides, hydroxyl cinnamic acids, cyanogenetic glucosides, pectic polysaccharides and other chemical constituents have been identified from flaxseed extracts [3,4]. There is an increasing interest in flaxseed in human nutrition as it gains popularity as a health food, a dietary supplement, and an ingredient in bread, muffins and breakfast cereals [5]. Therefore, it is essential to further research on the active constituents of flaxseeds. As part of an effort to assess the chemical diversity and biological activities of flaxseed meal, an ethanol extract of ⁎ Corresponding authors. Tel.: +86 10 60212110; fax: +86 10 63017757. E-mail addresses: [email protected] (C.-S. Yao), [email protected] (J.-G. Shi).
http://dx.doi.org/10.1016/j.fitote.2014.05.008 0367-326X/© 2014 Elsevier B.V. All rights reserved.
the flaxseed meal was investigated. We describe herein the isolation, and structural elucidation of 4 new compounds (1–4) (Fig. 1), together with 8 known ones. Among them, 1 was a new megastigmane derivative, 2 was a new methyl jasmonate glycoside derivative, and 3 and 4 were new C-28 steroids with 3β,5β-cis-dihydroxyl conformation. The absolute configuration of 1, 3, and 4 was determined by observing their induced circular dichroism after addition of Mo2(OAc)4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. All the isolates were assessed against nitrogen oxide (NO) production in macrophage induced by lipopolysaccharide (LPS), but none of them showed active at a concentration of 10−5 M. 2. Experimental details 2.1. General experimental procedures Optical rotations were measured on a P2000 polarimeter (JASCO, Tokyo, Japan), and UV spectra were obtained on a JASCO P650 spectrometer (JASCO). CD spectra were recorded on a JASCO J-815 CD spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1H and 125, or 150 MHz for 13C, respectively, on Varian INOVA
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L. Song et al. / Fitoterapia 97 (2014) 15–22
2
1
3
4 Fig. 1. Structures of compounds 1–4.
500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, Germany), in acetone-d6 or methanol-d4, with solvent peaks as references. ESIMS and HRESIMS data were measured using an AccuToFCS JMST100CS spectrometer (Agilent Technologies, Ltd, Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, People's Republic of China), and Pharmadex LH-20 (Amersham Biosciences, Inc., Shanghai, China). Preparative TLC separation was performed with high-performance silica gel TLC plates (HSGF254, glass precoated, Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China). HPLC separation was performed on an instrument consisting of a Waters 515 pump, a Waters 2487 dual λ absorbance detector, and a Knauer Smartline RI detector 2300 with a YMC semi-preparative column (250 × 10 mm i.d.) packed with C18 (5 μM). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating.
2.2. Plant material Linseed meal (L. usitatissimum L) was provided by Gansu Puyuan Pharmaceutical Technology Co., Ltd. (Gansu Province, People's Republic of China, May, 2009), which was identified by Prof. Wang-zhi Song (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China). A voucher specimen (No. ID-S-2540) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
2.3. Extraction and isolation Linseed meal (4.8 kg) was dried, ground and extracted (45 min each) with 70% EtOH/H2O (3 × 10 L) under condition of ultrasonic. The combined aqueous EtOH extracts were evaporated to near dryness under vacuum, and the resulting residue (500 g) was then suspended in H2O. After removal of insoluble solid, the water soluble fraction was partitioned successively with petroleum ether and EtOAc. The water solution was then absorbed on macroporous resin (SP825, 3 L), and eluted with H2O, 40% EtOH/H2O, 60% EtOH/H2O, 70% EtOH/H2O, and 95% EtOH/H2O, respectively. 40% EtOH/H2O fraction (45 g) was subjected to silica gel CC (80 × 10 cm, 200–300 mesh) eluted with a CHCl3/MeOH (80:20; 70:30; 60:40; 50:50; 30:70; 10:90, v/v) gradient system to yield ten fractions, LW-I-LW-X. LW-VIII (6.684 g) fraction was loaded on MPLC, eluted with a gradient of increasing MeOH (90:10; 80:20; 70:30, v/v) in H2O, to produce three subfractions, LW-VIII-A-LW-VIII-C. LW-VIII-B (4.0 g) was subjected to an ODS column, and eluted with MeOH–H2O (0:100; 5:95; 10:90; 20:80; 30:70, v/v) to afford seven subfractions, LW-VIII-B1-LW-VIII-B7. Compound 12 (20.8 mg) was obtained from fraction LW-VIII-B1 by recrystallization in MeOH. LW-VIII-C (1.239 g) was subjected to Sephadex LH-20 using a mobile phase of MeOH–H2O (1:1) to produce three subfractions, LW-VIII-C1-LW-VIII-C3. LW-VIII-C3 (311.4 mg) was separated by normal silica gel CC, and eluted with CHCl3–MeOH–H2O (40:5:0.5; 40:10:0.5; 40:20:0.5; 45:35:0.5; 50:40:0.5; 50:50:0.5, v/v) to afford ten subfractions LW-VIII-C3-a1-LW-VIII- C3-a10. LW-VIII-C3-a2 (90 mg) was purified by semi-preparative Rp-HPLC using CH3CN–H2O (12:88 v/v) as mobile phase to obtain compound 5 (3.21 mg). By following the same procedure, compound 2 (2.97 mg) was obtained from LW-VIII-C3-a4 (25 mg).
L. Song et al. / Fitoterapia 97 (2014) 15–22
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Table 1 1 H and 13C NMR spectral data of compounds 1 and 2a. 1
2 δH
Position 1 2a 2b 3 4a 4b 5 6 7 8 9 10a 10b 11 12 13 a
1.60 1.33 3.74 1.60 1.33 1.88
(m) (m) (ddt, 9.1, 6.3, 4.6) (m) (m) (m)
5.68 5.72 4.11 3.45 3.39 0.92 0.83 0.76
(d, 15.0) (d, 15.0) (dt, 7.8, 4.2) (dd, 11.4, 4.2) (dd, 11.4, 7.2) (s) (s) (d, 6.6)
δC
Position
δH
40.4s 45.9t 45.9t 67.5d 39.9t 39.9t 35.5d 78.3s 130.9d 136.6d 74.3d 67.7t 67.7t 25.2q 25.8q 16.5q
1 2 3 4a 4b 5a 5b 1′a 1′b 2′ 3′ 4′ 5′a 5′b
2.63 1.92 3.95 1.52 2.04 1.34 1.92 1.92 2.04 5.48 5.48 2.38 3.56 3.90
Measured in MeOH-d4 at 600 MHz for 1H NMR and 150 MHz for
13
(m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m)
δC
Position
δH
37.9d 51.6d 77.7d 33.1t 33.1t 29.1t 29.1t 26.1t 26.1t 127.8d 131.1d 29.3t 70.3t 70.3t
1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b 1″ 2″a 2″b OMe
4.27 3.16 3.34 3.27 3.27 3.67 3.87
δC (d, 7.8) (m) (m) (m) (m) (dd, 12.0, 5.4) (m)
2.26 (dd, 15.6, 9.0) 2.43 (dd, 15.6, 7.2) 3.66 (s)
104.4d 75.1d 78.1d 71.7d 78.0d 62.8t 62.8t 175.6s 36.1t 36.1t 52.0q
C NMR, with assignments confirmed by DEPT, 1H–1H COSY, NOESY, HMQC and HMBC.
LW-IV (245.4 mg) was chromatographed on silica gel, eluted with CHCl3–MeOH–H2O (85:5:0.5; 60:30:0.5; 50:50:0.5, v/v), to give three fractions LW-IV-A-LW-IV-C. LW-IV-B (70 mg) was purified repeatedly by semi-preparative Rp-HPLC with MeOH– H2O (17:83) as eluent to yield compound 1 (2.26 mg).
95% EtOH/H2O fraction (5.622 g) was separated into fifteen subfractions of NLW-A-NLW-O by silica gel CC, eluted with a mixture of CHCl3–MeOH of increasing polarity (100:1; 95:5; 90:10; 85:15; 80:20, v/v). NLW-C (200 mg) was divided into five subfractions (NLW-C-1-NLW-C-5) by high-performance silica
Table 2 1 H and 13C NMR spectral data of compounds 3, 3a, 4, and 4aa. No.
3
3a
δH 1a 1b 2a 2b 3 4a 4b 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22 23 24 25 26 27 28 a
1.96 1.65 1.65 2.05 3.79 1.96 1.96
δC (m) (m) (m) (m) (m) (m) (m)
4
δH
δC
δH
39.3 39.3 34.7 34.7 65.8 36.6 36.6 86.7 135.4 130.5 79.6 142.1 36.9 129.1
1.65 1.88 1.25 2.06 3.74 1.88 1.94
5.45 (dd, 6.0, 1.8)
33.5 33.5 31.5 31.5 66.1 37.1 37.1 83.0 136.7 131.2 78.5 144.6 38.8 s 119.4
2.27 (dd, 14.0, 6.0)
41.9
29.6
44.2 49.2 21.5
44.6
1.79 (m) 1.54 (m) 2.05 (m)
29.6
28.5
6.29 (d, 8.4) 6.62 (d, 8.4)
1.41 (m) 0.77 (s) 1.08 (s) 2.02 (m) 1.02 (d, 6.6) 5.22 (m) 5.28 (m) 1.88 (m) 1.47 (m) 0.85 (d, 6.6) 0.83 (d, 6.6) 0.93 (d, 6.6)
56.6 13.3 25.8 40.7 21.1 136.4 133.0 43. 33.8 19.9 20.3 18.0
3.75–3.90 (m)
6.42 (d, 8.4) 6.17 (d, 8.4)
5.34 (m)
23.3
55.9 12.8 18.0 39.7 20.9 135.5 132.1 42.7 32.9 19.5 19.8 17.8
0.60 (s) 1.18 (s)
5.08–5.34 (m) 5.08–5.34 (m)
Measured in CD3COCD3 at 600 MHz for 1H NMR and 150 MHz for
13
4a [CDCl3] δC (m) (m) (m) (m) (m) (m) (m)
6.22d (8.4) 6.48d (8.4) 1.39 (m) 1.49 1.49 1.25 1.94
(m) (m) (m) (m)
1.49 (m) 1.27m 2.08m 1.25 (m) 1.49 (m) 1.25 (m) 0.84 (s) 0.88 (s) 1.94 (m) 0.83d (6.6) 5.19dd (15.3, 8.4) 5.24 dd (15.3, 8.4) 1.94 (m) 1.44 (m) 0.82d (6.6) 0.91d (6.6) 1.01d (6.6)
35.67 35.67 29.35 29.35 66.25 38.00 38.00 82.37 131.14 136.44 79.42 52.44 21.28 21.28 40.23 40.23 45.16 52.67 29.35 29.35 23.98 23.98 56.98 13.17 18.47 40.55 19.93 132.84 135.50 43.68 33.80 20.29 18.02 21.28
δH
3.94 (m)
6.22 (d, 8.4) 6.48 (d, 8.4)
0.82 (s) 0.88 (s) 0.94d (6.4) 5.12 (dd, 15.4, 8.1) 5.20 (dd, 15.4, 7.3)
0.84d (6.4) 0.80d (6.4) 0.99d (7.2)
δC 36.84 36.84 29.98 29.98 66.27 34.64 34.64 79.36 130.67 135.40 82.12 51.04 36.82 20.56 20.56 39.26 39.26 44.49 51.61 28.56 28.56 23.32 23.32 56.13 12.80 18.01 39.64 19.57 132.2 135.1 42.70 32.99 19.88 20.82 17.50
C NMR, with assignments confirmed by DEPT, 1H–1H COSY, NOESY, HMQC and HMBC.
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L. Song et al. / Fitoterapia 97 (2014) 15–22
gel preparative TLC plates with petroleum ether–acetone (25:1) as mobile phase. NLW-C-4 (18.9 mg) was then subjected to semi-preparative Rp-HPLC with MeOH–H2O (95:5) as mobile phase to afford compound 10 (8.84 mg). NLW-D (2.01 g) was applied to silica gel CC, eluted with petroleum ether–acetone (50:1; 50:5; 50:10; 50:15; 50:20, v/v), to give ten fractions NLW-D-A–NLW-D-J. NLW-D-B (51.9 mg) was subjected to semi-preparative Rp-HPLC eluted with MeOH– H2O (90:10) to afford compound 7 (4.82 mg). Compound 6 (180 mg) was obtained from NLW-D-E (285.6 mg) by silica gel CC using a gradient of increasing acetone (50:5; 50: 10, v/v) in petroleum ether as eluent. Fraction NLW-D-J (282.7 mg) was applied to CC over Sephadex LH-20, eluted with petroleum ether–CHCl3–MeOH (5:5:1), to afford three subfractions NLW-D-J-1-NLW-D-J-3. NLW-D-J-2 (40 mg) was purified repeatedly by semi-preparative Rp-HPLC eluted with MeOH–H2O (97:3) to yield compound 11 (4.9 mg). NLW-D-J-3 (72 mg) was subjected to semi-preparative Rp-HPLC, eluted with MeOH–H2O (90:10), to afford compounds 8 (3.47 mg), and 9 (3.77 mg). Fraction NLW-D-H (356.2 mg) was loaded on Sephadex LH-20 with petroleum ether– CHCl3–MeOH (5:5:1) as eluent to give four subfractions of NLW-D-H1-NLW-D-H4, of which NLW-D-H3 (37.94 mg) was repeatedly subjected to semi-preparative Rp-HPLC, eluted with MeOH–H2O (85:15), to yield compounds 3 (1.11 mg), and 4 (2.38 mg), respectively. (3S,5R,6S,9R)-3,6,9,10-Terahydroxy-5,6-dihydro-β-ionol) (1): Light brown amorphous powder; [α]20 D −45.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 203 (3.93) nm; IR νmax 3380, 2972, 2934, 2868, 1677, 1460, 1434, 1368, 1319, 1204, 1188, 1139, 1076, 1027, 984, 910, 876, 839, 802, 760, 722, 629 cm−1; CD (MeOH) 233 (Δε +0.22) nm, Mo2(OCOCF3)4-induced CD (MeOH) 314 (Δε +0.40), 275 (Δε −0.11) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data (see Table 1); (+)-ESIMS m/z 267 [M + Na]+, (−)-ESIMS m/z 279 [M + Cl]−, 487[2M–H]−; HR-ESI-MS [M + Na]+ m/z 267.1566 (calcd for C13H24O4Na, 267.1567). (1S,2R,3R)-Methyl 2-[5-(β-D-glucopyranosyloxy)-2-penten1-yl]-3-hydroxyl cyclo pentane acetate (2): White amorphous powder; [α]20 D −8.2 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 202 (4.38), 224 (3.62) nm; IR νmax 3392, 3009, 2941, 1731, 1655, 1438, 1371, 1280, 1199, 1167, 1079, 1040, 897, 707, 630 cm−1; 1 H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data (see Table 1); (+)-ESIMS m/z 427 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 427.1954 (clcd for C19H32O9Na, 427.1939). (22E)-Ergosta-6,9,22-triene-3β,5β,8α-triol (3): Light yellow amorphous powder; [α]20 D 32.8 (c 0.09, CHCl3); UV (CHCl3) λmax
a
(log ε) 240 (3.54) nm; IR νmax: 3522, 2957, 2924, 2873, 1734, 1460, 1377, 1284, 1132, 1076, 1033, 976, 930, 901, 860, 819, 791 cm−1; CD (n-hexane) 212 (Δε +1.49), 198 (Δε −1.46) nm, Mo2(OAc)4 -induced CD (DMSO) 293 (Δε + 0.44), 380 (Δε + 0.06) nm; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR (CD3COCD3, 150 MHz) spectroscopic data (see Table 2); ESIMS m/z: 451 [M + Na]+; 467 [M + K]+, 463 [M + Cl]−; HR-ESI-MS [M + Na]+ m/z: 451.3191 (calcd for C28H44O3Na, 451.3188). (22E)-Ergosta-6,22-diene-3β,5β,8α-triol (4): Light yellow amorphous powder; [α]20 D − 22.9 (c 0.20, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.12) nm; IR νmax 3528, 3407, 2956, 2869, 1737, 1459, 1443, 1378, 1330, 1292, 1241, 1163, 1107, 1075, 1042, 1017, 986, 938, 915, 882, 832, 778, 723, 698, 653 cm−1; CD (n-hexane) 217 (Δε − 57.70) nm, Mo2(OAc)4-induced CD (DMSO) 380 (Δε + 0.02) nm; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR (CD3COCD3, 150 MHz) spectroscopic data (see Table 2); ESIMS m/z: 453 [M + Na]+; HR-ESI-MS [M + Na]+ m/z: 430.3428 (calcd for C28H46O3Na, 430.3447). 2.4. Inhibition assay of NO production in macrophage See Ref. [6]. 3. Results and discussion Compound 1 had the molecular formula C13H24O4, as indicated by its quasi-molecular ion peak at m/z 267.1566 [M + Na]+ (calcd for C13H24O4Na, 267.1567) in the HRESIMS spectrum, combined with the NMR data. Its IR spectrum showed the presence of hydroxyl (3380 cm−1) and olefinic (1677 cm−1) functionalities. The 1H NMR spectrum of 1 (Table 1) displayed signals for three methyl groups [δH 0.76 (3H, d, J = 6.6 Hz, H3-13), 0.92 (3H, s, H3-11), 0.83 (3H, s, H3-12)], two pair of methylene protons [δH 1.33 (2H, m, H-2b, 4b), 1.60 (2H, m, H-2a, 4a)], a methine protons [δH 1.88 (1H, m, H-5)], an oxygenated methylene protons [δH 3.45 (1H, dd, J = 11.4, 4.2 Hz, H-10a), 3.39 (1H, dd, J = 11.4, 7.2 Hz, H-10b)], two oxygenated methine protons [δH 3.74 (1H, m, H-3), 4.11 (1H, dt, J = 7.8, 4.2 Hz, H-9)], and two olefinic protons [δH 5.68 (1H, d, J = 15.0 Hz, H-7), 5.72 (1H, d, J = 15.0 Hz, H-8)]. The 13C NMR and DEPT spectra (Table 1) of 1 showed 13 carbon signals ascribable to three methyls [δC 16.5 (C-13), 25.2 (C-11), 25.8 (C-12)], three methylenes [δC 45.9 (C-2), 39.9 (C-4), 67.7 (C-10)] including an oxygenated one (C-10), three methines [δC 67.5 (C-3), 35.5 (C-5), 74.3 (C-9)] including two oxygenated ones (C-3 and C-9), two sp2 methines [δC 130.9 (C-7), 136.6 (C-8)], and
b Fig. 2. Key 1H–1H COSY (a), HMBC (b) and NOESY (c) correlations of 1.
c
L. Song et al. / Fitoterapia 97 (2014) 15–22
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Fig. 3. CD spectrum (a) and Mo2(OAc)4 induced CD spectrum (b) of 1.
two quaternary carbons [δC 40.4 (C-1), 78.3 (C-6)]. The above evidences suggested that 1 possessed the megastigmane skeleton, similar to the aglycone of lauroside D [7]. The difference between them was the replacement of a methyl group in the aglycone of lauroside D by a hydroxymethyl group in 1 [δH 3.45 (1H, dd, J = 11.4, 4.2Hz, H-10a), 3.39 (1H, dd, J = 11.4, 7.2Hz, H-10b); δC 67.7 (C-10)]. The 1H–1H COSY and HSQC spectra (Fig. 2) displayed the correlations of H2-/ H-3, H-3/H2-4, H2-4/H-5, H-5/H3-13 and H-7/H-8, H-8/H-9, H-9/H2-10, together with the HMBC correlations of H2-4, H3-13, H-7, and H-8 to C-6 further confirmed the above conjecture. Therefore, the planar structure of 1 was determined as Fig. 1. The relative configuration of 1 was deduced from analysis of the coupling constants and the NOESY spectrum (Fig. 2), which were both consistent with a chair conformation for the cyclohexane ring. The strong NOESY correlations between H-5 and H3-11 indicated that H-5 and H3-11 were axial bonds and assigned as β-oriented. The 3,4-dihydroxyl-1-butenyl side chain at C-6 was determined to be β-oriented based on the strong NOESY correlations of H-7 to H-5β and H3-11β. The β-orientation of H-3 was deduced from the NOESY correlations of H-3 to H-5 and H-11, as well as the larger coupling constants (J = 9.1, 6.3, 4.6 Hz) between H-3 with H-2α, H-2β and H-4α, H-4β. CH3-13 was determined to take an equatorial orientation from the NOESY correlations between H-5 with H-7 and H3-11, in combination with the configuration analysis. Accordingly, the configuration of C-3, C-5 and C-6 was
determined as 3S,5R,6S. In addition, the larger coupling constant (J = 15.0 Hz) between H-7 and H-8 indicates that the geometry of the Δ7(8) double bond is E. In order to determine the configuration of C-9, the Mo2(OAc)4-induced CD (DMSO) spectrum was obtained (Fig. 3). The positive cotton effect at 314 nm (Δε +0.40) observed in the Mo2(OAc)4induced CD (DMSO) spectrum permitted the assignment of the 9R configuration for 1 [8–10]. Thus, the structure of 1 was determined as (3S,5R,6S,9R)-3,6,9,10-tetrahydroxy-5,6-dihydro-β-ionol, which was named linustam A. Compound 2 was isolated as a white amorphous powder. Its HRESIMS at m/z 427.1954 [M + Na]+ (calcd for C19H32O9Na, 427.1939) revealed the molecular formula of C19H32O9, which indicated the presence of four degrees of unsaturation. The UV spectrum exhibited the absorption bands at λmax 202, 224 nm, which indicated the absence of long conjugated system in 2. Its IR spectrum showed absorptions for hydroxyl (3400 cm−1), olefinic bond (1655 cm−1), and carbonyl group (1731 cm−1). The NMR spectrum of 2 (Table 1) displayed an anomeric proton at δH 4.27 (1H, d, J = 7.8 Hz, H-1‴) and an anomeric carbon at δC 104.4 ppm (C-1‴), together with five characteristic carbons at δC 60–80 ppm and protons at δH 3.5–5.0 ppm, which demonstrated the presence of a glycosyl group in 2. Besides, 1H NMR spectrum of 2 also showed signals for a cis double bond [δH 5.48 (2H, m, H-2′, 3′)], an oxygenated methine [δH 3.95 (1H, m, H-3)], an oxygenated methylene [δH 3.90 (1H, m, H-5′b), 3.56 (1H, m, H-5′ a)], a methoxyl [δH 3.66 (3H, s)], and twelve overlap protons at
b
a Fig. 4. Selected NOESY (a) and HMBC (b) correlations of 2.
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L. Song et al. / Fitoterapia 97 (2014) 15–22
3a
3 Fig. 5. The conformations of compounds 3a and 3.
δH 1.8–3.0 ppm. The 13C NMR and DEPT spectra showed carbon signals ascribable to a double bond [δC 127.8 (C-2′), 131.1 (C-3′)], six methylenes [δC 33.1 (C-4), 29.1 (C-5), 26.1 (C-1′), 29.3 (C-4′), 70.3 (C-5′), 36.1 (C-2″)] including an oxygenated one (C-5′), three methines [δC 37.9 (C-1), 51.6 (C-2), 77.7 (C-3)] including an oxygenated one (C-3), a carbonyl group [δC 175.6 (C-1″)], and a methoxyl (52.0). These evidences suggested that 2 should be a methyl jasmonate glucoside derivative. Comparison of the NMR spectral data indicated that 2 had the similar skeleton as methyl jasmonate glucosides [11,12], the major difference was the replacement of the carbonyl group in the latter by a hydroxyl in the former. Consequently, compound 2 was determined as a methyl jasmonate glycoside derivative as shown in Fig. 1. In HMBC spectrum (Fig. 4), the correlations of OCH3/C-1″; H-5′/ C-1‴; H-1′/C-1, C-3; H-2″/C-2, C-5, in combination with their
chemical shifts, verified the connections of methoxyl and C-1″, C-1‴ and C-5′, C-2 and C-1′, and C-2″and C-1. In the NOESY experiment of 2 (Fig. 4), the strong correlations of H-3 with H-2″a, H-1′, H-2′, and H-4′ indicated that H-3, methyl acetate side chain, and pentenyl side chain were cofacial, and that H-3, methyl acetate side chain occupied axial positions, whereas pentenyl side chain was an equatorial orientation, which suggested that the stereochemistry of 2 should be determined as 1S, 2R, 3R configuration. Accordingly, the structure of 2 was identified as methyl (1S,2R,3R)-2-[5-(β-D-glucopyranosyloxy)-2-penten-1-yl]-3-hydroxyl cyclopentane acetate, which was named linustam B. Compound 3 was isolated as a light yellow amorphous powder. Its HRESIMS m/z 451.3191 [M + Na]+ (calcd for C28H44O3Na, 451.3188), in combination with its NMR
Fig. 6. CD spectrum (a) and Mo-induced CD spectrum (b) of 3.
L. Song et al. / Fitoterapia 97 (2014) 15–22
21
Fig. 7. CD spectrum (a) and Mo-induced CD spectrum (b) of 4.
spectral data (Table 2), prompted the molecular formula of C28H44O3. Its IR spectrum revealed the presence of hydroxyl (3522 cm−1), olefinic bond (1650 cm−1), and saturated aliphatic protons (2957, 2924, and 2873 cm−1). The 1H NMR spectrum of 3 showed the protons due to a cis olefinic bond [δH 6.29 (1H, d, J = 8.4 Hz, H-6), 6.62 (1H, d, J = 8.4 Hz, H-7)], a trisubstituted olefinic bond [δH 5.45 (1H, dd, J = 6.0, 1.8 Hz, H-11)], a trans olefinic bond [δH 5.22 (1H, m, H-22),5.28 (1H, m, H-23)], an oxygenated methine [δH 3.79 (1H, m, H-3)], and six methyls [δH 0.77 (3H, s, H3-18),1.08 (3H, s, H3-19), 1.02 (1H, d, J = 6.6 Hz, H3-21), 0.85 (1H, d, J = 6.6 Hz, H3-26), 0.83 (1H, d, J = 6.6 Hz, H3-27), 0.93 (1H, d, J = 6.6 Hz, H3-28)]. The 13C NMR and DEPT spectra of 3 revealed 28 carbon signals ascribable to six methyls, six methylenes, eleven methines including three oxygenate methines and five sp2 carbons, and five quaternary carbons including two oxygenated quaternary carbons and a sp2 carbon. These data suggested that compound 3 was a steroid with 28 carbons, of which the structure was similar to that of DHOE (3a) reported in the literature [13]. This structure was further substantiated by 2D NMR experiments involving HSQC, HMBC, 1H–1H COSY and NOESY. Careful comparison of the NMR data of 3 with those of 3a (Table 2) revealed a close structural similarity between them, except for ring A and its own substituted groups. The upfield shifted values (δC 83.0 and 119.4) in 3 with respect to 3a (δC 86.7 and 129.1) for C-5 and C-11 let us conceive that the hydroxyl at C-5 in 3 could take a β-orientation, instead of a α-orientation in 3a. That is, ring A and ring B should be cis-fused in 3 (Fig. 5). The obvious difference of the chemical shifts at C-1, C-2, and CH3-19 between 3 (δC 33.5, 31.5, 25.8) and 3a (δC 39.3, 34.7, 18.0) further verified the conjecture. In the case of cis conformation of 3, the steric hindrance caused by 5-hydroxyl and CH3-19 with axial orientation resulted in the upfield shifted values of C-5 and C-11 when compared with 3a. According to the sector rule of chiral 1,3 diol [14], in the case of chiral Mo-complexes with rigid 1,3-diols, the sign of the cotton effect occurring around 400 nm is the same as the sign of the sector in which the majority of the molecule is located. In terms of 3,5-dihydroxycholestane, the conformer with syn-parallel orientation of 3α,5α-dihydroxyl groups
corresponds to a negative cotton effect, but the other conformer with 3β,5β-dihydroxyl groups corresponds to a positive cotton effect at around 400 nm. In order to further prove the cis conformation of 3,5-diol, the CD and Mo2(OAc)4-induced CD (DMSO) spectra of 3 were obtained (Fig. 6), respectively. The positive cotton effect at 380 nm (Δε + 0.06) observed in the Mo2(OAc)4-induced CD (DMSO) spectrum confirmed the cis configuration of 3β,5β-dihydroxyl groups. In addition, the resemblance of the NMR data between 3 and 3a in C, D-ring, in consideration of biogenesis, suggested a cis-fused relationship between the C ring and D-ring of 3, similar to that of 3a. Thus, the structure of 3 was determined as (22E)-ergosta-6,9,22-triene-3β,5β,8α-triol, and was named linustam C. Compound 4 was obtained as a light yellow amorphous powder. Its HRESIMS at m/z 430.3428 [M]+ (calcd for C28H46O3Na, 430.3447) demonstrated the molecular formula of C28H46O3. The 1H NMR and 13C NMR spectral data strongly resembled those of (22E)-ergosta-6,22-diene-3β,5α,8α-triol (4a) (Table 2) reported in the literature [15]. However, careful comparison of its NMR spectral data with those of 4a revealed that the chemical shifts of C-5 and C-8 at δC 82.37 and 79.42 in 4 were replaced by δC 79.36 and 82.12 in 4a, which were confirmed by HSQC, and the HMBC correlations of H-4, H3-19, H-7 with C-5, and H-6, H-11, H-14 with C-8. Referred to compound 3, the hydroxyls at C-3 and C-5 of 4 were suggested to take a β orientation, as well as ring A and ring B suggested to be cis-fused. Application of the same methods as described in 3, the positive cotton effect at 380 nm (Δε +0.02) observed in the Mo2(OAc)4-induced CD (DMSO) spectrum (Fig. 7) of 4, combined with the sector rule of chiral 1,3 diol, supported the cis configuration of ring A and ring B. Similarly, the resemblance of the spectral data of C ring and D ring between 4 and 4a, in consideration of the biogenesis, suggested a tran-fused relationship of C ring and D ring of 4, similar to that of 4a. Thus, the structure of compound 4a was revised as (22E)-ergosta-6,22-diene-3β,5β,8α-triol. The known compounds were identified by comparison of the spectroscopic data with those reported as methylβ-D-glucopyranosyl tuberonate (5) [11], sitosterol (6),
22
L. Song et al. / Fitoterapia 97 (2014) 15–22
stigmast-7-oxo-5-ene (7) [16], stigmast-5-ene-3,16-diol (8) [17], 7α-hydroxyl sitosterol (9) [18], 24-methylene cyloartenol (10) [19], 7-oxo-β-sitosterol (11) [20], and sucrose (12). All the isolates were assessed against nitrogen oxide (NO) production in macrophage induced by lipopolysaccharide (LPS), but were inactive at a concentration of 10−5 M. The positive control dexamethasone (DEX) gave an 82.2% inhibition at the same condition. Acknowledgment We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for measuring the IR, UV, NMR, and MS spectra. The authors are grateful to Dr. L. Li and Professor Yikang Si for the testing of CDs. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.05.008. References [1] Buranov AU, Ross KA, Mazza G. Isolation and characterization of lignins extracted from flax shives using pressurized aqueous ethanol. Bioresour Technol 2010;101:7446–55. [2] Prasad K. Regression of hypercholesterolemic atherosclerosis in rabbits by secoisolarici-resinol diglucoside isolated from flaxseed. Atherosclerosis 2008;197:34–42. [3] Struijs K, Vinchen JP, Verhoef R, Voragen AG, Gruppen H. Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls. Phytochemistry 2008;69:1250–60. [4] Zhang CY, Zhang BG, Yang XW. Current status of investigations on chemical constituents and pharmacological effects of flaxseed (Linum usitatissimum). Chin J New Drug 2005;14:525–30.
[5] Meagher LP, Beecher GR, Flanagan VP, Li BW. Isolation and characterization of the lignans, isolariciresinol and pinoresinol, in flaxseed meal. J Agric Food Chem 1999;47:3173–80. [6] Zhao F, Wang SJ, Lin S, Zhu CG, Yuan SP, Ding XY, et al. Anthraquinones from the roots of Knoxia valerianoides. J Asian Nat Prod Res 2011;13:1023–9. [7] Marino SD, Borbone N, Zollo F, Ianaro A, Meglio PD, Iorizzi M. Megastigmane and phenolic components from Laurus nobilis L. leaves and their inhibitory effects on nitric oxide production. J Agric Food Chem 2004;52:7525–31. [8] Bari LD, Pescitelli G, Pratelli C, Pini D, Salvadori P. Determination of absolute configuration of acyclic 1,2-diols with Mo2(OAc)4. 1. Snatzke's method revisited. J Org Chem 2001;66:4819–25. [9] Liu J, Du D, Si YK, Lv HN, Wu XF, Li Y, et al. Application of dimolybdenum reagent Mo2(OAc)4 for determination of the absolute configuration of vicdiols. Chin J Org 2010;30:1270–8. [10] Politi M, Tommasi ND, Pescitelli G, Bari LD, Morelli I, Braca A. Structure and absolute configuration of new diterpenes from Lavandula multifida. J Nat Prod 2002;65:1742–5. [11] Matsuura H, Yoshihara T, Ichihara A, Kikuta Y, Koda Y. Tuber-forming substances in Jerusalem artichoke (Helianthus tuberosus L.). Biosci Biotechnol Biochem 1993;57:1253–6. [12] Inoue M, Kitahara T. Synthesis of both the enantiomers of methyl tuberonate, natural methyl β-D-glucopyranosyloxy jasmonate and its epimer. Tetrahedron 1999;55:4621–30. [13] Ponce MA, Ramirez JA, Galagovsky LR, Gros EG, Erra-Balsells R. A new look into the reaction between ergosterol and singlet oxygen in vitro. Photochem Photobiol Sci 2002;1:749–56. [14] Frelek J, Klimek A, Ruskowska P. Dinuclear transition metal complexes as auxiliary chromophores in chiroptical studies on bioactive compounds. Curr Org Chem 2003;7:1081–104. [15] Feng JT, Shi YP. Steroids from Saussurea ussuriensis. Pharmazie 2005; 60:464–7. [16] Ahmad MS, Ansari IA, Ansari SA, Moinuddin G. Synthesis of some steroidal tetrazoles from stigmastane series. Indian J Chem Sect B 1984; 11:1110–2. [17] Anjaneyulu ASR, Sagar KS, Prakash CVS. Two new steroid derivatives from the soft coral Sinularia gibberosa of Andaman & Nicobar Islands. Indian J Chem Sect B 1996;35B:819–25. [18] Zhang X, Geoffroy P, Miesch M, Diane JD, Raul F, Dalal AW, et al. Gran-scale chromatographic purification of β-sitosterol synthesis and characterization of β-sitosterol oxides. Steroids 2005;70:886–95. [19] Ikuta A, Itokawa H. Triterpenoids of Paeonia japonica callus tissue. Phytochemistry 1988;27:2813–5. [20] Pettit GR, Numata A, Gragg GM, Herald DL, Takada T, Iwamoto C, et al. Isolation and structures of schleicherastatins 1–7 and schleicheols 1 and 2 from the teak forest medicinal tree Schleichera oleosa. J Nat Prod 2000;63:72–8.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Chemical constituents from the linseed meal Li Song, Xian-Fen Wang, Yan Wu, Wen-Yi He, Chun-Suo Yao ⁎, Jian-Gong Shi ⁎ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 22 March 2014 Accepted in revised form 13 May 2014 Available online 24 May 2014 Keywords: Linum usitatissimum L Linseed meal Linaceae Linustam
a b s t r a c t One megastigmane derivative 1, one methyl jasmonate glycoside derivative 2, and two C-28 steroids with 3β,5β-cis-dihydroxyl conformation 3 and 4, together with eight known compounds 5–12 were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L). Structures of 1–4 were elucidated by spectroscopic methods including NMR, HRESIMS, and Mo2(OAc)4-induced CD. The absolute configuration of 1 and 3 was determined by observing their induced circular dichroism after addition of Mo2(OAc)4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. The structure of 4a was corrected as 4 by an extensive analysis of its 1D and 2D NMR, in combination with the Mo2(OAc)4-induced CD in DMSO. The effect of all the isolates on nitric oxide (NO) generation by stimulated macrophages was evaluated, and none of them showed active. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Flaxseed (Linum usitatissimum L), a herbaceous plant belonging to Linaceae family, is an important industrial crop grown worldwide for its fiber and oilseed [1]. Flaxseed contains 35% of its mass as oil, of which 55% is α-linolenic acid (ω-3 fatty acid) and 15–18% is linoleic acid [2]. Concentrations of lignans up to 3% (w/w) have been reported in flaxseed, making flax one of the richest edible sources of lignans. In contrast to most plants in which free lignans are present, the lignans in flaxseeds are incorporated into an oligomeric structure. Flaxseed lignans showed multi-faced bioactivities, and attracted a lot of interests for the researchers all over the world. Besides lignans, flavonoids, cyclic peptides, hydroxyl cinnamic acids, cyanogenetic glucosides, pectic polysaccharides and other chemical constituents have been identified from flaxseed extracts [3,4]. There is an increasing interest in flaxseed in human nutrition as it gains popularity as a health food, a dietary supplement, and an ingredient in bread, muffins and breakfast cereals [5]. Therefore, it is essential to further research on the active constituents of flaxseeds. As part of an effort to assess the chemical diversity and biological activities of flaxseed meal, an ethanol extract of ⁎ Corresponding authors. Tel.: +86 10 60212110; fax: +86 10 63017757. E-mail addresses: [email protected] (C.-S. Yao), [email protected] (J.-G. Shi).
http://dx.doi.org/10.1016/j.fitote.2014.05.008 0367-326X/© 2014 Elsevier B.V. All rights reserved.
the flaxseed meal was investigated. We describe herein the isolation, and structural elucidation of 4 new compounds (1–4) (Fig. 1), together with 8 known ones. Among them, 1 was a new megastigmane derivative, 2 was a new methyl jasmonate glycoside derivative, and 3 and 4 were new C-28 steroids with 3β,5β-cis-dihydroxyl conformation. The absolute configuration of 1, 3, and 4 was determined by observing their induced circular dichroism after addition of Mo2(OAc)4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. All the isolates were assessed against nitrogen oxide (NO) production in macrophage induced by lipopolysaccharide (LPS), but none of them showed active at a concentration of 10−5 M. 2. Experimental details 2.1. General experimental procedures Optical rotations were measured on a P2000 polarimeter (JASCO, Tokyo, Japan), and UV spectra were obtained on a JASCO P650 spectrometer (JASCO). CD spectra were recorded on a JASCO J-815 CD spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1H and 125, or 150 MHz for 13C, respectively, on Varian INOVA
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L. Song et al. / Fitoterapia 97 (2014) 15–22
2
1
3
4 Fig. 1. Structures of compounds 1–4.
500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, Germany), in acetone-d6 or methanol-d4, with solvent peaks as references. ESIMS and HRESIMS data were measured using an AccuToFCS JMST100CS spectrometer (Agilent Technologies, Ltd, Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, People's Republic of China), and Pharmadex LH-20 (Amersham Biosciences, Inc., Shanghai, China). Preparative TLC separation was performed with high-performance silica gel TLC plates (HSGF254, glass precoated, Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China). HPLC separation was performed on an instrument consisting of a Waters 515 pump, a Waters 2487 dual λ absorbance detector, and a Knauer Smartline RI detector 2300 with a YMC semi-preparative column (250 × 10 mm i.d.) packed with C18 (5 μM). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating.
2.2. Plant material Linseed meal (L. usitatissimum L) was provided by Gansu Puyuan Pharmaceutical Technology Co., Ltd. (Gansu Province, People's Republic of China, May, 2009), which was identified by Prof. Wang-zhi Song (Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China). A voucher specimen (No. ID-S-2540) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
2.3. Extraction and isolation Linseed meal (4.8 kg) was dried, ground and extracted (45 min each) with 70% EtOH/H2O (3 × 10 L) under condition of ultrasonic. The combined aqueous EtOH extracts were evaporated to near dryness under vacuum, and the resulting residue (500 g) was then suspended in H2O. After removal of insoluble solid, the water soluble fraction was partitioned successively with petroleum ether and EtOAc. The water solution was then absorbed on macroporous resin (SP825, 3 L), and eluted with H2O, 40% EtOH/H2O, 60% EtOH/H2O, 70% EtOH/H2O, and 95% EtOH/H2O, respectively. 40% EtOH/H2O fraction (45 g) was subjected to silica gel CC (80 × 10 cm, 200–300 mesh) eluted with a CHCl3/MeOH (80:20; 70:30; 60:40; 50:50; 30:70; 10:90, v/v) gradient system to yield ten fractions, LW-I-LW-X. LW-VIII (6.684 g) fraction was loaded on MPLC, eluted with a gradient of increasing MeOH (90:10; 80:20; 70:30, v/v) in H2O, to produce three subfractions, LW-VIII-A-LW-VIII-C. LW-VIII-B (4.0 g) was subjected to an ODS column, and eluted with MeOH–H2O (0:100; 5:95; 10:90; 20:80; 30:70, v/v) to afford seven subfractions, LW-VIII-B1-LW-VIII-B7. Compound 12 (20.8 mg) was obtained from fraction LW-VIII-B1 by recrystallization in MeOH. LW-VIII-C (1.239 g) was subjected to Sephadex LH-20 using a mobile phase of MeOH–H2O (1:1) to produce three subfractions, LW-VIII-C1-LW-VIII-C3. LW-VIII-C3 (311.4 mg) was separated by normal silica gel CC, and eluted with CHCl3–MeOH–H2O (40:5:0.5; 40:10:0.5; 40:20:0.5; 45:35:0.5; 50:40:0.5; 50:50:0.5, v/v) to afford ten subfractions LW-VIII-C3-a1-LW-VIII- C3-a10. LW-VIII-C3-a2 (90 mg) was purified by semi-preparative Rp-HPLC using CH3CN–H2O (12:88 v/v) as mobile phase to obtain compound 5 (3.21 mg). By following the same procedure, compound 2 (2.97 mg) was obtained from LW-VIII-C3-a4 (25 mg).
L. Song et al. / Fitoterapia 97 (2014) 15–22
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Table 1 1 H and 13C NMR spectral data of compounds 1 and 2a. 1
2 δH
Position 1 2a 2b 3 4a 4b 5 6 7 8 9 10a 10b 11 12 13 a
1.60 1.33 3.74 1.60 1.33 1.88
(m) (m) (ddt, 9.1, 6.3, 4.6) (m) (m) (m)
5.68 5.72 4.11 3.45 3.39 0.92 0.83 0.76
(d, 15.0) (d, 15.0) (dt, 7.8, 4.2) (dd, 11.4, 4.2) (dd, 11.4, 7.2) (s) (s) (d, 6.6)
δC
Position
δH
40.4s 45.9t 45.9t 67.5d 39.9t 39.9t 35.5d 78.3s 130.9d 136.6d 74.3d 67.7t 67.7t 25.2q 25.8q 16.5q
1 2 3 4a 4b 5a 5b 1′a 1′b 2′ 3′ 4′ 5′a 5′b
2.63 1.92 3.95 1.52 2.04 1.34 1.92 1.92 2.04 5.48 5.48 2.38 3.56 3.90
Measured in MeOH-d4 at 600 MHz for 1H NMR and 150 MHz for
13
(m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m) (m)
δC
Position
δH
37.9d 51.6d 77.7d 33.1t 33.1t 29.1t 29.1t 26.1t 26.1t 127.8d 131.1d 29.3t 70.3t 70.3t
1‴ 2‴ 3‴ 4‴ 5‴ 6‴a 6‴b 1″ 2″a 2″b OMe
4.27 3.16 3.34 3.27 3.27 3.67 3.87
δC (d, 7.8) (m) (m) (m) (m) (dd, 12.0, 5.4) (m)
2.26 (dd, 15.6, 9.0) 2.43 (dd, 15.6, 7.2) 3.66 (s)
104.4d 75.1d 78.1d 71.7d 78.0d 62.8t 62.8t 175.6s 36.1t 36.1t 52.0q
C NMR, with assignments confirmed by DEPT, 1H–1H COSY, NOESY, HMQC and HMBC.
LW-IV (245.4 mg) was chromatographed on silica gel, eluted with CHCl3–MeOH–H2O (85:5:0.5; 60:30:0.5; 50:50:0.5, v/v), to give three fractions LW-IV-A-LW-IV-C. LW-IV-B (70 mg) was purified repeatedly by semi-preparative Rp-HPLC with MeOH– H2O (17:83) as eluent to yield compound 1 (2.26 mg).
95% EtOH/H2O fraction (5.622 g) was separated into fifteen subfractions of NLW-A-NLW-O by silica gel CC, eluted with a mixture of CHCl3–MeOH of increasing polarity (100:1; 95:5; 90:10; 85:15; 80:20, v/v). NLW-C (200 mg) was divided into five subfractions (NLW-C-1-NLW-C-5) by high-performance silica
Table 2 1 H and 13C NMR spectral data of compounds 3, 3a, 4, and 4aa. No.
3
3a
δH 1a 1b 2a 2b 3 4a 4b 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16a 16b 17 18 19 20 21 22 23 24 25 26 27 28 a
1.96 1.65 1.65 2.05 3.79 1.96 1.96
δC (m) (m) (m) (m) (m) (m) (m)
4
δH
δC
δH
39.3 39.3 34.7 34.7 65.8 36.6 36.6 86.7 135.4 130.5 79.6 142.1 36.9 129.1
1.65 1.88 1.25 2.06 3.74 1.88 1.94
5.45 (dd, 6.0, 1.8)
33.5 33.5 31.5 31.5 66.1 37.1 37.1 83.0 136.7 131.2 78.5 144.6 38.8 s 119.4
2.27 (dd, 14.0, 6.0)
41.9
29.6
44.2 49.2 21.5
44.6
1.79 (m) 1.54 (m) 2.05 (m)
29.6
28.5
6.29 (d, 8.4) 6.62 (d, 8.4)
1.41 (m) 0.77 (s) 1.08 (s) 2.02 (m) 1.02 (d, 6.6) 5.22 (m) 5.28 (m) 1.88 (m) 1.47 (m) 0.85 (d, 6.6) 0.83 (d, 6.6) 0.93 (d, 6.6)
56.6 13.3 25.8 40.7 21.1 136.4 133.0 43. 33.8 19.9 20.3 18.0
3.75–3.90 (m)
6.42 (d, 8.4) 6.17 (d, 8.4)
5.34 (m)
23.3
55.9 12.8 18.0 39.7 20.9 135.5 132.1 42.7 32.9 19.5 19.8 17.8
0.60 (s) 1.18 (s)
5.08–5.34 (m) 5.08–5.34 (m)
Measured in CD3COCD3 at 600 MHz for 1H NMR and 150 MHz for
13
4a [CDCl3] δC (m) (m) (m) (m) (m) (m) (m)
6.22d (8.4) 6.48d (8.4) 1.39 (m) 1.49 1.49 1.25 1.94
(m) (m) (m) (m)
1.49 (m) 1.27m 2.08m 1.25 (m) 1.49 (m) 1.25 (m) 0.84 (s) 0.88 (s) 1.94 (m) 0.83d (6.6) 5.19dd (15.3, 8.4) 5.24 dd (15.3, 8.4) 1.94 (m) 1.44 (m) 0.82d (6.6) 0.91d (6.6) 1.01d (6.6)
35.67 35.67 29.35 29.35 66.25 38.00 38.00 82.37 131.14 136.44 79.42 52.44 21.28 21.28 40.23 40.23 45.16 52.67 29.35 29.35 23.98 23.98 56.98 13.17 18.47 40.55 19.93 132.84 135.50 43.68 33.80 20.29 18.02 21.28
δH
3.94 (m)
6.22 (d, 8.4) 6.48 (d, 8.4)
0.82 (s) 0.88 (s) 0.94d (6.4) 5.12 (dd, 15.4, 8.1) 5.20 (dd, 15.4, 7.3)
0.84d (6.4) 0.80d (6.4) 0.99d (7.2)
δC 36.84 36.84 29.98 29.98 66.27 34.64 34.64 79.36 130.67 135.40 82.12 51.04 36.82 20.56 20.56 39.26 39.26 44.49 51.61 28.56 28.56 23.32 23.32 56.13 12.80 18.01 39.64 19.57 132.2 135.1 42.70 32.99 19.88 20.82 17.50
C NMR, with assignments confirmed by DEPT, 1H–1H COSY, NOESY, HMQC and HMBC.
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gel preparative TLC plates with petroleum ether–acetone (25:1) as mobile phase. NLW-C-4 (18.9 mg) was then subjected to semi-preparative Rp-HPLC with MeOH–H2O (95:5) as mobile phase to afford compound 10 (8.84 mg). NLW-D (2.01 g) was applied to silica gel CC, eluted with petroleum ether–acetone (50:1; 50:5; 50:10; 50:15; 50:20, v/v), to give ten fractions NLW-D-A–NLW-D-J. NLW-D-B (51.9 mg) was subjected to semi-preparative Rp-HPLC eluted with MeOH– H2O (90:10) to afford compound 7 (4.82 mg). Compound 6 (180 mg) was obtained from NLW-D-E (285.6 mg) by silica gel CC using a gradient of increasing acetone (50:5; 50: 10, v/v) in petroleum ether as eluent. Fraction NLW-D-J (282.7 mg) was applied to CC over Sephadex LH-20, eluted with petroleum ether–CHCl3–MeOH (5:5:1), to afford three subfractions NLW-D-J-1-NLW-D-J-3. NLW-D-J-2 (40 mg) was purified repeatedly by semi-preparative Rp-HPLC eluted with MeOH–H2O (97:3) to yield compound 11 (4.9 mg). NLW-D-J-3 (72 mg) was subjected to semi-preparative Rp-HPLC, eluted with MeOH–H2O (90:10), to afford compounds 8 (3.47 mg), and 9 (3.77 mg). Fraction NLW-D-H (356.2 mg) was loaded on Sephadex LH-20 with petroleum ether– CHCl3–MeOH (5:5:1) as eluent to give four subfractions of NLW-D-H1-NLW-D-H4, of which NLW-D-H3 (37.94 mg) was repeatedly subjected to semi-preparative Rp-HPLC, eluted with MeOH–H2O (85:15), to yield compounds 3 (1.11 mg), and 4 (2.38 mg), respectively. (3S,5R,6S,9R)-3,6,9,10-Terahydroxy-5,6-dihydro-β-ionol) (1): Light brown amorphous powder; [α]20 D −45.5 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 203 (3.93) nm; IR νmax 3380, 2972, 2934, 2868, 1677, 1460, 1434, 1368, 1319, 1204, 1188, 1139, 1076, 1027, 984, 910, 876, 839, 802, 760, 722, 629 cm−1; CD (MeOH) 233 (Δε +0.22) nm, Mo2(OCOCF3)4-induced CD (MeOH) 314 (Δε +0.40), 275 (Δε −0.11) nm; 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data (see Table 1); (+)-ESIMS m/z 267 [M + Na]+, (−)-ESIMS m/z 279 [M + Cl]−, 487[2M–H]−; HR-ESI-MS [M + Na]+ m/z 267.1566 (calcd for C13H24O4Na, 267.1567). (1S,2R,3R)-Methyl 2-[5-(β-D-glucopyranosyloxy)-2-penten1-yl]-3-hydroxyl cyclo pentane acetate (2): White amorphous powder; [α]20 D −8.2 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 202 (4.38), 224 (3.62) nm; IR νmax 3392, 3009, 2941, 1731, 1655, 1438, 1371, 1280, 1199, 1167, 1079, 1040, 897, 707, 630 cm−1; 1 H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz) spectroscopic data (see Table 1); (+)-ESIMS m/z 427 [M + Na]+; HR-ESI-MS [M + Na]+ m/z 427.1954 (clcd for C19H32O9Na, 427.1939). (22E)-Ergosta-6,9,22-triene-3β,5β,8α-triol (3): Light yellow amorphous powder; [α]20 D 32.8 (c 0.09, CHCl3); UV (CHCl3) λmax
a
(log ε) 240 (3.54) nm; IR νmax: 3522, 2957, 2924, 2873, 1734, 1460, 1377, 1284, 1132, 1076, 1033, 976, 930, 901, 860, 819, 791 cm−1; CD (n-hexane) 212 (Δε +1.49), 198 (Δε −1.46) nm, Mo2(OAc)4 -induced CD (DMSO) 293 (Δε + 0.44), 380 (Δε + 0.06) nm; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR (CD3COCD3, 150 MHz) spectroscopic data (see Table 2); ESIMS m/z: 451 [M + Na]+; 467 [M + K]+, 463 [M + Cl]−; HR-ESI-MS [M + Na]+ m/z: 451.3191 (calcd for C28H44O3Na, 451.3188). (22E)-Ergosta-6,22-diene-3β,5β,8α-triol (4): Light yellow amorphous powder; [α]20 D − 22.9 (c 0.20, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.12) nm; IR νmax 3528, 3407, 2956, 2869, 1737, 1459, 1443, 1378, 1330, 1292, 1241, 1163, 1107, 1075, 1042, 1017, 986, 938, 915, 882, 832, 778, 723, 698, 653 cm−1; CD (n-hexane) 217 (Δε − 57.70) nm, Mo2(OAc)4-induced CD (DMSO) 380 (Δε + 0.02) nm; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR (CD3COCD3, 150 MHz) spectroscopic data (see Table 2); ESIMS m/z: 453 [M + Na]+; HR-ESI-MS [M + Na]+ m/z: 430.3428 (calcd for C28H46O3Na, 430.3447). 2.4. Inhibition assay of NO production in macrophage See Ref. [6]. 3. Results and discussion Compound 1 had the molecular formula C13H24O4, as indicated by its quasi-molecular ion peak at m/z 267.1566 [M + Na]+ (calcd for C13H24O4Na, 267.1567) in the HRESIMS spectrum, combined with the NMR data. Its IR spectrum showed the presence of hydroxyl (3380 cm−1) and olefinic (1677 cm−1) functionalities. The 1H NMR spectrum of 1 (Table 1) displayed signals for three methyl groups [δH 0.76 (3H, d, J = 6.6 Hz, H3-13), 0.92 (3H, s, H3-11), 0.83 (3H, s, H3-12)], two pair of methylene protons [δH 1.33 (2H, m, H-2b, 4b), 1.60 (2H, m, H-2a, 4a)], a methine protons [δH 1.88 (1H, m, H-5)], an oxygenated methylene protons [δH 3.45 (1H, dd, J = 11.4, 4.2 Hz, H-10a), 3.39 (1H, dd, J = 11.4, 7.2 Hz, H-10b)], two oxygenated methine protons [δH 3.74 (1H, m, H-3), 4.11 (1H, dt, J = 7.8, 4.2 Hz, H-9)], and two olefinic protons [δH 5.68 (1H, d, J = 15.0 Hz, H-7), 5.72 (1H, d, J = 15.0 Hz, H-8)]. The 13C NMR and DEPT spectra (Table 1) of 1 showed 13 carbon signals ascribable to three methyls [δC 16.5 (C-13), 25.2 (C-11), 25.8 (C-12)], three methylenes [δC 45.9 (C-2), 39.9 (C-4), 67.7 (C-10)] including an oxygenated one (C-10), three methines [δC 67.5 (C-3), 35.5 (C-5), 74.3 (C-9)] including two oxygenated ones (C-3 and C-9), two sp2 methines [δC 130.9 (C-7), 136.6 (C-8)], and
b Fig. 2. Key 1H–1H COSY (a), HMBC (b) and NOESY (c) correlations of 1.
c
L. Song et al. / Fitoterapia 97 (2014) 15–22
19
Fig. 3. CD spectrum (a) and Mo2(OAc)4 induced CD spectrum (b) of 1.
two quaternary carbons [δC 40.4 (C-1), 78.3 (C-6)]. The above evidences suggested that 1 possessed the megastigmane skeleton, similar to the aglycone of lauroside D [7]. The difference between them was the replacement of a methyl group in the aglycone of lauroside D by a hydroxymethyl group in 1 [δH 3.45 (1H, dd, J = 11.4, 4.2Hz, H-10a), 3.39 (1H, dd, J = 11.4, 7.2Hz, H-10b); δC 67.7 (C-10)]. The 1H–1H COSY and HSQC spectra (Fig. 2) displayed the correlations of H2-/ H-3, H-3/H2-4, H2-4/H-5, H-5/H3-13 and H-7/H-8, H-8/H-9, H-9/H2-10, together with the HMBC correlations of H2-4, H3-13, H-7, and H-8 to C-6 further confirmed the above conjecture. Therefore, the planar structure of 1 was determined as Fig. 1. The relative configuration of 1 was deduced from analysis of the coupling constants and the NOESY spectrum (Fig. 2), which were both consistent with a chair conformation for the cyclohexane ring. The strong NOESY correlations between H-5 and H3-11 indicated that H-5 and H3-11 were axial bonds and assigned as β-oriented. The 3,4-dihydroxyl-1-butenyl side chain at C-6 was determined to be β-oriented based on the strong NOESY correlations of H-7 to H-5β and H3-11β. The β-orientation of H-3 was deduced from the NOESY correlations of H-3 to H-5 and H-11, as well as the larger coupling constants (J = 9.1, 6.3, 4.6 Hz) between H-3 with H-2α, H-2β and H-4α, H-4β. CH3-13 was determined to take an equatorial orientation from the NOESY correlations between H-5 with H-7 and H3-11, in combination with the configuration analysis. Accordingly, the configuration of C-3, C-5 and C-6 was
determined as 3S,5R,6S. In addition, the larger coupling constant (J = 15.0 Hz) between H-7 and H-8 indicates that the geometry of the Δ7(8) double bond is E. In order to determine the configuration of C-9, the Mo2(OAc)4-induced CD (DMSO) spectrum was obtained (Fig. 3). The positive cotton effect at 314 nm (Δε +0.40) observed in the Mo2(OAc)4induced CD (DMSO) spectrum permitted the assignment of the 9R configuration for 1 [8–10]. Thus, the structure of 1 was determined as (3S,5R,6S,9R)-3,6,9,10-tetrahydroxy-5,6-dihydro-β-ionol, which was named linustam A. Compound 2 was isolated as a white amorphous powder. Its HRESIMS at m/z 427.1954 [M + Na]+ (calcd for C19H32O9Na, 427.1939) revealed the molecular formula of C19H32O9, which indicated the presence of four degrees of unsaturation. The UV spectrum exhibited the absorption bands at λmax 202, 224 nm, which indicated the absence of long conjugated system in 2. Its IR spectrum showed absorptions for hydroxyl (3400 cm−1), olefinic bond (1655 cm−1), and carbonyl group (1731 cm−1). The NMR spectrum of 2 (Table 1) displayed an anomeric proton at δH 4.27 (1H, d, J = 7.8 Hz, H-1‴) and an anomeric carbon at δC 104.4 ppm (C-1‴), together with five characteristic carbons at δC 60–80 ppm and protons at δH 3.5–5.0 ppm, which demonstrated the presence of a glycosyl group in 2. Besides, 1H NMR spectrum of 2 also showed signals for a cis double bond [δH 5.48 (2H, m, H-2′, 3′)], an oxygenated methine [δH 3.95 (1H, m, H-3)], an oxygenated methylene [δH 3.90 (1H, m, H-5′b), 3.56 (1H, m, H-5′ a)], a methoxyl [δH 3.66 (3H, s)], and twelve overlap protons at
b
a Fig. 4. Selected NOESY (a) and HMBC (b) correlations of 2.
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L. Song et al. / Fitoterapia 97 (2014) 15–22
3a
3 Fig. 5. The conformations of compounds 3a and 3.
δH 1.8–3.0 ppm. The 13C NMR and DEPT spectra showed carbon signals ascribable to a double bond [δC 127.8 (C-2′), 131.1 (C-3′)], six methylenes [δC 33.1 (C-4), 29.1 (C-5), 26.1 (C-1′), 29.3 (C-4′), 70.3 (C-5′), 36.1 (C-2″)] including an oxygenated one (C-5′), three methines [δC 37.9 (C-1), 51.6 (C-2), 77.7 (C-3)] including an oxygenated one (C-3), a carbonyl group [δC 175.6 (C-1″)], and a methoxyl (52.0). These evidences suggested that 2 should be a methyl jasmonate glucoside derivative. Comparison of the NMR spectral data indicated that 2 had the similar skeleton as methyl jasmonate glucosides [11,12], the major difference was the replacement of the carbonyl group in the latter by a hydroxyl in the former. Consequently, compound 2 was determined as a methyl jasmonate glycoside derivative as shown in Fig. 1. In HMBC spectrum (Fig. 4), the correlations of OCH3/C-1″; H-5′/ C-1‴; H-1′/C-1, C-3; H-2″/C-2, C-5, in combination with their
chemical shifts, verified the connections of methoxyl and C-1″, C-1‴ and C-5′, C-2 and C-1′, and C-2″and C-1. In the NOESY experiment of 2 (Fig. 4), the strong correlations of H-3 with H-2″a, H-1′, H-2′, and H-4′ indicated that H-3, methyl acetate side chain, and pentenyl side chain were cofacial, and that H-3, methyl acetate side chain occupied axial positions, whereas pentenyl side chain was an equatorial orientation, which suggested that the stereochemistry of 2 should be determined as 1S, 2R, 3R configuration. Accordingly, the structure of 2 was identified as methyl (1S,2R,3R)-2-[5-(β-D-glucopyranosyloxy)-2-penten-1-yl]-3-hydroxyl cyclopentane acetate, which was named linustam B. Compound 3 was isolated as a light yellow amorphous powder. Its HRESIMS m/z 451.3191 [M + Na]+ (calcd for C28H44O3Na, 451.3188), in combination with its NMR
Fig. 6. CD spectrum (a) and Mo-induced CD spectrum (b) of 3.
L. Song et al. / Fitoterapia 97 (2014) 15–22
21
Fig. 7. CD spectrum (a) and Mo-induced CD spectrum (b) of 4.
spectral data (Table 2), prompted the molecular formula of C28H44O3. Its IR spectrum revealed the presence of hydroxyl (3522 cm−1), olefinic bond (1650 cm−1), and saturated aliphatic protons (2957, 2924, and 2873 cm−1). The 1H NMR spectrum of 3 showed the protons due to a cis olefinic bond [δH 6.29 (1H, d, J = 8.4 Hz, H-6), 6.62 (1H, d, J = 8.4 Hz, H-7)], a trisubstituted olefinic bond [δH 5.45 (1H, dd, J = 6.0, 1.8 Hz, H-11)], a trans olefinic bond [δH 5.22 (1H, m, H-22),5.28 (1H, m, H-23)], an oxygenated methine [δH 3.79 (1H, m, H-3)], and six methyls [δH 0.77 (3H, s, H3-18),1.08 (3H, s, H3-19), 1.02 (1H, d, J = 6.6 Hz, H3-21), 0.85 (1H, d, J = 6.6 Hz, H3-26), 0.83 (1H, d, J = 6.6 Hz, H3-27), 0.93 (1H, d, J = 6.6 Hz, H3-28)]. The 13C NMR and DEPT spectra of 3 revealed 28 carbon signals ascribable to six methyls, six methylenes, eleven methines including three oxygenate methines and five sp2 carbons, and five quaternary carbons including two oxygenated quaternary carbons and a sp2 carbon. These data suggested that compound 3 was a steroid with 28 carbons, of which the structure was similar to that of DHOE (3a) reported in the literature [13]. This structure was further substantiated by 2D NMR experiments involving HSQC, HMBC, 1H–1H COSY and NOESY. Careful comparison of the NMR data of 3 with those of 3a (Table 2) revealed a close structural similarity between them, except for ring A and its own substituted groups. The upfield shifted values (δC 83.0 and 119.4) in 3 with respect to 3a (δC 86.7 and 129.1) for C-5 and C-11 let us conceive that the hydroxyl at C-5 in 3 could take a β-orientation, instead of a α-orientation in 3a. That is, ring A and ring B should be cis-fused in 3 (Fig. 5). The obvious difference of the chemical shifts at C-1, C-2, and CH3-19 between 3 (δC 33.5, 31.5, 25.8) and 3a (δC 39.3, 34.7, 18.0) further verified the conjecture. In the case of cis conformation of 3, the steric hindrance caused by 5-hydroxyl and CH3-19 with axial orientation resulted in the upfield shifted values of C-5 and C-11 when compared with 3a. According to the sector rule of chiral 1,3 diol [14], in the case of chiral Mo-complexes with rigid 1,3-diols, the sign of the cotton effect occurring around 400 nm is the same as the sign of the sector in which the majority of the molecule is located. In terms of 3,5-dihydroxycholestane, the conformer with syn-parallel orientation of 3α,5α-dihydroxyl groups
corresponds to a negative cotton effect, but the other conformer with 3β,5β-dihydroxyl groups corresponds to a positive cotton effect at around 400 nm. In order to further prove the cis conformation of 3,5-diol, the CD and Mo2(OAc)4-induced CD (DMSO) spectra of 3 were obtained (Fig. 6), respectively. The positive cotton effect at 380 nm (Δε + 0.06) observed in the Mo2(OAc)4-induced CD (DMSO) spectrum confirmed the cis configuration of 3β,5β-dihydroxyl groups. In addition, the resemblance of the NMR data between 3 and 3a in C, D-ring, in consideration of biogenesis, suggested a cis-fused relationship between the C ring and D-ring of 3, similar to that of 3a. Thus, the structure of 3 was determined as (22E)-ergosta-6,9,22-triene-3β,5β,8α-triol, and was named linustam C. Compound 4 was obtained as a light yellow amorphous powder. Its HRESIMS at m/z 430.3428 [M]+ (calcd for C28H46O3Na, 430.3447) demonstrated the molecular formula of C28H46O3. The 1H NMR and 13C NMR spectral data strongly resembled those of (22E)-ergosta-6,22-diene-3β,5α,8α-triol (4a) (Table 2) reported in the literature [15]. However, careful comparison of its NMR spectral data with those of 4a revealed that the chemical shifts of C-5 and C-8 at δC 82.37 and 79.42 in 4 were replaced by δC 79.36 and 82.12 in 4a, which were confirmed by HSQC, and the HMBC correlations of H-4, H3-19, H-7 with C-5, and H-6, H-11, H-14 with C-8. Referred to compound 3, the hydroxyls at C-3 and C-5 of 4 were suggested to take a β orientation, as well as ring A and ring B suggested to be cis-fused. Application of the same methods as described in 3, the positive cotton effect at 380 nm (Δε +0.02) observed in the Mo2(OAc)4-induced CD (DMSO) spectrum (Fig. 7) of 4, combined with the sector rule of chiral 1,3 diol, supported the cis configuration of ring A and ring B. Similarly, the resemblance of the spectral data of C ring and D ring between 4 and 4a, in consideration of the biogenesis, suggested a tran-fused relationship of C ring and D ring of 4, similar to that of 4a. Thus, the structure of compound 4a was revised as (22E)-ergosta-6,22-diene-3β,5β,8α-triol. The known compounds were identified by comparison of the spectroscopic data with those reported as methylβ-D-glucopyranosyl tuberonate (5) [11], sitosterol (6),
22
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stigmast-7-oxo-5-ene (7) [16], stigmast-5-ene-3,16-diol (8) [17], 7α-hydroxyl sitosterol (9) [18], 24-methylene cyloartenol (10) [19], 7-oxo-β-sitosterol (11) [20], and sucrose (12). All the isolates were assessed against nitrogen oxide (NO) production in macrophage induced by lipopolysaccharide (LPS), but were inactive at a concentration of 10−5 M. The positive control dexamethasone (DEX) gave an 82.2% inhibition at the same condition. Acknowledgment We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for measuring the IR, UV, NMR, and MS spectra. The authors are grateful to Dr. L. Li and Professor Yikang Si for the testing of CDs. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.05.008. References [1] Buranov AU, Ross KA, Mazza G. Isolation and characterization of lignins extracted from flax shives using pressurized aqueous ethanol. Bioresour Technol 2010;101:7446–55. [2] Prasad K. Regression of hypercholesterolemic atherosclerosis in rabbits by secoisolarici-resinol diglucoside isolated from flaxseed. Atherosclerosis 2008;197:34–42. [3] Struijs K, Vinchen JP, Verhoef R, Voragen AG, Gruppen H. Hydroxycinnamic acids are ester-linked directly to glucosyl moieties within the lignan macromolecule from flaxseed hulls. Phytochemistry 2008;69:1250–60. [4] Zhang CY, Zhang BG, Yang XW. Current status of investigations on chemical constituents and pharmacological effects of flaxseed (Linum usitatissimum). Chin J New Drug 2005;14:525–30.
[5] Meagher LP, Beecher GR, Flanagan VP, Li BW. Isolation and characterization of the lignans, isolariciresinol and pinoresinol, in flaxseed meal. J Agric Food Chem 1999;47:3173–80. [6] Zhao F, Wang SJ, Lin S, Zhu CG, Yuan SP, Ding XY, et al. Anthraquinones from the roots of Knoxia valerianoides. J Asian Nat Prod Res 2011;13:1023–9. [7] Marino SD, Borbone N, Zollo F, Ianaro A, Meglio PD, Iorizzi M. Megastigmane and phenolic components from Laurus nobilis L. leaves and their inhibitory effects on nitric oxide production. J Agric Food Chem 2004;52:7525–31. [8] Bari LD, Pescitelli G, Pratelli C, Pini D, Salvadori P. Determination of absolute configuration of acyclic 1,2-diols with Mo2(OAc)4. 1. Snatzke's method revisited. J Org Chem 2001;66:4819–25. [9] Liu J, Du D, Si YK, Lv HN, Wu XF, Li Y, et al. Application of dimolybdenum reagent Mo2(OAc)4 for determination of the absolute configuration of vicdiols. Chin J Org 2010;30:1270–8. [10] Politi M, Tommasi ND, Pescitelli G, Bari LD, Morelli I, Braca A. Structure and absolute configuration of new diterpenes from Lavandula multifida. J Nat Prod 2002;65:1742–5. [11] Matsuura H, Yoshihara T, Ichihara A, Kikuta Y, Koda Y. Tuber-forming substances in Jerusalem artichoke (Helianthus tuberosus L.). Biosci Biotechnol Biochem 1993;57:1253–6. [12] Inoue M, Kitahara T. Synthesis of both the enantiomers of methyl tuberonate, natural methyl β-D-glucopyranosyloxy jasmonate and its epimer. Tetrahedron 1999;55:4621–30. [13] Ponce MA, Ramirez JA, Galagovsky LR, Gros EG, Erra-Balsells R. A new look into the reaction between ergosterol and singlet oxygen in vitro. Photochem Photobiol Sci 2002;1:749–56. [14] Frelek J, Klimek A, Ruskowska P. Dinuclear transition metal complexes as auxiliary chromophores in chiroptical studies on bioactive compounds. Curr Org Chem 2003;7:1081–104. [15] Feng JT, Shi YP. Steroids from Saussurea ussuriensis. Pharmazie 2005; 60:464–7. [16] Ahmad MS, Ansari IA, Ansari SA, Moinuddin G. Synthesis of some steroidal tetrazoles from stigmastane series. Indian J Chem Sect B 1984; 11:1110–2. [17] Anjaneyulu ASR, Sagar KS, Prakash CVS. Two new steroid derivatives from the soft coral Sinularia gibberosa of Andaman & Nicobar Islands. Indian J Chem Sect B 1996;35B:819–25. [18] Zhang X, Geoffroy P, Miesch M, Diane JD, Raul F, Dalal AW, et al. Gran-scale chromatographic purification of β-sitosterol synthesis and characterization of β-sitosterol oxides. Steroids 2005;70:886–95. [19] Ikuta A, Itokawa H. Triterpenoids of Paeonia japonica callus tissue. Phytochemistry 1988;27:2813–5. [20] Pettit GR, Numata A, Gragg GM, Herald DL, Takada T, Iwamoto C, et al. Isolation and structures of schleicherastatins 1–7 and schleicheols 1 and 2 from the teak forest medicinal tree Schleichera oleosa. J Nat Prod 2000;63:72–8.
