Fitoterapia 95 (2014) 220–228
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New flavonoids from Campylotropis hirtella with immunosuppressive activity Xiaoping Li a,d, Bixia Xuan a, Qingyao Shou b, Zhengwu Shen a,c,⁎ a b c d
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, United States School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China Suzhou Polytechnic Institute of Agriculture, Suzhou 215008, PR China
a r t i c l e
i n f o
Article history: Received 18 November 2013 Accepted in revised form 26 March 2014 Available online 5 April 2014 Keywords: Campylotropis hirtella Coumaronochromone Isoflavanone Isoflavone Flavonol Immunosuppressive activity
a b s t r a c t In an effort to identify natural compounds with immunosuppressive activity, nine new flavonoids, including one isoflav-3-ene derivative (1), one coumaronochromone (2), two isoflavanones (3, 4), one isoflavone derivative (6), one isoflavone (7), three flavonols (8, 9, 10), as well as one known compound, hydroisoflavone C (5), were isolated from the roots of Campylotropis hirtella. The structures of these compounds were elucidated by extensive spectroscopic measurements. All of the compounds were assessed for immunosuppressive activity. Among the isolates, compound 2 showed good inhibitory activity against mitogen-induced splenocyte proliferation with an IC50 of 0.28 μM and relatively low cytotoxicity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Campylotropis hirtella (Franch.) Schindl. (family Leguminosae) (Fig. 1) is an undershrub of approximately 1 m in height distributed widely in the subtropical zones of China, such as the Yunnan, Sichuan and Guizhou provinces. The roots of this species have been used in traditional Chinese medicine for the treatment of irregular menstruation, dysmenorrhea, metrorrhagia, metrostaxis, as well as gastric ulcers, either alone or in combinations [1]. The species has previously been reported to contain lignans, sesquilignans, dilignans and coumarins in its roots by Yao et al. and some of the compounds showed inhibitory activity on prostate specific antigen secretion in LNCaP cells [2–4].Our group's earlier efforts have led to the isolation of a bunch of new flavonoids and their derivatives, most of which possess immunosuppressive activity [5–9]. The diversity of flavonoid structures and intriguing bioactivity of the flavonoids ⁎ Corresponding author. Tel.: +86 21 63846590; fax: +86 21 54650067. E-mail address: [email protected] (Z. Shen).
http://dx.doi.org/10.1016/j.fitote.2014.03.028 0367-326X/© 2014 Elsevier B.V. All rights reserved.
from this species attracted our attention and prompted us to perform a further chemical investigation; herein we report the isolation and structure elucidation of nine new flavonoids including one isoflav-3-ene derivative (1), one coumaronochromone (2), two isoflavanones (3, 4), one isoflavone derivative (6), one isoflavone (7), three flavonols (8, 9, 10), as well as one known compound, hydroisoflavone C (5) (Fig. 2). All of the isolates were assessed for their cytotoxicity and inhibitory activities against mitogen-induced splenocyte proliferation. Compound 2 was found to be the most active compound with an IC50 of 0.28 μM and relatively low cytotoxicity, while compounds 6, 7 and 8 show moderate activity. 2. Experimental 2.1. General experimental procedures UV spectra were acquired with a Shimadzu UV–Vis 2201 spectrometer, IR spectra were acquired with a Shimadzu FTIR-8400S Spectrometer, and optical rotations were acquired
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221
Fig. 1. The aerial part and roots of C. hirtella.
with an Autopol IV A2120 automatic polarimeter. CD spectra were acquired with a JASCO DIP-360J-500C polarimeter at 20 °C. ESI-MS was acquired with an Agilent 1100 1946D LC-MS spectrometer, HR-ESIMS was acquired with a Q-TOF Micro LC-MS-MS spectrometer, and NMR spectra were acquired with a Varian INOVA 400 spectrometer using TMS as the internal standard. Column chromatographic separations were carried out using silica gel H60 (300–400 mesh; Qingdao Haiyang Chemical Group Corporation), RP-C18 (50 μm; Merck) and Sephadex LH-20 (Amersham Pharmacia Biotech, Canada). Cyclosporine A (CsA, purity 99%, Sandimmun, 50 mg/mL) was manufactured by Novartis Pharma AG, Switzerland. All other chemical reagents were obtained from commercial vendors. 2.2. Plant material The roots of C. hirtella (Franch.) Schindl. were collected from Chuxiong, Yunnan Province, People's Republic of China in November 2009 and authenticated by Professor Xiling Li of Shanghai University of Traditional Chinese Medicine. A voucher specimen (no. 200911013) has been deposited in the herbarium of the Shanghai University of Traditional Chinese Medicine. 2.3. Extraction and isolation 1. The air-dried and comminuted roots of C. hirtella (2.0 kg) were extracted twice (each for 7 days) with 95% EtOH (2 × 5 L) at room temperature. The EtOH extract (80.0 g) was suspended in water (0.8 L) and extracted in successful steps using hexane (3 × 1 L) and EtOAc (3 × 1.5 L); the EtOAc portion was evaporated under reduced pressure to afford a crude extract (32.3 g). The crude EtOAc extract was subjected on a silica gel column (5 × 70 cm, 300 g silica gel, 200–300 mesh) eluted with a gradient of hexane/ EtOAc (30:1–0:1), to give seven fractions (A–G). Fraction B (4.8 g) was subjected to a silica gel column (4 × 60 cm, 200 g silica gel, 300–400 mesh) eluted with a gradient of hexane/EtOAc (8:2–0:1) to give a subfraction BII (328 mg), which was further applied to preparative HPLC (Merck C18
column, 5 μm, 125 × 25 mm; gradient elution with MeCN/ H2O containing 0.1% TFA from 5/3 to 9/1 for 15 min; UV detection at 254 nm; flow rate 14 mL/min;) to give compound 7 (14 mg). Fraction C (4.3 g) was divided into seven subfractions (CI–CVII) by a silica gel column (4 × 60 cm, 200 g silica gel, 200–300 mesh) eluted with a gradient of hexane/EtOAc (6:1–0:1). Compound 3 (18.0 mg) and compound 4 (13.5 mg) were obtained from subfraction CI (265 mg) and CV (396 mg), respectively, with the same method of preparative HPLC as compound 7. Subfraction CVII (567 mg) was purified by a C18 column (3 × 70 cm, 80 g C18 material, 50 μm) eluted with a gradient of MeOH/H2O (3:7–9:1) to give compound 5 (10.2 mg) and compound 6 (3.3 mg). Fraction D (4.0 g) was separated by using a silica gel column (4 × 60 cm, 200 g silica gel, 200–300 mesh) eluted with a gradient of hexane/ethyl acetate (7:1–0:1) to give five subfractions (DI–DV). After further purification using a C18 column (3 × 50 cm, 60 g C18 material, 50 μm) with a gradient of MeOH–H2O (4:6–9:1), Compounds 1 (4.7 mg) and 8 (13.7 mg) were obtained from subfraction DI (216 mg) while compound 2 (17.1 mg) was from subfraction DIV (327 mg); Fraction G (3.7 g) was separated using a Sephadex LH-20 column (3 × 60 cm, 100 g; mobile phase: CHCl3/ MeOH, 3:1) to give compound 9 (10.0 mg) and compound 10 (7.0 mg). 2.3.1. Compound (1) Yellow oil; [a]25 D 0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 269 (4.13), 383 (5.01) nm; IR (KBr) νmax 3304, 2926, 1639, 1614, 1450, 1379, 1263, 1182, 1163, 1091, 835 cm−1; 1H and 13C NMR (Table 1); HRESIMS m/z 399.1402 [M + Na]+ (calcd for C20H24O7Na, 399.1414); ESIMS m/z 375 [M − H]−. 2.3.2. Compound (2) Colourless needles (MeOH), mp: 274–276 °C; UV (MeOH) λmax (log ε) 258 (4.31), 289 (2.95) nm; IR (KBr) νmax 3517, 3122, 2918, 1623, 1448, 1344, 1286, 1114, 1070, 1037, 823 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 397.1277 [M + H]+ (calcd for C22H21O7, 397.1282); ESIMS m/z 397 [M + H]+.
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Fig. 2. Structures of compounds 1–10.
2.3.3. Compound (3) Colourless oil; [a]25 D +1.5° (c 0.3, MeOH); UV (MeOH) λmax (log ε) 294 (4.72) nm; IR (KBr) νmax 3392, 2968, 2923, 1637, 1602, 1496, 1448, 1286, 1215, 1159, 1076, 1047, 902, 827 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 459.2041 [M − H]− (calcd for C25H31O8, 459.2024); ESIMS m/z 459 [M − H]−. 2.3.4. Compound (4) Colourless oil; [a]25 D +4.0° (c 0.47, MeOH); UV (MeOH) λmax (log ε) 294 (4.91) nm; IR (KBr) νmax 3440, 2968, 2927, 1637, 1458, 1363, 1278, 1215, 1157, 1053, 827 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 473.2197 [M − H]− (calcd for C26H33O8, 473.2181); ESIMS m/z 473 [M − H]−. 2.3.5. Compound (6) Colourless oil; UV (MeOH) λmax (log ε) 261 (4.23), 299 (0.97) nm; IR (KBr) νmax 3588, 2927, 2858, 1662, 1618, 1560, 1458, 1317, 1197, 1124, 1058, 819 cm −1; 1H and 13C NMR
(Table 2); HRESIMS m/z 451.2076 [M + Na]+ (calcd for C25H32O6Na, 451.2091); ESIMS m/z 429 [M + H]+.
2.3.6. Compound (7) Colourless needles (MeOH), mp 163–164 °C; [a]25 D 0 (c 0.5, MeOH); UV (MeOH): λmax (log ε) 260 (4.12) nm; IR (KBr) νmax 3377, 2974, 1649, 1610, 1508, 1448, 1363, 1263, 1197, 1047, 825 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 463.1711 [M + Na]+ (calcd for C25H28O7Na, 463.1727); ESIMS m/z 441 [M + H]+.
2.3.7. Compound (8) Colourless oil; [a]25 D +7.0° (c 0.74, MeOH); UV (MeOH): λmax (log ε) 290 (5.01) nm; IR (KBr) νmax 3392, 2970, 2937, 1629, 1508, 1465, 1363, 1261, 1163, 1085, 1022, 831 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 441.1921 [M − H]− (calcd for C25H29O7, 441.1919); ESIMS m/z 441 [M − H]−.
X. Li et al. / Fitoterapia 95 (2014) 220–228
223
Table 1 NMR spectroscopic data of compounds 1–4 (δ values in ppm, J values in Hz). No.
1
2
δC 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 1″′ 2″′ 3″′ 4″′ 5′-Me 3″-Me 3″′-Me 5-OMe 7-OMe 2′-OMe
65.3 117.8 125.9 157.6 117.0 160.0 99.3 154.6 108.1 169.5 112.3 162.0 105.9
3
δH
δC
4.86, 4.89, ABq (13.2)
163.2
70.5
99.7 172.6 158.3 111.9 161.7 97.3 155.1 122.3 114.5 143.0 99.7 144.6 145.0 106.7 22.6 123.0 131.8 18.4
47.0 198.2 162.1 109.6 164.2 94.6 161.4 102.6 114.7 154.3 117.6 155.6 107.2 127.3 16.9 42.7 70.5 29.0 18.0 42.1 70.6 29.2
7.45, s
6.27, s
5.96, s
18.3 43.6 69.8 28.8
2.67, t (8.0) 1.69, t (8.0)
26.4 28.8
1.81, s 1.24, s
62.7
3.80, s
1.24, s
1
δH
4
7.13, s
7.07, s
7.35, s 3.30, d (7.2) 5.06, t (6.8) 1.73, s
26.2
1.61, s
62.8 57.2
3.77, s 3.91, s
1, 3 in acetone-d6, 2 in DMSO-d6, 4 in CD3OD; H NMR at 400 MHz,
δC
29.0 29.2
δH
δC
a 4.47, dd (5.2, 11.2) b 4.60, t (11.2) 4.20, dd (5.2, 8.8)
δH
71.3
4.37, dd (3.1, 6.0) 4.27, dd (6.4, 9.2)
1.24, s
45.9 198.3 161.6 109.2 164.8 94.1 161.7 102.6 119.5 158.1 122.9 156.4 111.1 127.0 17.0 42.2 70.6 27.8 19.4 42.8 70.6 27.7
1.24, s 1.24, s
27.8 27.7
1.24, s 1.24, s
61.6
3.75, s
6.00, s
6.39, 6.83, 2.66, 1.66,
d (8.4) d (8.4) t (8.0) t (8.0)
1.24, s 2.78, t (6.8) 1.75, t (6.8)
5.94, s
6.53, d (8.4) 6.76, d (8.4) 2.60, t (8.0) 1.63, t (8.0) 1.24, s 2.71, dd (8.8, 17.6) 1.74, t (8.0) 1.24, s
13
C NMR at 100 MHz.
2.3.8. Compound (9) Colourless oil; [a]25 D +2.0° (c 0.25, MeOH); UV (MeOH) λmax (log ε) 285 (4.78) nm; IR (KBr) νmax 3411, 2972, 2918, 1637, 1496, 1382, 1272, 1161, 1124, 1051, 827 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 469.1875 [M − H]− (calcd for C26H29O8, 469.1868); ESIMS m/z 469 [M − H]−. 2.3.9. Compound (10) Colourless oil; [a]25 D +6.0° (c 0.15, MeOH); UV (MeOH) λmax (log ε) 285 (4.63) nm; IR (KBr) νmax 3384, 2923, 1627, 1490, 1375, 1272, 1255, 1182, 1124, 1097, 827 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 469.1870 [M − H]− (calcd for C26H29O8, 469.1868); ESIMS m/z 469 [M − H]−. 2.4. Lymphocyte proliferation test Inbred 7–9-week-old BALB/c mice (IACUC approval no. 2012-02-ZJP-13) were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Science. Mice were sacrificed and their spleens were removed aseptically. A single spleen cell suspension was prepared and cell debris and clumps were removed. Erythrocytes were lysed with Tris-buffered ammonium chloride (0.155 M NH4Cl and 16.5 mM Tris, pH 7.2). Mononuclear cells were washed and resuspended in RPMI 1640 media (containing 10% FBS) supplemented with penicillin (100 IU/mL), and streptomycin (100 μg/mL). For in vitro assays, the spleens of five mice were pooled for cell separation. The isolated compounds were dissolved in pure DMSO as a stock solution,
stored at 4 °C and diluted as needed with RPMI-1640 media supplemented with 10% FBS. The final concentration of DMSO in the culture medium was less than 0.01%, with no influence on the assays. Splenocytes were cultured in triplicate for 48 h in 96-well plates with 5 μg/mL of concanavalin A (ConA) or 10 μg/mL of lipopolysaccharide (LPS) plus [compounds 1–10, cyclosporine A (CsA)]. Cells were pulsed with 0.5 μCi/well of [3H]-thymidine for 8 h, harvested onto glass filters and counted for incorporated radioactivity using a Beta Scintillation Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA). Cytotoxicity was assessed using the MTT assay. Briefly, splenocytes were cultured in triplicate for 48 h with compounds [compounds 1–10, cyclosporine A (CsA)]. The cells cultured with media alone were used as a control. MTT (5 mg/mL) reagent was added 4 h before the end of the culture, and the supernatants were discarded. Then, the cells were lysed with DMSO. The OD values were read at 570 nm, and the percentage of cell death was calculated [6,8]. 3. Results and discussion Compound 1 was isolated as a yellow oil. Its HRESIMS showed an ion peak at m/z 399.1402 [M + Na]+, corresponding to a molecular formula of C20H24O7. A maximum UV absorption at 269 nm and 383 nm indicated that 1 had a conjugated benzene ring. In the 1H and 13C NMR spectra (Table 1), the proton signals of an oxygen-bearing methylene [δH 4.86, 4.89 (2H, ABq, J = 13.2 Hz)], an olefinic proton at δH 7.45 (1H, s) and an aromatic proton at δH 6.27 (1H, s) as well
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Table 2 NMR spectroscopic data of compounds 6–10 (δ values in ppm, J values in Hz). 6
7
8
9
10
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
2 3 4 5 6 7 8 9 10 1′ 2′
153.6 128.3 182.3 159.8 111.9 162.5 93.0 156.2 105.2 70.4 33.8
8.15, s
153.5 123.6 181.0 162.9 99.1 164.4 93.8 158.4 105.4 122.4 130.5
8.11, s
83.9 72.4 197.7 164.3 96.4 167.2 95.4 163.5 100.9 128.4 129.6
5.06, d (11.6) 4.64, d (11.2)
84.4 73.7 198.3 162.0 105.0 165.5 95.3 161.6 101.2 129.9 128.6
5.07, d (11.6) 4.64, m
83.8 72.6 197.7 161.4 104.4 164.9 94.7 161.0 100.6 129.4 128.7
5.07, d (11.6) 4.64, m
3′
30.8
4′ 5′
69.3 30.8
6′
33.8
1″ 2″ 3″ 4″
21.3 122.4 134.7 39.9
5″
26.7
6″ 7″
124.5 130.9
8″ 9″ 10″ 11″ 6-Me 2″-Me 3″-Me 7″-Me 10″-Me
17.0
15.6 25.2
6.46, s
a 2.27, t (9.2) b 1.71, m a 1.76, m b 1.60, m 3.61, m a 1.76, m b 1.60, m a 2.27, t (9.2) b 1.71, m 3.34, m 5.27, t (6.8) 1.95, m
6.41, d (1,6)
7.35, d (1.6)
127.9 155.3 114.8 128.0 28.3 122.7 136.2 37.0
2.05, m (overlapped) 5.06, t (6.0)
30.0 77.7 72.2
1.55, s
25.2
1.77, s 1.60, s
6–10 in acetone-d6; 1H NMR at 400 MHz,
6.28, d (1.6)
15.7 24.4
5.98, d (1.9) 5.95, d (1.9)
7.33, d (1.9)
127.9
6.89, d (8.0) 7.26, dd (1.6, 8.4) 3.37, d (7.2) 5.43, t (7.2) a 2.08, m (overlapped) b 2.31, m a 1.37, m b 1.70, m 3.28,d (10.0)
1.10, s
1.74, s 1.10, s
6.03, s
7.67, s
125.3
6.03, s
7.67, s
125.3
155.7 114.8
6.89, d (8.0)
153.9 116.9
6.80, d (8.4)
153.1 116.5
6.80, d (8.4)
126.9
7.24, brd (8.4)
129.9
7.36, brd (8.4)
128.4
7.35, brd (8.4)
28.2 122.6 136.2 40.4
3.37, d (7.6) 5.39, t (7.2) 2.04, m
22.7
1.52, m
43.6 69.8
1.44, m
28.9
1.13, s
15.4 28.9
80.9 73.8 69.6
39.6 21.9 125.3 131.8 17.6 7.0 18.6
3.71, d (8.4) 4.63, m
1.74, 1.84, 2.21, 5.19,
m m m t (7.2)
80.6 76.8 68.7
31.6
3.65, d (7.6) 4.63, m
1.76, 1.54, 2.11, 5.06,
m m m m
1.63, s 1.99, s 1.22, s
21.7 124.7 131.1 16.9 6.4 22.7
1.53, s 1.98, s 1.46, s
1.68, s
25.1
1.61, s
1.71,s 1.13,s 25.8
13
C NMR at 100 MHz.
as the carbon signals of a trisubstituted double bond (δC 125.9, 117.8) and an oxygen-bearing carbon at δC 65.3 suggested the presence of a 2H-chromene core [10,11]. The signals of a methoxy group at [δH 3.80 (3H, s), δC 62.7] and a 3-hydroxy-3-methylbutyl group [δH 1.69 (2H, t, J = 8.0 Hz, H-2″), δC 43.6; δH 2.67 (2H, t, J = 8.0 Hz, H-1″), δC 18.3; δH 1.24 (6H, s)] were also observed in the NMR spectra. The methoxy group was unambiguously attached to C-5 by the HMBC correlations of H-4 (δH 7.45) and -OMe (δH 3.80) with C-5 (δC 157.6) and the assignment of the 3-hydroxy3-methylbutyl group was achieved by the correlations of H-1″ (δH 2.67) with C-5, C-6 and C-7. Thus, a moiety of 7-hydroxyl-5-methoxy 6-(3-hydroxyl-3-methylbutyl)-2Hchromene was established. The remaining signals were seen arising from a vinylic proton adjacent to a carbonyl group at δH 5.96 (1H, s), a carbonyl group at δC 169.5, a trisubstituted double bond (δC 112.3, 162.0), a sp3 carbon bearing two oxygens at 105.9 as well as a tertiary methyl at δH 1.81. The above signals suggested a unit of γ-methyl, γ-hydroxyl-α,β-unsaturated γ-lactone [12,13] and also supported by the analysis of the HMBC spectrum. The
linkage of the unit of γ-methyl,γ-hydroxyl-α,β-unsaturated γ-lactone with the 2H-chromene was achieved by the HMBC correlations of H-2 with C-4′ as well as H-3′ with C-3. Thus, the structure of 1 was elucidated as depicted. Optical activity of this compound was absent, which indicated that compound 1 might be a racemate the same as pratenol A [12]. The α,β-unsaturated γ-lactone of a B ring instead of a benzene ring is extremely rare in flavonoids and the origin of the α,β-unsaturated γ-lactone could be rationalized biogenetically and traced back to a B ring of 2,4-dihydroxylbenzene. After oxidation, decarboxylation, and cyclization, the moiety of γ-methyl,γ-hydroxyl-α,β-unsaturated γ-lactone could be formed, finally. The proposed biosynthetic pathway of compound 1 was illustrated in Fig. 5. Compound 2 was obtained as a colourless needle. The HRESIMS showed a peak at m/z 397.1277 [M + H]+, corresponding to the molecular formula of C22H20O7. The maximum UV absorption at 258 nm and 289 nm as well as the absence of a characteristic proton signal for 2-H (usually appearing at δH 8) (Table 1) indicated a coumaronochromone nature of 2 similar to hirtellanine A [7]. Typical α,β-unsaturated carbonyl
X. Li et al. / Fitoterapia 95 (2014) 220–228
225
Fig. 3. Key HMBC correlations of 1, 2, 3, 6, and 9.
signals (δC 172.6, 163.2, 99.7) (Table 1) from the 13C NMR spectrum supported the presence of a coumaronochromone skeleton. In the 1H NMR spectrum, two methoxy groups [δH 3.77 (3H, s, 5-OMe), 3.91 (3H, s, 7-OMe)], two hydroxyl groups [δH 9.29 (2H, brs)] as well as a 3-methylbut-2-enyl group [δH 1.61 (3H, s), 1.73 (3H, s), 3.30 (2H, d, J = 7.2 Hz) and 5.06 (1H, t, J = 6.8 Hz)] were observed. The HMBC correlations of the proton at δH 7.13 with C-6, C-7, C-9 and C-10 allowed the unequivocal assignment of this proton at C-8. The correlations of H-1″ (δH 3.30) to C-5, C-6 and C-7 as well as of the two methoxy groups (δH 3.77, 3.91) with C-5 and C-7 enabled the assignment of the 3-methylbut-2-enyl group at C-6 and the two methoxy groups at C-5 and C-7 (Fig. 3). Two isolated aromatic proton signals (δH 7.35, 7.07) and the signals of two adjacent hydroxyl groups (δH 9.29), as well as two carbon signals (δC 144.6, 145.0), suggested 4′- and 5′-dihydroxyl substitutions on the B ring. Thus, the structure of compound 2 was established as 4′,5′-dihydroxy-5,7-dimethoxy6-(3-methylbut-2-enyl) coumaronochromone.
Compound 3 was isolated as a colourless oil. The HRESIMS showed a peak at m/z 459.2041 [M − H]−, corresponding to the molecular formula C25H32O8. The IR spectrum indicated the presence of hydroxyl and carbonyl groups (3392, 1637 cm- 1). The 1H NMR spectrum showed three proton signals [δH 4.20 (1H, dd, J = 5.2, 8.8 Hz), 4.47 (1H, dd, J = 5.2, 11.2 Hz) and 4.60 (1H, t, J = 11.2 Hz)] (Table 1), which are typical H-3 and H2-2 signals of an isoflavanone [14]. The carbon signals [δC 70.5 (C-2), 47.0 (C-3), and 198.2 (C-4)] further confirmed the isoflavanone skeleton. The 1H NMR spectrum also indicated the presence of three aromatic protons including one assigned to H-8 of ring A and two ortho-coupled aromatic protons [δH 6.39 (1H, d, J = 8.4 Hz) and 6.83 (1H, d, J = 8.4 Hz)] on ring B; Two 3-hydroxyl3-methylbultyl groups [δH 2.78 (2H, t, J = 6.8 Hz), 2.66 (2H, t, J = 8.0 Hz), 1.75 (2H, t, J = 6.8 Hz), 1.66 (2H, t, J = 8.0 Hz), 1.24 (12H, s)] [11] were also observed. The HMBC correlations (Fig. 3) of H-1″′ (δH 2.78) with C-2′, C-3′ and C-4′ and of H-3 (δH 4.20) with C-2′ suggested the assignments
Fig. 4. Key NOESY (H–H) correlations of 9 and 10.
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Fig. 5. Proposed biosynthetic pathway of compound 1.
of one of the 3-hydroxyl-3-methylbutyl groups at C-3′ and two hydroxyl groups at C-2′ and C-4′, respectively. The other 3-hydroxyl-3-methylbutyl group was assigned at C-6 unequivocally by the HMBC correlations of H-1″ (δH 2.66) with C-2′, C-3′ and C-4′. Thus, the structure of 3 was identified as 6,3′-di(3-hydroxy-3-methylbutyl)-5,7,2′, 4′-tetrahydroxyisoflavanone. The chiral analysis suggested a racemate of compound 3 by the separation of the two enantiomers with reversed configurations at C-3 (see SI). Compound 4 was isolated as a colourless oil. The molecular formula of C26H34O8 was established by its HRESIMS (m/z 473.2197 [M − H]−). The 1H NMR spectrum (Table 1) of 4 differed from that of 3 only by the presence of a singlet at δH 3.75, which represented a methoxy group. The signal at δC 61.6 in the 13C NMR spectrum (Table 1) of 4 further confirmed the presence of a methoxy group. The HMBC correlation of δH 3.75 (OMe) with δC 158.1 (C-2′) allowed the assignment of the methoxy group at C-2′. In the CD spectrum, the positive Cotton effect at 310 nm and the negative Cotton effect at 294 nm suggested an R configuration of C-3 [15,16]. Based on the analysis above, the structure of 4 was elucidated as 3(R)-6,3′-di(3-hydroxy-3-methylbutyl)-2′-methoxyl-5,7,4′trihydroxyisoflavanone.
Compound 6, a colourless oil, had a molecular formula of C25H32O6, which was established from its HR-ESIMS at m/z 451.2076 [M + Na]+. The 1H NMR spectrum (Table 2) showed the typical H-2 signal of an isoflavone at δH 8.15 (1H, s), an aromatic proton at δH 6.46 (1H, s) assigned to H-8 of ring B as well as a geranyl group [δH 1.55, 1.60, 1.77 (3H, each, s), 5.06 (1H, t, J = 6.0 Hz), 5.27 (1H, t, J = 6.8 Hz), 3.34 (2H, m), 2.05 (2H, m, overlapped), and 1.95 (2H, m)]. However, except the chromenone moiety, no aromatic protons and carbons could be observed in NMR spectra, indicating a modified B ring. The presence of two pairs of symmetric methylenes, one oxygen-bearing methine, one oxygen-bearing quaternary carbon as well as the homonuclear coupling correlations of 2′ (6′)H/3′ (5′)H, 3′ (5′)H/4′ H suggested a moiety of 1,6-dihydroxyl-cyclohexane. The linkage with the chromenone moiety was defined by HMBC correlations of H-2 with C-1′ and of H-2′ (H-6′) with C-3. The NOESY correlations between H-2 and H-2′ as well as the analysis of the coupling constants of H-2′ and H-4′ placed OH-1′ in equatorial position and OH-4′ in axial position. Thus, the structure of compound 6 was determined to be a novel isoflavone derivative, 3-(1′,4′-dihydroxycyclohexyl)-6-geranyl5,7,-dihydroxyisoflavone.
Table 3 Cytotoxicity and immunosuppressive activities of compounds 1–10a. Compounds
Cytotoxicity
Proliferative responses of lymphocytes
CC50 (μM)
IC50 (μM)
SIb
IC50 (μM)
SIb
N100 N40 N100 75.8 N100 29.23 15.09 N10 73.32 48.22 1.08
53.70 0.28 79.13 18.12 53.16 3.25 4.52 4.34 20.18 3.48 0.01
N1.86 N142.86 N1.26 4.18 N1.88 8.99 29.02 N2.30 3.63 13.86 N108
14.96 1.55 29.12 6.66 25.76 2.68 2.38 4.82 20.83 3.52 0.97
N6.68 N25.81 N3.43 11.38 N3.88 10.91 6.34 N2.07 3.52 13.70 N1.11
ConA
1 2 3 4 5 6 7 8 9 10 CsA a b
LPS
The data shown here were from a representative experiment repeated three times with similar results. Selectivity index (SI) was determined as the CC50/IC50 value.
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Compound 7 was obtained as a colourless needle. The molecular formula C25H28O7 was obtained from its HR-ESIMS (m/z 463.1711 [M + Na]+). Analysis of the 1H and 13C NMR spectra suggested that compound 7 possessed an isoflavone skeleton with 5,7-dihydroxyl substitutions (δC 99.1 and 93.8; δH 6.28 and 6.41, d, J = 1.6 Hz). The proton signals [δH 1.10 (6H, s), 1.74 (3H, s), 5.43 (t, J = 7.2 Hz), 2.31 (1H, m), 2.08 (1H, m, overlapped), 1.70 (1H, m), and 1.37 (1H,m)], as well as the carbon signals (δC 77.7, 72.2), indicated the presence of a 6,7-dihydroxy-3,7-dimethyl-2(E)-octenyl group [17]. The E configuration of the double bond was confirmed by the upfield signals of the 3″-Me (δC 15.7) due to γ-gauche effects. In addition, three protons with an ABX spin system [δH 6.89 (d, J = 8.0 Hz), 7.26 (dd, J = 1.6, 8.4 Hz), and 7.35 (d, J = 1.6 Hz)] on the B ring were observed. In the HMBC spectrum, the proton corresponding to δC 130.5 in the HSQC was assigned to H-2′ (δH 7.35) based on its correlation with C-3, and correlations of H-1″ (δH 3.37) with C-2′, C-3′ and C-4′ allowed us to assign the 6,7-odihydroxy-3,7-dimethyl-2(E)octenyl group at C-3′. No Cotton effect was observed in the CD spectrum, which indicated that 7 might be a racemate. This was confirmed by the separation of the two enantiomers with a chiral column (see SI). Thus, the structure of 7 was established as 5,7,4′-trihydroxy-3′-[6,7-dihydroxy-3,7dimethyl-2(E)-octenyl] isoflavone. Compound 8 was obtained as a colourless oil. The molecular formula was determined to be C25H30O7 by HRESIMS analysis (m/z 441.1919 [M − H]−). In the 1H NMR data (Table 2), the signals of an AB system [δH 5.06 (1H, d, J = 11.6 Hz), 4.64 (1H, d, J = 11.2 Hz)], were assigned to the trans-diaxial H-2 and H-3 of a flavonol [18]. This assignment was also supported by the 13C NMR signals of C-2 and C-3 (δC 83.9, 72.4). The analysis of NMR data (δC 95.4, 96.4; δH 5.95; 5.98, d, J = 1.9 Hz) suggested a 5,7-dihydroxy substitution of ring A. The proton signals [δH 1.13 (6H, s), 5.39 (1H, t, J = 7.2 Hz), 2.04 (2H, m), 1.52 (2H, m) and 1.44 (2H, m)], as well as a quaternary carbon signal [δC 69.8 (C-7″)], demonstrated the presence of a 7-hydroxy-3,7-dimethyl-2(E)-octenyl group [7,19]. In addition, three protons appearing in an ABX spin system [δH 6.89 (d, J = 8.0 Hz), 7.24 (brd, J = 8.4 Hz), 7.33 (d, J = 1.9 Hz)] on the B ring as well as the HMBC correlations of H-1″ (δH 3.37) with C-2′, C-3′ and C-4′ allowed us to unequivocally assign the 7-hydroxy-3,7-dimethyl-2(E)-octenyl group at C-3′. Consequently, the remaining hydroxyl group could only be located at C-4′. Hence, the structure of compound 8 was concluded to be 5,7,4′-trihydroxy-3′-[7-hydroxy-3,7-dimethyl-2(E)-octenyl] flavonol. The positive Cotton effect at 328 nm and a negative Cotton effect at 293 nm in the CD spectrum suggested a 2R, 3R absolute configuration for compound 8 [20]. Thus, the structure of 8 was confirmed to be 2R,3R-3′-[7-hydroxy-3,7-dimethyl2(E)-octenyl]-2,3-trans-5,7,4′-trihydroxy-flavonol. Compound 9 was isolated as a colourless oil. An iron at m/z 469.1870 [M − H]− in the HRESIMS indicated a molecular formula of C26H30O8. The 1H NMR spectrum showed the proton signals of the trans-diaxial H-2 and H-3 [δH 5.07 (1H, d, J = 11.6 Hz) and 4.64 (1H, m)], an aromatic proton of H-8 at δH 6.03 as well as a methyl group assigned to C-6 from the interpretation of the HMBC spectrum. The above information suggested a 5,7-dihydroxyl-flavonol with a 6-methyl group. Three proton signals [δH 7.67 (s), 6.80 (d, J = 8.4 Hz) and 7.36 (brd, J = 8.4 Hz)] were assigned to the aromatic protons of a
227
3′, 4′-disubstituted B ring. NMR signals at δC 80.9, 69.6 and 73.8 as well as δH 4.63 (m) and 3.71 (d, J = 8.4 Hz) indicated a B ring fused dihydropyran group with a glycol unit. The HMBC correlations of H-4″ (δH 4.63) with C-2′, C-3′, and C-4′ implied the fusion of the dihydropyran group with ring B through C-3″ and C-4″. A methyl group was observed with attachment to the oxygen-bearing carbon (C-2″) from the HMBC correlations of 2″-Me (δH 1.22) with C-3″, C-2″ and C-7″ (Fig. 3). The remaining signals suggested a 4-methyl-3-pentenyl group as the second substituent at C-2″, based on the HMBC correlations of H-7″ (δH 1.74) with C-2″, C-3″ and C-8″. The NOESY spectrum (Fig. 4) showed a correlation between H-4″ and 2″-Me, indicating that these protons were on the same side of the molecule. The NOESY correlation between H-3″ and H-7″ placed them on the opposite side of the molecule. Therefore, the structure of compound 9 was established as depicted. Compound 10, isolated as colourless oil, had a peak at m/z 469.1875[M − H]−, corresponding to a molecular formula of C26H30O8. The 1H and 13C NMR spectra (Table 2) of 10 were very similar to those of 9 except the shifts of carbon signals from δC 18.6 (C-2″ Me), 73.8 (C-3″), 39.6 (C-7″) in 9 shift to δC 22.7 (C-2″ Me), 76.8 (C-3″), 31.6 (C-7″) in 10, respectively. In the NOESY spectrum (Fig. 4), the correlations of 2″-Me with H-3″ as well as of H-4″ with H-7″ indicated the reversed configurations of the two hydroxyl-bearing carbons (C-4″, 5″) compared with compound 9. In conclusion, compound 10 was established as depicted. Compound 1 is a novel isoflav-3-ene derivative possessing an α,β-unsaturated γ-lactone moiety, which is extremely scarce in the form of natural products. Similar compounds sharing the same skeleton have only been isolated from Red Clover (Trifolium pretense) [12]. Compound 5 was identified as hydroisoflavone C by comparing its physical and spectroscopic data with the literature values [17]. It is worth noting that compounds 5 and 6 are isoflavone derivatives bearing a cyclohexane moiety, which have never been identified from the plant resources with only several molecules including compound 5 reported from a bacterial strain [21]. The immunosuppressive activity of all the compounds was tested on mitogen-induced splenocyte proliferation and the cytotoxicity on splenic lymphocytes. The results (Table 3) indicated that compound 2 showed significant B lymphocyte inhibitory activity (IC50: 0.28 μM) and T lymphocyte inhibitory activity (IC50: 1.55 μM) with relatively low cytotoxicity. The immunosuppressive activity of compound 2 was very similar to that of hirtellanine A with even lower cytotoxicity. As compound 2 and hirtellanine A share the same coumaronochromone structure, further research into structure/activity relationships in this class might reveal compounds of medicinal utility. Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Original spectra for compounds 1–10; chiral HPLC analysis of compounds 3 and 7 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote.2014. 03.028.
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References [1] Chinese Pharmacopoeia Committee. Part 1. Pharmacopoeia of the People's Republic of China. Beijing: People's Hygiene Press; 1977. p. 30–1. [2] Han HY, Wen P, Liu HW, Wang NL, Yao XS. Coumarins from Campylotropis hirtella (Franch) Schindl. and their inhibitory activity on prostate specific antigen secreted from LNCaP Cells. Chem Pharm Bull 2008;56:1338–41. [3] Han HY, Wang XH, Wang NL, Ling MT, Wong YC, Yao XS. Lignans isolated from Campylotropis hitella (Franch.) Schindl. decreased prostate specific antigen and androgen receptor expression in LNCaP cells. J Agric Food Chem 2008;56:6928–35. [4] Han HY, Liu HW, Wang NL, Yao XS. Sesquilignans and dilignans from Campylotropis hitella (Franch.) Schindl. Nat Prod Res 2008;22:990–5. [5] Shou QY, Tan Q, Shen ZW. Isoflavonoids from the roots of Campylotropis hirtella. Planta Med 2010;76:803–8. [6] Shou QY, Tan Q, Shen ZW. Geranylated flavonoids from the roots of Campylotropis hirtella and their immunosuppressive activities. J Agric Food Chem 2009;57:6712–9. [7] Shou QY, Tan Q, Shen ZW. Hirtellanines A and B, a pair of isomeric isoflavonoid derivatives from Campylotropis hirtella and their immunosuppressive activities. Bioorg Med Chem Lett 2009;19:3389–91. [8] Tan Q, Zhang S, Shen ZW. Flavonoids from the roots of Campylotropis hirtella. Planta Med 2011;77:1811–7. [9] Zhang S, Tan Q, Shou QY, Qin GW, Shen ZW. Two new isoflavonones from Radix Campylotropis hirtella. Acta Chim Sin 2010;68:2227–30. [10] Miyase T, Sano M, Nakai H, Muraoka M, Nakazawa M, Suzuki M, et al. Antioxidants from Lespedeza homoloba. (I). Phytochemistry 1999; 52:303–10. [11] Seo JY, Lee YS, Kim HJ, Lim SS, Lim JS, Lee IA, et al. Dehydroglyasperin C isolated from licorice caused Nrf2-mediated induction of detoxifying enzymes. J Agric Food Chem 2010;58:1603–8.
[12] Yoshihara T, Sakuma T, Ichihara A. Yellow fluorescent stress compounds, Pratenols A and B, from Red Clover (Trifolium pratense) infected by Kabatiella caulivora. Biosci Biotechnol Biochem 1992;56:1955–8. [13] Park HS, Ohama T. Biotransformation of a herb plant metabolite by a cell disruptant of Chlamydomonas reinhardtii. Biosci Biotechnol Biochem 2009;73:2803–5. [14] Miyase T, Sano M, Yoshino K, Nonaka K. Antioxidants from Lespedeza homoloba. (II). Phytochemistry 1999;52:311–9. [15] Yahara S, Ogata T, Saijo R, Konishi R, Yamahara J, Miyahara K, et al. Isoflavan and related compounds from Dalbergia odorifera. Chem Pharm Bull 1989;37:979–87. [16] Galeffic C, Rasoanaivo P, Federici E, Palazzino G, Nicoletti MR, Rasolondratovo B. Two prenylated isoflavanones from Millettia pervilleana. Phytochemistry 1997;45:189–92. [17] Ibrahim AK, Radwan MM, Ahmed SA, Slade D, Ross SA, Elsohly MA, et al. Microbial metabolism of cannflavin A and B isolated from Cannabis sativa. Phytochemistry 2010;71:1014–9. [18] Benavides A, Bassarello C, Montoro P, Vilegas W, Piacente S, Pizza C. Flavonoids and isoflavonoids from Gynerium sagittatum. Phytochemistry 2007;68:1277–84. [19] Asai T, Hara N, Kobayashi S, Kohshima S, Fujimoto Y. Geranylated flavanones from the secretion on the surface of the immature fruits of Paulownia tomentosa. Phytochemistry 2008;69:1234–41. [20] Nonaka GI, Goto Y, Kinjo JE, Nohara T, Nishioka I. Tannins and related compounds. LII. Studies on the constituents of the leaves of Thujopsis dolabrata SIEB. et ZUCC. Chem Pharm Bull 1987;35:1105–8. [21] Tchize Ndejouong BLS, Sattler I, Dahse HM, Kothe E, Hertweck C. Isoflavones with unusually modified B-rings and their evaluation as antiproliferative agents. Bioorg Med Chem Lett 2009;19:6473–6.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New flavonoids from Campylotropis hirtella with immunosuppressive activity Xiaoping Li a,d, Bixia Xuan a, Qingyao Shou b, Zhengwu Shen a,c,⁎ a b c d
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, United States School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, PR China Suzhou Polytechnic Institute of Agriculture, Suzhou 215008, PR China
a r t i c l e
i n f o
Article history: Received 18 November 2013 Accepted in revised form 26 March 2014 Available online 5 April 2014 Keywords: Campylotropis hirtella Coumaronochromone Isoflavanone Isoflavone Flavonol Immunosuppressive activity
a b s t r a c t In an effort to identify natural compounds with immunosuppressive activity, nine new flavonoids, including one isoflav-3-ene derivative (1), one coumaronochromone (2), two isoflavanones (3, 4), one isoflavone derivative (6), one isoflavone (7), three flavonols (8, 9, 10), as well as one known compound, hydroisoflavone C (5), were isolated from the roots of Campylotropis hirtella. The structures of these compounds were elucidated by extensive spectroscopic measurements. All of the compounds were assessed for immunosuppressive activity. Among the isolates, compound 2 showed good inhibitory activity against mitogen-induced splenocyte proliferation with an IC50 of 0.28 μM and relatively low cytotoxicity. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Campylotropis hirtella (Franch.) Schindl. (family Leguminosae) (Fig. 1) is an undershrub of approximately 1 m in height distributed widely in the subtropical zones of China, such as the Yunnan, Sichuan and Guizhou provinces. The roots of this species have been used in traditional Chinese medicine for the treatment of irregular menstruation, dysmenorrhea, metrorrhagia, metrostaxis, as well as gastric ulcers, either alone or in combinations [1]. The species has previously been reported to contain lignans, sesquilignans, dilignans and coumarins in its roots by Yao et al. and some of the compounds showed inhibitory activity on prostate specific antigen secretion in LNCaP cells [2–4].Our group's earlier efforts have led to the isolation of a bunch of new flavonoids and their derivatives, most of which possess immunosuppressive activity [5–9]. The diversity of flavonoid structures and intriguing bioactivity of the flavonoids ⁎ Corresponding author. Tel.: +86 21 63846590; fax: +86 21 54650067. E-mail address: [email protected] (Z. Shen).
http://dx.doi.org/10.1016/j.fitote.2014.03.028 0367-326X/© 2014 Elsevier B.V. All rights reserved.
from this species attracted our attention and prompted us to perform a further chemical investigation; herein we report the isolation and structure elucidation of nine new flavonoids including one isoflav-3-ene derivative (1), one coumaronochromone (2), two isoflavanones (3, 4), one isoflavone derivative (6), one isoflavone (7), three flavonols (8, 9, 10), as well as one known compound, hydroisoflavone C (5) (Fig. 2). All of the isolates were assessed for their cytotoxicity and inhibitory activities against mitogen-induced splenocyte proliferation. Compound 2 was found to be the most active compound with an IC50 of 0.28 μM and relatively low cytotoxicity, while compounds 6, 7 and 8 show moderate activity. 2. Experimental 2.1. General experimental procedures UV spectra were acquired with a Shimadzu UV–Vis 2201 spectrometer, IR spectra were acquired with a Shimadzu FTIR-8400S Spectrometer, and optical rotations were acquired
X. Li et al. / Fitoterapia 95 (2014) 220–228
221
Fig. 1. The aerial part and roots of C. hirtella.
with an Autopol IV A2120 automatic polarimeter. CD spectra were acquired with a JASCO DIP-360J-500C polarimeter at 20 °C. ESI-MS was acquired with an Agilent 1100 1946D LC-MS spectrometer, HR-ESIMS was acquired with a Q-TOF Micro LC-MS-MS spectrometer, and NMR spectra were acquired with a Varian INOVA 400 spectrometer using TMS as the internal standard. Column chromatographic separations were carried out using silica gel H60 (300–400 mesh; Qingdao Haiyang Chemical Group Corporation), RP-C18 (50 μm; Merck) and Sephadex LH-20 (Amersham Pharmacia Biotech, Canada). Cyclosporine A (CsA, purity 99%, Sandimmun, 50 mg/mL) was manufactured by Novartis Pharma AG, Switzerland. All other chemical reagents were obtained from commercial vendors. 2.2. Plant material The roots of C. hirtella (Franch.) Schindl. were collected from Chuxiong, Yunnan Province, People's Republic of China in November 2009 and authenticated by Professor Xiling Li of Shanghai University of Traditional Chinese Medicine. A voucher specimen (no. 200911013) has been deposited in the herbarium of the Shanghai University of Traditional Chinese Medicine. 2.3. Extraction and isolation 1. The air-dried and comminuted roots of C. hirtella (2.0 kg) were extracted twice (each for 7 days) with 95% EtOH (2 × 5 L) at room temperature. The EtOH extract (80.0 g) was suspended in water (0.8 L) and extracted in successful steps using hexane (3 × 1 L) and EtOAc (3 × 1.5 L); the EtOAc portion was evaporated under reduced pressure to afford a crude extract (32.3 g). The crude EtOAc extract was subjected on a silica gel column (5 × 70 cm, 300 g silica gel, 200–300 mesh) eluted with a gradient of hexane/ EtOAc (30:1–0:1), to give seven fractions (A–G). Fraction B (4.8 g) was subjected to a silica gel column (4 × 60 cm, 200 g silica gel, 300–400 mesh) eluted with a gradient of hexane/EtOAc (8:2–0:1) to give a subfraction BII (328 mg), which was further applied to preparative HPLC (Merck C18
column, 5 μm, 125 × 25 mm; gradient elution with MeCN/ H2O containing 0.1% TFA from 5/3 to 9/1 for 15 min; UV detection at 254 nm; flow rate 14 mL/min;) to give compound 7 (14 mg). Fraction C (4.3 g) was divided into seven subfractions (CI–CVII) by a silica gel column (4 × 60 cm, 200 g silica gel, 200–300 mesh) eluted with a gradient of hexane/EtOAc (6:1–0:1). Compound 3 (18.0 mg) and compound 4 (13.5 mg) were obtained from subfraction CI (265 mg) and CV (396 mg), respectively, with the same method of preparative HPLC as compound 7. Subfraction CVII (567 mg) was purified by a C18 column (3 × 70 cm, 80 g C18 material, 50 μm) eluted with a gradient of MeOH/H2O (3:7–9:1) to give compound 5 (10.2 mg) and compound 6 (3.3 mg). Fraction D (4.0 g) was separated by using a silica gel column (4 × 60 cm, 200 g silica gel, 200–300 mesh) eluted with a gradient of hexane/ethyl acetate (7:1–0:1) to give five subfractions (DI–DV). After further purification using a C18 column (3 × 50 cm, 60 g C18 material, 50 μm) with a gradient of MeOH–H2O (4:6–9:1), Compounds 1 (4.7 mg) and 8 (13.7 mg) were obtained from subfraction DI (216 mg) while compound 2 (17.1 mg) was from subfraction DIV (327 mg); Fraction G (3.7 g) was separated using a Sephadex LH-20 column (3 × 60 cm, 100 g; mobile phase: CHCl3/ MeOH, 3:1) to give compound 9 (10.0 mg) and compound 10 (7.0 mg). 2.3.1. Compound (1) Yellow oil; [a]25 D 0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 269 (4.13), 383 (5.01) nm; IR (KBr) νmax 3304, 2926, 1639, 1614, 1450, 1379, 1263, 1182, 1163, 1091, 835 cm−1; 1H and 13C NMR (Table 1); HRESIMS m/z 399.1402 [M + Na]+ (calcd for C20H24O7Na, 399.1414); ESIMS m/z 375 [M − H]−. 2.3.2. Compound (2) Colourless needles (MeOH), mp: 274–276 °C; UV (MeOH) λmax (log ε) 258 (4.31), 289 (2.95) nm; IR (KBr) νmax 3517, 3122, 2918, 1623, 1448, 1344, 1286, 1114, 1070, 1037, 823 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 397.1277 [M + H]+ (calcd for C22H21O7, 397.1282); ESIMS m/z 397 [M + H]+.
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X. Li et al. / Fitoterapia 95 (2014) 220–228
Fig. 2. Structures of compounds 1–10.
2.3.3. Compound (3) Colourless oil; [a]25 D +1.5° (c 0.3, MeOH); UV (MeOH) λmax (log ε) 294 (4.72) nm; IR (KBr) νmax 3392, 2968, 2923, 1637, 1602, 1496, 1448, 1286, 1215, 1159, 1076, 1047, 902, 827 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 459.2041 [M − H]− (calcd for C25H31O8, 459.2024); ESIMS m/z 459 [M − H]−. 2.3.4. Compound (4) Colourless oil; [a]25 D +4.0° (c 0.47, MeOH); UV (MeOH) λmax (log ε) 294 (4.91) nm; IR (KBr) νmax 3440, 2968, 2927, 1637, 1458, 1363, 1278, 1215, 1157, 1053, 827 cm −1; 1H and 13C NMR (Table 1); HRESIMS m/z 473.2197 [M − H]− (calcd for C26H33O8, 473.2181); ESIMS m/z 473 [M − H]−. 2.3.5. Compound (6) Colourless oil; UV (MeOH) λmax (log ε) 261 (4.23), 299 (0.97) nm; IR (KBr) νmax 3588, 2927, 2858, 1662, 1618, 1560, 1458, 1317, 1197, 1124, 1058, 819 cm −1; 1H and 13C NMR
(Table 2); HRESIMS m/z 451.2076 [M + Na]+ (calcd for C25H32O6Na, 451.2091); ESIMS m/z 429 [M + H]+.
2.3.6. Compound (7) Colourless needles (MeOH), mp 163–164 °C; [a]25 D 0 (c 0.5, MeOH); UV (MeOH): λmax (log ε) 260 (4.12) nm; IR (KBr) νmax 3377, 2974, 1649, 1610, 1508, 1448, 1363, 1263, 1197, 1047, 825 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 463.1711 [M + Na]+ (calcd for C25H28O7Na, 463.1727); ESIMS m/z 441 [M + H]+.
2.3.7. Compound (8) Colourless oil; [a]25 D +7.0° (c 0.74, MeOH); UV (MeOH): λmax (log ε) 290 (5.01) nm; IR (KBr) νmax 3392, 2970, 2937, 1629, 1508, 1465, 1363, 1261, 1163, 1085, 1022, 831 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 441.1921 [M − H]− (calcd for C25H29O7, 441.1919); ESIMS m/z 441 [M − H]−.
X. Li et al. / Fitoterapia 95 (2014) 220–228
223
Table 1 NMR spectroscopic data of compounds 1–4 (δ values in ppm, J values in Hz). No.
1
2
δC 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 1″′ 2″′ 3″′ 4″′ 5′-Me 3″-Me 3″′-Me 5-OMe 7-OMe 2′-OMe
65.3 117.8 125.9 157.6 117.0 160.0 99.3 154.6 108.1 169.5 112.3 162.0 105.9
3
δH
δC
4.86, 4.89, ABq (13.2)
163.2
70.5
99.7 172.6 158.3 111.9 161.7 97.3 155.1 122.3 114.5 143.0 99.7 144.6 145.0 106.7 22.6 123.0 131.8 18.4
47.0 198.2 162.1 109.6 164.2 94.6 161.4 102.6 114.7 154.3 117.6 155.6 107.2 127.3 16.9 42.7 70.5 29.0 18.0 42.1 70.6 29.2
7.45, s
6.27, s
5.96, s
18.3 43.6 69.8 28.8
2.67, t (8.0) 1.69, t (8.0)
26.4 28.8
1.81, s 1.24, s
62.7
3.80, s
1.24, s
1
δH
4
7.13, s
7.07, s
7.35, s 3.30, d (7.2) 5.06, t (6.8) 1.73, s
26.2
1.61, s
62.8 57.2
3.77, s 3.91, s
1, 3 in acetone-d6, 2 in DMSO-d6, 4 in CD3OD; H NMR at 400 MHz,
δC
29.0 29.2
δH
δC
a 4.47, dd (5.2, 11.2) b 4.60, t (11.2) 4.20, dd (5.2, 8.8)
δH
71.3
4.37, dd (3.1, 6.0) 4.27, dd (6.4, 9.2)
1.24, s
45.9 198.3 161.6 109.2 164.8 94.1 161.7 102.6 119.5 158.1 122.9 156.4 111.1 127.0 17.0 42.2 70.6 27.8 19.4 42.8 70.6 27.7
1.24, s 1.24, s
27.8 27.7
1.24, s 1.24, s
61.6
3.75, s
6.00, s
6.39, 6.83, 2.66, 1.66,
d (8.4) d (8.4) t (8.0) t (8.0)
1.24, s 2.78, t (6.8) 1.75, t (6.8)
5.94, s
6.53, d (8.4) 6.76, d (8.4) 2.60, t (8.0) 1.63, t (8.0) 1.24, s 2.71, dd (8.8, 17.6) 1.74, t (8.0) 1.24, s
13
C NMR at 100 MHz.
2.3.8. Compound (9) Colourless oil; [a]25 D +2.0° (c 0.25, MeOH); UV (MeOH) λmax (log ε) 285 (4.78) nm; IR (KBr) νmax 3411, 2972, 2918, 1637, 1496, 1382, 1272, 1161, 1124, 1051, 827 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 469.1875 [M − H]− (calcd for C26H29O8, 469.1868); ESIMS m/z 469 [M − H]−. 2.3.9. Compound (10) Colourless oil; [a]25 D +6.0° (c 0.15, MeOH); UV (MeOH) λmax (log ε) 285 (4.63) nm; IR (KBr) νmax 3384, 2923, 1627, 1490, 1375, 1272, 1255, 1182, 1124, 1097, 827 cm −1; 1H and 13C NMR (Table 2); HRESIMS m/z 469.1870 [M − H]− (calcd for C26H29O8, 469.1868); ESIMS m/z 469 [M − H]−. 2.4. Lymphocyte proliferation test Inbred 7–9-week-old BALB/c mice (IACUC approval no. 2012-02-ZJP-13) were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Science. Mice were sacrificed and their spleens were removed aseptically. A single spleen cell suspension was prepared and cell debris and clumps were removed. Erythrocytes were lysed with Tris-buffered ammonium chloride (0.155 M NH4Cl and 16.5 mM Tris, pH 7.2). Mononuclear cells were washed and resuspended in RPMI 1640 media (containing 10% FBS) supplemented with penicillin (100 IU/mL), and streptomycin (100 μg/mL). For in vitro assays, the spleens of five mice were pooled for cell separation. The isolated compounds were dissolved in pure DMSO as a stock solution,
stored at 4 °C and diluted as needed with RPMI-1640 media supplemented with 10% FBS. The final concentration of DMSO in the culture medium was less than 0.01%, with no influence on the assays. Splenocytes were cultured in triplicate for 48 h in 96-well plates with 5 μg/mL of concanavalin A (ConA) or 10 μg/mL of lipopolysaccharide (LPS) plus [compounds 1–10, cyclosporine A (CsA)]. Cells were pulsed with 0.5 μCi/well of [3H]-thymidine for 8 h, harvested onto glass filters and counted for incorporated radioactivity using a Beta Scintillation Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA). Cytotoxicity was assessed using the MTT assay. Briefly, splenocytes were cultured in triplicate for 48 h with compounds [compounds 1–10, cyclosporine A (CsA)]. The cells cultured with media alone were used as a control. MTT (5 mg/mL) reagent was added 4 h before the end of the culture, and the supernatants were discarded. Then, the cells were lysed with DMSO. The OD values were read at 570 nm, and the percentage of cell death was calculated [6,8]. 3. Results and discussion Compound 1 was isolated as a yellow oil. Its HRESIMS showed an ion peak at m/z 399.1402 [M + Na]+, corresponding to a molecular formula of C20H24O7. A maximum UV absorption at 269 nm and 383 nm indicated that 1 had a conjugated benzene ring. In the 1H and 13C NMR spectra (Table 1), the proton signals of an oxygen-bearing methylene [δH 4.86, 4.89 (2H, ABq, J = 13.2 Hz)], an olefinic proton at δH 7.45 (1H, s) and an aromatic proton at δH 6.27 (1H, s) as well
224
X. Li et al. / Fitoterapia 95 (2014) 220–228
Table 2 NMR spectroscopic data of compounds 6–10 (δ values in ppm, J values in Hz). 6
7
8
9
10
δC
δH
δC
δH
δC
δH
δC
δH
δC
δH
2 3 4 5 6 7 8 9 10 1′ 2′
153.6 128.3 182.3 159.8 111.9 162.5 93.0 156.2 105.2 70.4 33.8
8.15, s
153.5 123.6 181.0 162.9 99.1 164.4 93.8 158.4 105.4 122.4 130.5
8.11, s
83.9 72.4 197.7 164.3 96.4 167.2 95.4 163.5 100.9 128.4 129.6
5.06, d (11.6) 4.64, d (11.2)
84.4 73.7 198.3 162.0 105.0 165.5 95.3 161.6 101.2 129.9 128.6
5.07, d (11.6) 4.64, m
83.8 72.6 197.7 161.4 104.4 164.9 94.7 161.0 100.6 129.4 128.7
5.07, d (11.6) 4.64, m
3′
30.8
4′ 5′
69.3 30.8
6′
33.8
1″ 2″ 3″ 4″
21.3 122.4 134.7 39.9
5″
26.7
6″ 7″
124.5 130.9
8″ 9″ 10″ 11″ 6-Me 2″-Me 3″-Me 7″-Me 10″-Me
17.0
15.6 25.2
6.46, s
a 2.27, t (9.2) b 1.71, m a 1.76, m b 1.60, m 3.61, m a 1.76, m b 1.60, m a 2.27, t (9.2) b 1.71, m 3.34, m 5.27, t (6.8) 1.95, m
6.41, d (1,6)
7.35, d (1.6)
127.9 155.3 114.8 128.0 28.3 122.7 136.2 37.0
2.05, m (overlapped) 5.06, t (6.0)
30.0 77.7 72.2
1.55, s
25.2
1.77, s 1.60, s
6–10 in acetone-d6; 1H NMR at 400 MHz,
6.28, d (1.6)
15.7 24.4
5.98, d (1.9) 5.95, d (1.9)
7.33, d (1.9)
127.9
6.89, d (8.0) 7.26, dd (1.6, 8.4) 3.37, d (7.2) 5.43, t (7.2) a 2.08, m (overlapped) b 2.31, m a 1.37, m b 1.70, m 3.28,d (10.0)
1.10, s
1.74, s 1.10, s
6.03, s
7.67, s
125.3
6.03, s
7.67, s
125.3
155.7 114.8
6.89, d (8.0)
153.9 116.9
6.80, d (8.4)
153.1 116.5
6.80, d (8.4)
126.9
7.24, brd (8.4)
129.9
7.36, brd (8.4)
128.4
7.35, brd (8.4)
28.2 122.6 136.2 40.4
3.37, d (7.6) 5.39, t (7.2) 2.04, m
22.7
1.52, m
43.6 69.8
1.44, m
28.9
1.13, s
15.4 28.9
80.9 73.8 69.6
39.6 21.9 125.3 131.8 17.6 7.0 18.6
3.71, d (8.4) 4.63, m
1.74, 1.84, 2.21, 5.19,
m m m t (7.2)
80.6 76.8 68.7
31.6
3.65, d (7.6) 4.63, m
1.76, 1.54, 2.11, 5.06,
m m m m
1.63, s 1.99, s 1.22, s
21.7 124.7 131.1 16.9 6.4 22.7
1.53, s 1.98, s 1.46, s
1.68, s
25.1
1.61, s
1.71,s 1.13,s 25.8
13
C NMR at 100 MHz.
as the carbon signals of a trisubstituted double bond (δC 125.9, 117.8) and an oxygen-bearing carbon at δC 65.3 suggested the presence of a 2H-chromene core [10,11]. The signals of a methoxy group at [δH 3.80 (3H, s), δC 62.7] and a 3-hydroxy-3-methylbutyl group [δH 1.69 (2H, t, J = 8.0 Hz, H-2″), δC 43.6; δH 2.67 (2H, t, J = 8.0 Hz, H-1″), δC 18.3; δH 1.24 (6H, s)] were also observed in the NMR spectra. The methoxy group was unambiguously attached to C-5 by the HMBC correlations of H-4 (δH 7.45) and -OMe (δH 3.80) with C-5 (δC 157.6) and the assignment of the 3-hydroxy3-methylbutyl group was achieved by the correlations of H-1″ (δH 2.67) with C-5, C-6 and C-7. Thus, a moiety of 7-hydroxyl-5-methoxy 6-(3-hydroxyl-3-methylbutyl)-2Hchromene was established. The remaining signals were seen arising from a vinylic proton adjacent to a carbonyl group at δH 5.96 (1H, s), a carbonyl group at δC 169.5, a trisubstituted double bond (δC 112.3, 162.0), a sp3 carbon bearing two oxygens at 105.9 as well as a tertiary methyl at δH 1.81. The above signals suggested a unit of γ-methyl, γ-hydroxyl-α,β-unsaturated γ-lactone [12,13] and also supported by the analysis of the HMBC spectrum. The
linkage of the unit of γ-methyl,γ-hydroxyl-α,β-unsaturated γ-lactone with the 2H-chromene was achieved by the HMBC correlations of H-2 with C-4′ as well as H-3′ with C-3. Thus, the structure of 1 was elucidated as depicted. Optical activity of this compound was absent, which indicated that compound 1 might be a racemate the same as pratenol A [12]. The α,β-unsaturated γ-lactone of a B ring instead of a benzene ring is extremely rare in flavonoids and the origin of the α,β-unsaturated γ-lactone could be rationalized biogenetically and traced back to a B ring of 2,4-dihydroxylbenzene. After oxidation, decarboxylation, and cyclization, the moiety of γ-methyl,γ-hydroxyl-α,β-unsaturated γ-lactone could be formed, finally. The proposed biosynthetic pathway of compound 1 was illustrated in Fig. 5. Compound 2 was obtained as a colourless needle. The HRESIMS showed a peak at m/z 397.1277 [M + H]+, corresponding to the molecular formula of C22H20O7. The maximum UV absorption at 258 nm and 289 nm as well as the absence of a characteristic proton signal for 2-H (usually appearing at δH 8) (Table 1) indicated a coumaronochromone nature of 2 similar to hirtellanine A [7]. Typical α,β-unsaturated carbonyl
X. Li et al. / Fitoterapia 95 (2014) 220–228
225
Fig. 3. Key HMBC correlations of 1, 2, 3, 6, and 9.
signals (δC 172.6, 163.2, 99.7) (Table 1) from the 13C NMR spectrum supported the presence of a coumaronochromone skeleton. In the 1H NMR spectrum, two methoxy groups [δH 3.77 (3H, s, 5-OMe), 3.91 (3H, s, 7-OMe)], two hydroxyl groups [δH 9.29 (2H, brs)] as well as a 3-methylbut-2-enyl group [δH 1.61 (3H, s), 1.73 (3H, s), 3.30 (2H, d, J = 7.2 Hz) and 5.06 (1H, t, J = 6.8 Hz)] were observed. The HMBC correlations of the proton at δH 7.13 with C-6, C-7, C-9 and C-10 allowed the unequivocal assignment of this proton at C-8. The correlations of H-1″ (δH 3.30) to C-5, C-6 and C-7 as well as of the two methoxy groups (δH 3.77, 3.91) with C-5 and C-7 enabled the assignment of the 3-methylbut-2-enyl group at C-6 and the two methoxy groups at C-5 and C-7 (Fig. 3). Two isolated aromatic proton signals (δH 7.35, 7.07) and the signals of two adjacent hydroxyl groups (δH 9.29), as well as two carbon signals (δC 144.6, 145.0), suggested 4′- and 5′-dihydroxyl substitutions on the B ring. Thus, the structure of compound 2 was established as 4′,5′-dihydroxy-5,7-dimethoxy6-(3-methylbut-2-enyl) coumaronochromone.
Compound 3 was isolated as a colourless oil. The HRESIMS showed a peak at m/z 459.2041 [M − H]−, corresponding to the molecular formula C25H32O8. The IR spectrum indicated the presence of hydroxyl and carbonyl groups (3392, 1637 cm- 1). The 1H NMR spectrum showed three proton signals [δH 4.20 (1H, dd, J = 5.2, 8.8 Hz), 4.47 (1H, dd, J = 5.2, 11.2 Hz) and 4.60 (1H, t, J = 11.2 Hz)] (Table 1), which are typical H-3 and H2-2 signals of an isoflavanone [14]. The carbon signals [δC 70.5 (C-2), 47.0 (C-3), and 198.2 (C-4)] further confirmed the isoflavanone skeleton. The 1H NMR spectrum also indicated the presence of three aromatic protons including one assigned to H-8 of ring A and two ortho-coupled aromatic protons [δH 6.39 (1H, d, J = 8.4 Hz) and 6.83 (1H, d, J = 8.4 Hz)] on ring B; Two 3-hydroxyl3-methylbultyl groups [δH 2.78 (2H, t, J = 6.8 Hz), 2.66 (2H, t, J = 8.0 Hz), 1.75 (2H, t, J = 6.8 Hz), 1.66 (2H, t, J = 8.0 Hz), 1.24 (12H, s)] [11] were also observed. The HMBC correlations (Fig. 3) of H-1″′ (δH 2.78) with C-2′, C-3′ and C-4′ and of H-3 (δH 4.20) with C-2′ suggested the assignments
Fig. 4. Key NOESY (H–H) correlations of 9 and 10.
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X. Li et al. / Fitoterapia 95 (2014) 220–228
Fig. 5. Proposed biosynthetic pathway of compound 1.
of one of the 3-hydroxyl-3-methylbutyl groups at C-3′ and two hydroxyl groups at C-2′ and C-4′, respectively. The other 3-hydroxyl-3-methylbutyl group was assigned at C-6 unequivocally by the HMBC correlations of H-1″ (δH 2.66) with C-2′, C-3′ and C-4′. Thus, the structure of 3 was identified as 6,3′-di(3-hydroxy-3-methylbutyl)-5,7,2′, 4′-tetrahydroxyisoflavanone. The chiral analysis suggested a racemate of compound 3 by the separation of the two enantiomers with reversed configurations at C-3 (see SI). Compound 4 was isolated as a colourless oil. The molecular formula of C26H34O8 was established by its HRESIMS (m/z 473.2197 [M − H]−). The 1H NMR spectrum (Table 1) of 4 differed from that of 3 only by the presence of a singlet at δH 3.75, which represented a methoxy group. The signal at δC 61.6 in the 13C NMR spectrum (Table 1) of 4 further confirmed the presence of a methoxy group. The HMBC correlation of δH 3.75 (OMe) with δC 158.1 (C-2′) allowed the assignment of the methoxy group at C-2′. In the CD spectrum, the positive Cotton effect at 310 nm and the negative Cotton effect at 294 nm suggested an R configuration of C-3 [15,16]. Based on the analysis above, the structure of 4 was elucidated as 3(R)-6,3′-di(3-hydroxy-3-methylbutyl)-2′-methoxyl-5,7,4′trihydroxyisoflavanone.
Compound 6, a colourless oil, had a molecular formula of C25H32O6, which was established from its HR-ESIMS at m/z 451.2076 [M + Na]+. The 1H NMR spectrum (Table 2) showed the typical H-2 signal of an isoflavone at δH 8.15 (1H, s), an aromatic proton at δH 6.46 (1H, s) assigned to H-8 of ring B as well as a geranyl group [δH 1.55, 1.60, 1.77 (3H, each, s), 5.06 (1H, t, J = 6.0 Hz), 5.27 (1H, t, J = 6.8 Hz), 3.34 (2H, m), 2.05 (2H, m, overlapped), and 1.95 (2H, m)]. However, except the chromenone moiety, no aromatic protons and carbons could be observed in NMR spectra, indicating a modified B ring. The presence of two pairs of symmetric methylenes, one oxygen-bearing methine, one oxygen-bearing quaternary carbon as well as the homonuclear coupling correlations of 2′ (6′)H/3′ (5′)H, 3′ (5′)H/4′ H suggested a moiety of 1,6-dihydroxyl-cyclohexane. The linkage with the chromenone moiety was defined by HMBC correlations of H-2 with C-1′ and of H-2′ (H-6′) with C-3. The NOESY correlations between H-2 and H-2′ as well as the analysis of the coupling constants of H-2′ and H-4′ placed OH-1′ in equatorial position and OH-4′ in axial position. Thus, the structure of compound 6 was determined to be a novel isoflavone derivative, 3-(1′,4′-dihydroxycyclohexyl)-6-geranyl5,7,-dihydroxyisoflavone.
Table 3 Cytotoxicity and immunosuppressive activities of compounds 1–10a. Compounds
Cytotoxicity
Proliferative responses of lymphocytes
CC50 (μM)
IC50 (μM)
SIb
IC50 (μM)
SIb
N100 N40 N100 75.8 N100 29.23 15.09 N10 73.32 48.22 1.08
53.70 0.28 79.13 18.12 53.16 3.25 4.52 4.34 20.18 3.48 0.01
N1.86 N142.86 N1.26 4.18 N1.88 8.99 29.02 N2.30 3.63 13.86 N108
14.96 1.55 29.12 6.66 25.76 2.68 2.38 4.82 20.83 3.52 0.97
N6.68 N25.81 N3.43 11.38 N3.88 10.91 6.34 N2.07 3.52 13.70 N1.11
ConA
1 2 3 4 5 6 7 8 9 10 CsA a b
LPS
The data shown here were from a representative experiment repeated three times with similar results. Selectivity index (SI) was determined as the CC50/IC50 value.
X. Li et al. / Fitoterapia 95 (2014) 220–228
Compound 7 was obtained as a colourless needle. The molecular formula C25H28O7 was obtained from its HR-ESIMS (m/z 463.1711 [M + Na]+). Analysis of the 1H and 13C NMR spectra suggested that compound 7 possessed an isoflavone skeleton with 5,7-dihydroxyl substitutions (δC 99.1 and 93.8; δH 6.28 and 6.41, d, J = 1.6 Hz). The proton signals [δH 1.10 (6H, s), 1.74 (3H, s), 5.43 (t, J = 7.2 Hz), 2.31 (1H, m), 2.08 (1H, m, overlapped), 1.70 (1H, m), and 1.37 (1H,m)], as well as the carbon signals (δC 77.7, 72.2), indicated the presence of a 6,7-dihydroxy-3,7-dimethyl-2(E)-octenyl group [17]. The E configuration of the double bond was confirmed by the upfield signals of the 3″-Me (δC 15.7) due to γ-gauche effects. In addition, three protons with an ABX spin system [δH 6.89 (d, J = 8.0 Hz), 7.26 (dd, J = 1.6, 8.4 Hz), and 7.35 (d, J = 1.6 Hz)] on the B ring were observed. In the HMBC spectrum, the proton corresponding to δC 130.5 in the HSQC was assigned to H-2′ (δH 7.35) based on its correlation with C-3, and correlations of H-1″ (δH 3.37) with C-2′, C-3′ and C-4′ allowed us to assign the 6,7-odihydroxy-3,7-dimethyl-2(E)octenyl group at C-3′. No Cotton effect was observed in the CD spectrum, which indicated that 7 might be a racemate. This was confirmed by the separation of the two enantiomers with a chiral column (see SI). Thus, the structure of 7 was established as 5,7,4′-trihydroxy-3′-[6,7-dihydroxy-3,7dimethyl-2(E)-octenyl] isoflavone. Compound 8 was obtained as a colourless oil. The molecular formula was determined to be C25H30O7 by HRESIMS analysis (m/z 441.1919 [M − H]−). In the 1H NMR data (Table 2), the signals of an AB system [δH 5.06 (1H, d, J = 11.6 Hz), 4.64 (1H, d, J = 11.2 Hz)], were assigned to the trans-diaxial H-2 and H-3 of a flavonol [18]. This assignment was also supported by the 13C NMR signals of C-2 and C-3 (δC 83.9, 72.4). The analysis of NMR data (δC 95.4, 96.4; δH 5.95; 5.98, d, J = 1.9 Hz) suggested a 5,7-dihydroxy substitution of ring A. The proton signals [δH 1.13 (6H, s), 5.39 (1H, t, J = 7.2 Hz), 2.04 (2H, m), 1.52 (2H, m) and 1.44 (2H, m)], as well as a quaternary carbon signal [δC 69.8 (C-7″)], demonstrated the presence of a 7-hydroxy-3,7-dimethyl-2(E)-octenyl group [7,19]. In addition, three protons appearing in an ABX spin system [δH 6.89 (d, J = 8.0 Hz), 7.24 (brd, J = 8.4 Hz), 7.33 (d, J = 1.9 Hz)] on the B ring as well as the HMBC correlations of H-1″ (δH 3.37) with C-2′, C-3′ and C-4′ allowed us to unequivocally assign the 7-hydroxy-3,7-dimethyl-2(E)-octenyl group at C-3′. Consequently, the remaining hydroxyl group could only be located at C-4′. Hence, the structure of compound 8 was concluded to be 5,7,4′-trihydroxy-3′-[7-hydroxy-3,7-dimethyl-2(E)-octenyl] flavonol. The positive Cotton effect at 328 nm and a negative Cotton effect at 293 nm in the CD spectrum suggested a 2R, 3R absolute configuration for compound 8 [20]. Thus, the structure of 8 was confirmed to be 2R,3R-3′-[7-hydroxy-3,7-dimethyl2(E)-octenyl]-2,3-trans-5,7,4′-trihydroxy-flavonol. Compound 9 was isolated as a colourless oil. An iron at m/z 469.1870 [M − H]− in the HRESIMS indicated a molecular formula of C26H30O8. The 1H NMR spectrum showed the proton signals of the trans-diaxial H-2 and H-3 [δH 5.07 (1H, d, J = 11.6 Hz) and 4.64 (1H, m)], an aromatic proton of H-8 at δH 6.03 as well as a methyl group assigned to C-6 from the interpretation of the HMBC spectrum. The above information suggested a 5,7-dihydroxyl-flavonol with a 6-methyl group. Three proton signals [δH 7.67 (s), 6.80 (d, J = 8.4 Hz) and 7.36 (brd, J = 8.4 Hz)] were assigned to the aromatic protons of a
227
3′, 4′-disubstituted B ring. NMR signals at δC 80.9, 69.6 and 73.8 as well as δH 4.63 (m) and 3.71 (d, J = 8.4 Hz) indicated a B ring fused dihydropyran group with a glycol unit. The HMBC correlations of H-4″ (δH 4.63) with C-2′, C-3′, and C-4′ implied the fusion of the dihydropyran group with ring B through C-3″ and C-4″. A methyl group was observed with attachment to the oxygen-bearing carbon (C-2″) from the HMBC correlations of 2″-Me (δH 1.22) with C-3″, C-2″ and C-7″ (Fig. 3). The remaining signals suggested a 4-methyl-3-pentenyl group as the second substituent at C-2″, based on the HMBC correlations of H-7″ (δH 1.74) with C-2″, C-3″ and C-8″. The NOESY spectrum (Fig. 4) showed a correlation between H-4″ and 2″-Me, indicating that these protons were on the same side of the molecule. The NOESY correlation between H-3″ and H-7″ placed them on the opposite side of the molecule. Therefore, the structure of compound 9 was established as depicted. Compound 10, isolated as colourless oil, had a peak at m/z 469.1875[M − H]−, corresponding to a molecular formula of C26H30O8. The 1H and 13C NMR spectra (Table 2) of 10 were very similar to those of 9 except the shifts of carbon signals from δC 18.6 (C-2″ Me), 73.8 (C-3″), 39.6 (C-7″) in 9 shift to δC 22.7 (C-2″ Me), 76.8 (C-3″), 31.6 (C-7″) in 10, respectively. In the NOESY spectrum (Fig. 4), the correlations of 2″-Me with H-3″ as well as of H-4″ with H-7″ indicated the reversed configurations of the two hydroxyl-bearing carbons (C-4″, 5″) compared with compound 9. In conclusion, compound 10 was established as depicted. Compound 1 is a novel isoflav-3-ene derivative possessing an α,β-unsaturated γ-lactone moiety, which is extremely scarce in the form of natural products. Similar compounds sharing the same skeleton have only been isolated from Red Clover (Trifolium pretense) [12]. Compound 5 was identified as hydroisoflavone C by comparing its physical and spectroscopic data with the literature values [17]. It is worth noting that compounds 5 and 6 are isoflavone derivatives bearing a cyclohexane moiety, which have never been identified from the plant resources with only several molecules including compound 5 reported from a bacterial strain [21]. The immunosuppressive activity of all the compounds was tested on mitogen-induced splenocyte proliferation and the cytotoxicity on splenic lymphocytes. The results (Table 3) indicated that compound 2 showed significant B lymphocyte inhibitory activity (IC50: 0.28 μM) and T lymphocyte inhibitory activity (IC50: 1.55 μM) with relatively low cytotoxicity. The immunosuppressive activity of compound 2 was very similar to that of hirtellanine A with even lower cytotoxicity. As compound 2 and hirtellanine A share the same coumaronochromone structure, further research into structure/activity relationships in this class might reveal compounds of medicinal utility. Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Original spectra for compounds 1–10; chiral HPLC analysis of compounds 3 and 7 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote.2014. 03.028.
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