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New chalcone and pterocarpoid derivatives from the roots of Flemingia philippinensis with antiproliferative activity and apoptosis-inducing property
Fitoterapia 112 (2016) 222–228
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New chalcone and pterocarpoid derivatives from the roots of Flemingia philippinensis with antiproliferative activity and apoptosis-inducing property Wen-Jia Kang 1, Da-Hong Li 1, Tong Han, Lin Sun, Yan-Bin Fu, Chun-Mei Sai, Zhan-Lin Li ⁎, Hui-Ming Hua ⁎ Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e
i n f o
Article history: Received 19 April 2016 Received in revised form 11 June 2016 Accepted 13 June 2016 Available online 14 June 2016 Keywords: Flemingia philippinensis Leguminosae Chalcone Pterocarpoid Apoptosis
a b s t r a c t Investigation of the roots of Flemingia philippinensis resulted in the isolation of two new chalcones, flemiphilippinones B (1) and C (2), and one new pterocarpoid, demethylwedelolactone-11-methyl ether (3), together with 12 known compounds (4-15). The antiproliferative activity against PC-3 cells was evaluated and most compounds showed cytotoxicity, among which, compound 2 exhibited GI50 value of 14.12 μM. The antiproliferative activity of 2 against Bel-7402 and CaEs-17 cells was also measured, with GI50 values of 1.91 and 2.58 μM, respectively. Intensive mechanism study showed that 2 caused cell-cycle arrest at S/G2 phase and induced apoptosis in Bel-7402 cells through mitochondria-related pathway. © 2016 Published by Elsevier B.V.
Introduction The Flemingia (Leguminosae) genus, containing N40 species, is mainly distributed in tropical region of Africa, Asia, and Australia. About 20 species can be found in China, which are widely distributed from the east to the southwest. Several species are used as ethnodrugs for a long history to cure rheumatoid arthritis, lumbocrural pain, lumbar muscle strain, swollen feet, chronic nephritis, menopausal syndrome and so on [1]. Previous chemical investigations have demonstrated that the genus is a rich source of isoflavonoids, chalcones, flavones, terpenoids, and phenolics [2–6], exhibiting various biological activities [5–8]. Among them, isoflavonoids are the major bioactive ingredients and have been obtained from the roots, stems, flowers and seeds of the Flemingia plants, some of which exhibit anti-cancer, antioxidant and chemical protective activities [4,5]. Flemingia philippinensis (Merr. et Rolfe) Li is a medicinal valuable species distributing in the southwest of China and its dried roots have long been used as folk medicine for the treatment of rheumatoid arthritis and swelling of throat. It is also being used to relieve pain and induce sleep. The previous studies on chemical constituents of the roots and dried leaves of F. philippinensis afforded isoflavones, flavanones, flavonoid glycosides, and steroids [9–13]. It has been found that some isolated compounds showed anti-cancer, antioxidant, immunosuppressive, estrogenic and antiestrogenic activities [13–16]. ⁎ Corresponding authors. E-mail addresses: [email protected] (Z.-L. Li), [email protected] (H.-M. Hua). 1 Da-Hong Li and Wen-Jia Kang contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2016.06.003 0367-326X/© 2016 Published by Elsevier B.V.
In this paper, the chemical constituents of the roots of F. philippinensis were isolated by various kinds of column chromatography (CC) on silica gel, Sephadex LH-20, and ODS, and also preparative thin layer chromatography (PTLC) to yield fifteen compounds (Fig. 1), including three new ones, flemiphilippinones B (1), C (2), and demethylwedelolactone-11-methyl ether (3). The structures were identified by the physico-chemical characters and the spectroscopic analysis. By comparison with the published data, the known compounds were identified as coumestrol (4) [17], lupinalbin A (5) [18], formononetin (6) [19], biochanin A (7) [20], 5,7-dihydroxy-6,8diprenylchromone (8) [21], eriosematin (9) [21], isoeriosematin (10) [21], auriculasin (11) [22], flemiphilippinin A (12) [23,24], (2R)flemichin D (13) [25], 5,2′,4′-trihydroxy-8,5′-di-(3-methylbut-2enyl)-6,7-(2,2-dimethylpyrano)flavanone (14) [12], and flemiphilippinin D (15) [10]. Herein, we report the isolation and structural elucidation of three new compounds, as well as the antiproliferative activity of the obtained compounds against PC-3 cell line by MTT method. Structure-activity relationship (SAR) was also concluded. Antiproliferative activity against Bel-7402 and CaEs-17 cells and apoptosis related mechanism of new compound 2 in Bel-7402 cells concerning apoptosis-inducing effects and cell cycle arrest were also disclosed.
Materials and methods The UV spectra were recorded with a Shimadzu UV-2201 spectrometer. HR ESIMS was measured on a Bruker Micro-TOFQ-Q mass
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Fig. 1. The structures of compounds 1–15 from F. philippinensis.
spectrometer. NMR data were obtained from Bruker AV-600 NMR spectrometer using TMS as an internal standard. Silica gel (200–300 mesh) performed for column chromatography was purchased from Qingdao Ocean Chemical Factory and ODS (50 μm) was purchased from YMC Co. Ltd., Kyoto Japan. The Sephadex LH-20 was purchased from GE Healthcare. A Shimadzu SPD-20A series equipped with an YMC C18 column (250 × 20 mm, 5 μm) was used for HPLC analysis. 5-fluorouracil (5-Fu, purity N 99% by HPLC) was purchased from Aladdin. All the organic solvents were purchased from Yuwang Chemicals Industries, Ltd., China, and used without further purification. PC-3 (human prostate cancer) cell line was obtained from the American Type Culture Collection.
The roots of F. philippinensis were collected from Guangxi province, China, in 2008 and were identified by Prof. Qishi Sun, Shenyang Pharmaceutical University, Shenyang, China. The voucher sample (HHM200807) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China. The dry roots of F. philippinensis (20.6 kg) were extracted with ethanol (3 × 20 L 95:5, v/v) under reflux for three times (2 h, each) and concentrated under reduced pressure to give black-brown gum of 1626 g. The residue was suspended in water and partitioned with petroleum ether (3 × 3 L) and CHCl3 (3 × 3 L), successively. The CHCl3 soluble part (390 g) was chromatographed on silica gel CC (i.d. 10 × 150 cm) eluting
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with a PE/acetone gradient system from 100:0 to 0:100 to give 5 fractions Fr.A–E based on their TLC characteristic. Fr.C (PE/acetone 85:15, 32 g) was performed on silica gel CC (i.d. 4 × 80 cm, PE/acetone gradient system from 100:0 to 0:100) to give 5 fractions Fr.C1–C5. Fr.C1 (317 mg) was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to afford compounds 6 (5 mg) and 13 (35 mg). Fr.C2 was recrystallized with PE/acetone to get 11 (25 mg). Fr.C3 was performed on silica gel CC (i.d. 1 × 50 cm, PE/acetone 8:1) to give 14 (40 mg). Fr.C4 was recrystallized with PE/acetone to get 12 (30 mg). Silica gel CC (i.d. 4 × 80 cm) eluting with PE/acetone gradient system from 100:0 to 0:100 of Fr.D (PE/acetone 85:15, 20 g) gave 5 major fractions Fr.D1–D5. Fr.D1 (127 mg) then submitted to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to give 5 (10 mg). Fr.D2 (116 mg) was chromatographed on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to obtain 7 (10 mg). Fr.D3 was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to give 3 (2 mg), and 4 (10 mg). Fr.D4 (76 mg) was separated on ODS CC (i.d. 1.5 × 60 cm) eluting with MeOH/H2O 75:25 to yield 1 (3 mg). Fr.D5 was applied to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to afford 15 (30 mg). The PE soluble part (33 g) was fractionated by silica gel column chromatography (CC, i.d. 5 × 120 cm) and eluted with a PE/acetone gradient system from 100:0 to 0:100 to give 4 fractions Fr.F–I based on their TLC characteristic. Fr.F (PE/acetone 98:2, 421 mg) was separated on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and followed by PTLC (20 × 20 cm, PE/acetone 7:1) to obtain 2 (18 mg). Fr.G (PE/acetone 97:3, 267 mg) was performed on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and then PTLC (20 × 20 cm, PE/acetone 5:1) to obtain 9 (5 mg). Fr.H (PE/acetone 95:5, 325 mg) was separated on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to get 10 (13 mg). Fr.I (PE/acetone 95:5, 265 mg) was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and PTLC (20 × 20 cm, cyclohexane/acetone 5:1) to afford 8 (5 mg). PC-3 cells were plated in a 96-well plate and the test samples were dissolved in DMSO to obtain stock solution. After 24 h spreading, different dilutions of compound 2 were added into each well at 37 °C incubating for 4 days. The medium was removed and cells were then treated with MTT solution for an additional 4 h. After discarding culture
Table 2 NMR dataa and HMBC correlations of compound 3 (in DMSO-d6). 1
No
H
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5,7,12-OH 11-OCH3 a
6.37 (1H, s) 6.40 (1H, s) 7.30 (1H, s)
7.21 (1H, s)
10.82 (1H, s), 10.44 (1H, s), 9.50 (1H, s) 3.88 (3H, s)
13
C
158.0 101.2 159.7 95.5 155.2 95.0 161.3 99.2 155.5 101.7 146.9 146.3 99.2 149.4 113.8
HMBC
C-5, C-8 C-4, C-6, C-7 C-11, C-12, C-15
C-11, C-12, C-14
56.2
Chemical shifts (δ) were expressed in ppm and J in Hz.
medium, the cells were dissolved in 200 μL DMSO and the optical density (OD) at 570 nm was measured by a microplate reader spectrophotometer. The concentration of a compound (purity N 98% by HPLC) inhibiting half of the cell growth was calculated. The results were obtained from three independent experiments. Bel-7402 cells (5.0 × 103 cells/well) were plated in 6-well plates and incubated with 2 at 37 °C for 24 h, and stained with PI. Cellular DNA content, for cell cycle distribution analysis, was measured using a flow cytometer (FACS Calibur Becton-Dickinson). Cells were incubated with 2 for 72 h and apoptosis was analyzed using Annexin-V and PI double staining by flow cytometry according to the manufacturer's instructions in order to detect apoptotic cells. Bel-7402 cells were cultured in six-well plates after treatment with 2 or vehicle for 48 h. The cells were stained with the lipophilic cationic dye JC-1, according to the manufacturer's instruction (Keygen, KGA601). The percentage of cells
Table 1 NMR dataa of compounds 1 and 2. 1 (in CDCl3) No 1 2 3 4 5 6 C=O α β 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴ 2′–OH OCH3 a
2 (in CDCl3) 1
H
7.15 (1H, d, J = 8.6 Hz) 6.82 (1H, d, J = 8.6 Hz) 6.82 (1H, d, J = 8.6 Hz) 7.15 (1H, d, J = 8.6 Hz) 3.33 (1H, m), 3.25 (1H, m) 2.96 (2H, t, J = 7.6 Hz)
2.98 (1H, m) 3.08 (1H, dd, J = 15.1, 9.5 Hz) 4.73 (1H, d, J = 9.6 Hz) 1.22 (3H, s) 1.33 (3H, s) 3.04 (2H, d, J = 9.2 Hz) 4.76 (1H, d, J = 9.2 Hz) 1.31 (3H, s) 1.22 (3H, s) 13.30 (1H, s) 3.78 (3H, s)
Chemical shifts (δ) were expressed in ppm and J in Hz.
13
C
133.0 129.3 114.0 157.9 114.0 129.3 212.8 44.4 29.7 99.5 159.6 105.7 161.8 96.5 163.7 27.2 92.5 72.1 24.0 26.0 26.8 92.0 71.8 26.0 24.0 55.4
No 1 2 3 4 5 6 C=O α β 1′ 2′ 3′ 4′ 5′ 6′ 1″, 1‴ 2″, 2‴ 3″, 3‴ 4″, 4‴ 5″, 5‴ 1″″ 2″″ 3″″ 4″″ 5″″ OH OCH3
1
H
7.20 (1H, d, J = 8.6 Hz) 6.83 (1H, d, J = 8.6 Hz) 6.83 (1H, d, J = 8.6 Hz) 7.20 (1H, d, J = 8.6 Hz) 3.30 (2H, t, J = 7.8 Hz) 2.87 (2H, t, J = 7.8 Hz)
2.70 (1H, dd, J = 13.8, 7.5 Hz) 2.50 (1H, dd, J = 13.8, 7.5 Hz) 4.78 (2H, d, J = 7.5 Hz) 1.57 (3H, s) 1.57 (3H, s) 6.46 (1H, d, J = 10.1 Hz) 5.35 (1H, d, J = 10.1 Hz) 1.44 (3H, s) 1.44 (3H, s) 18.88 (1H, s) 3.78 (3H, s)
13
C
133.6 129.6 113.9 158.0 113.9 129.6 202.1 42.1 30.4 108.0 195.4 57.2 172.0 106.2 186.3 37.8 118.2 134.9 25.9 18.2 114.6 123.4 81.3 28.9 28.9 55.4
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Fig. 2. Key HMBC correlations of compounds 1 and 2.
Table 3 GI50a values of fifteen compounds isolated from F. philippinensis against PC-3 cells. Compound
GI50 ± SD (μM)
Compound
GI50 ± SD (μM)
1 2 3 4 5 6 7 8
NTb 14.12 ± 0.64 NT N100 42.76 ± 2.64 32.84 ± 0.33 64.25 ± 2.32 N100
9 10 11 12 13 14 15 5-Fuc
N100 N100 8.33 ± 0.12 10.13 ± 0.30 22.73 ± 0.85 27.45 ± 1.03 10.88 ± 0.34 39.37 ± 2.76
a GI50 is the concentration of a compound inhibiting half of the cell growth. The results given are means ± SD of three independent experiments carried out in duplicate. b NT = not test. c Positive control.
with healthy or collapsed mitochondrial membrane potentials was monitored by flow cytometry analysis. Flemiphilippinone B (1): yellow oil; UV (MeOH) λmax: 291 and 222 nm; 1H and 13C NMR data (measured in CDCl3) see Table 1; ESIMS: m/z = 457.3 [M + H]+, 479.2 [M + Na]+, 455.1 [M-H]−; HR ESIMS: m/z = 457.2240 (calcd. for C H32O7H+: 457.2226). Flemiphilippinone C (2): yellow oil; UV (MeOH) λmax: 245, 271, 221, and 202 nm; 1H and 13C NMR data (measured in CDCl3) see Table 1; ESIMS: m/z = 491.3 [M + H]+, 513.3 [M + Na]+, 489.1 [M-H]−; HR ESIMS: m/z = 513.2616 (calcd. for C31H38O5Na+: 513.2617). Demethylwedelolactone-11-methyl ether (3): white amorphous powder; UV (MeOH) λmax: 348 and 254 nm; 1H and 13C NMR data (measured in DMSO-d6) see Table 2; HR ESIMS: m/z = 315.0501 [M + H]+ (calcd. for C16H10O7H+, 315.0499).
Fig. 3. Cell cycle effects of compound 2 in Bel-7402 cells.
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Results and discussion Compound 1 was obtained as yellow oil and designated with a molecular formula of C26H32O7 based on quasi-molecular ion at m/z 457.2240 [M + H]+ (cacld. 457.2226) in its HRESIMS. The UV absorptions at 291 and 222 nm indicated that 1 established the presence of an o-hydroxyl aromatic carbonyl moiety [14]. The 1H NMR spectrum (Table 1) displayed 4 methyl singlets at δ 1.33 (3H, s), 1.31 (3H, s), and 1.22 (6H, s), two oxygenated methine doublets at δ 4.73 (1H, d, J = 9.6 Hz) and 4.76 (1H, d, J = 9.2 Hz), two aromatic proton doublets at δ 7.15 (2H, d, J = 8.6 Hz) and 6.82 (2H, d, J = 8.6 Hz) for a para-disubstituted benzene ring (ring B), two methylenes at δ 3.33 (1H, m), 3.25 (1H, m), and 2.96 (2H, t, J = 7.8 Hz) attributed to -CO-CH2-CH2-Ar and a methoxy signal at δ 3.78 (3H, s), as well as a downfield signal of a chelated hydroxyl group at δ 13.30 (1H, s). The 13C NMR spectrum (Table 1) of 1 showed 26 signals which disclosed the presence of a fully substituted aromatic ring (δ 96.5, 99.5, 105.7, 159.6, 161.8 and 163.7), a para-disubstituted benzene and a carbonyl carbon at δ 212.8. Base on the above data, compound 1 was inferred to be a dihydrochalcone with prenyl-related functionalities. The 1 H and 13 C NMR data were assigned by the HSQC and HMBC spectra (Fig. 2). The HMBC correlations of H-1″ with C-2′, C-3′, C4′, C-2″, and C-3″, and of H3–4″ and H3–5″ with C-2″ and C-3″ confirmed the presence of a furan ring substituted with an isopropanol, which was fused to C-3′ and C-4′. Similar method ensured that the other furan ring was attached to C-5′ and C-6′. The phenolic hydroxyl of 2′–OH at δ 13.30 showed correlations with C-1′, C-2′ and C-3′. The above data established the tricyclic structure in the left hand. The methoxy proton signal at δ 3.78 correlated with C-4 suggesting the
methoxy located at C-4. Therefore, 1 was determined as shown and named flemiphilippinone B. Compound 2 was isolated as yellow oil. Its molecular formula was deduced to be C31H38O5 by HR ESIMS (m/z 513.2616 [M + Na]+). The UV spectrum showed absorption maximum at 245, 271, 221, and 202 nm. The 1H NMR spectrum of 2 (Table 1) exhibited signals for a para-disubstituted aromatic ring at δ 7.20 (2H, d, J = 8.6 Hz), and 6.83 (2H, d, J = 8.6 Hz), a dimethylchromene moiety at δ 6.46 (1H, d, J = 10.1 Hz), 5.35 (1H, d, J = 10.1 Hz), and 1.44 (6H, s), two prenyl groups at δ 4.78 (2H, t, J = 7.5 Hz), 2.70 (2H, dd, J = 13.8, 7.5 Hz), 2.50 (2H, dd, J = 13.8, 7.5 Hz) and δ 1.57 (12H, s, Me × 4), a methoxy group at δ 3.78 and an enol hydroxyl at δ 18.88. The 13C NMR spectrum exhibited 31 carbon signals, including two ketone carbonyl carbons (δ 202.1 and 195.4). Comparison of 1H and 13C NMR data of 2 with 1 indicated that they possess the same 4-methoxy hydrocinnamoyl. The structure of 2 was deduced from detailed analysis of 1H and 13C NMR data (Table 1) together with 2D-NMR experiments (Fig. 2). The left moiety was established by the HMBC correlations from the proton at δ 6.46 of a dimethylchromene to C-4′ (δ 172.0), C-5′ (δ 106.2) and C-6′ (δ 186.3), from H-1″ and H-1‴ of two prenyls at δ 2.70, 2.50 to C-2′ (δ 195.4), C3′ (δ 57.2), and C-4′ (δ 172.0). The long-range correlations of H-β at δ 2.87 (2H, t, J = 7.8 Hz) to a carbonyl carbon at δ 202.1, and of H-α at δ 3.30 (2H, t, J = 7.8 Hz) to the carbonyl carbon (δ 202.1) and C-1 (δ 133.6) determined the linkage of two moieties via C-1′ and the carbonyl carbon. Thus, the structure of compound 2 was assigned as shown and named flemiphilippinone C. Compound 3 was obtained as white amorphous powder with the molecular formula C16H10O7 determined by a quasi-molecular ion
Fig. 4. Apoptotic property of compound 2 in Bel-7402 cells.
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peak at m/z 315.0501 [M + H]+ in its HR ESIMS. It exhibited UV absorption bands at 348 and 254 nm. The 1H NMR spectrum of 3 (Table 2) exhibited signals for three phenolic hydroxyl protons at δ 10.82, 10.44, and 9.50, and four aromatic proton singlets at δ 7.30, 7.21, 6.40, and 6.37, as well as a methoxy signal at δ 3.88. The 13C NMR spectrum (Table 2) revealed 16 carbon resonances, including a methoxyl group at δ 56.2 and 15 sp2 carbons. The 1H and 13C NMR chemical shifts were closely similar to those of the known compound wedelolactone [26], with the difference of H-6 and H-8 appearing at a higher field and H-10 appearing at a lower field, indicating the methoxy located at C-11 instead of C-7. The above deduction was supported by the HMBC correlations from H-8 (δ 6.40) to C-4 (δ 95.5), C-6 (δ 95.0) and C-7 (δ 161.3), from H-6 (δ 6.37) to C-5 (δ 155.2), and C-8 (δ 99.2), from H-10 (δ 7.30) to C-11 (δ 146.9), C-12 (δ 146.3) and C-15 (δ 113.8), and from H-13 (δ 7.21) to C-11, C-12 and C-14 (δ 149.4), as well as between the methoxy signal δ 3.88 and C-11. Thus, compound 3 was established as demethylwedelolactone-11-methyl ether. All the isolated compounds were tested for the growth inhibitory effects against PC-3 cell line using the MTT assay; the results were listed in Table 3. Among them, 11 exhibited the most potent in vitro antiproliferative effect with a GI50 value of 8.33 μM. The flavonoids (2, 5–7, and 11– 15) showed moderate inhibitory effects, while the chromone derivatives (8–10) showed no activity. Compared with 9, B ring with 3′,4′-dihydroxy group in the isoflavonone structure (11) would play an important role in inhibiting the growth of PC-3 cells. Isoflavonone 12 processing 3′,4′-dihydroxy group also showed strong antiproliferative activity with a GI50 value of 10.13 μM. Flavanones 13, 14, and 15 demonstrated good antiproliferative effects with GI50 values ranging from 10.88 to 27.45 μM, of which 15 with two prenyls is more active than the related compound 13 with dimethylchromene formed by a prenyl group. It was noteworthy that novel compound 2 also exhibited potent effect with a GI50 value of
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14.12 μM. Furthermore, the cytotoxicity of 2 against Bel-7402 (human hepatoma) and CaEs-17 (human gastric cancer) cell lines was evaluated and the GI50 values were 1.91 and 2.58 μM, respectively. Intensive mechanism study of 2 was carried out in Bel-7402 cells. Bel-7402 cells were treated with compound 2 at concentrations of 0.5, 1.0, and 2.0 μM, which resulted in accumulation of 34.03, 45.08, and 48.61% of cells at the S phase, respectively, compared with the untreated cells 22.30% (Fig. 3). There were almost no changes of G1 phase cells with 42.34, 38.14, and 41.86% cells, and the decline of G2 phase cells were observed of 23.63, 16.78 and 9.53%, respectively. So, compound 2 influenced cell-cycle progression of Bel-7402 cells at low micromolar concentrations and arrested at S/G2 phase. In order to examine the involvement of apoptosis in the loss of cancer cell viability of compound 2 in Bel-7402 cells, an annexin V-FITC/ propidium iodide (PI) binding assay was carried out. Bel-7402 cells were treated with different concentrations of 2 and percentages of apoptotic cells were determined by flow cytometry. As shown in Figs. 4, 2 exhibited potent dose-dependent activity in the induction of apoptosis. Treatment of Bel-7402 cells with 2 at 0.5, 1.0, and 1.5 μM, apoptotic cell rates (early and late) were 18.86, 35.45, and 59.79%, as compared with 8.09% in an untreated vehicle control, indicating that 2 was able to induce apoptotic cell death in Bel-7402 cells. Apoptosis could result in the loss of mitochondrial membrane potential. To determine plausible pathways that compound 2 triggered cell apoptosis, Bel-7402 cells were incubated with 0, 0.5, 1.5, and 2.0 μM of 2 prior to staining with the lipophilic mitochondrial probe JC-1. The number of cells with collapsed mitochondrial membrane potentials in different groups was determined by flow cytometry analysis (Fig. 5), yielding 2.87, 9.01, 19.53, and 28.49% apoptotic cells, respectively. These results showed that incubation with 2 increased the number of cells with collapsed mitochondrial membrane potentials.
Fig. 5. Effect of mitochondrial membrane potentials by compound 2 in Bel-7402 cells.
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Conclusions The chemical investigation of the roots of F. philippinensis led to the isolation of three new (1–3) and twelve known compounds (4–15). The structures of 1–3 were elucidated on the basis of their spectroscopic data. The results of in vitro antiproliferative activity showed that 2 and 11–15 exhibited strong growth inhibitory effects against PC-3 cells with GI50 values b 30 μM. The GI50 values of 2 against Bel-7402 and CaEs-17 cells were 1.91 and 2.58 μM, respectively. Intensive mechanism study of 2 was also carried out, and Bel-7402 cells were treated with compound 2 at concentrations of 0, 0.5, 1.0, and 2.0 μM, which revealed that 2 caused cell cycle arrest at S/G2 phase in a dose-dependent manner at low micromolar concentrations. The annexin V-FITC/PI binding assay and mitochondrial probe JC-1 assay revealed that the loss of cancer cell viability by 2 was involved of apoptosis. Conflict of interest
[6] [7]
[8]
[9] [10] [11] [12] [13]
[14]
The authors declare no conflict of interest. [15]
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31570350), General Scientific Research Projects of Department of Education in Liaoning Province (L2014382), and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.
[16]
[17]
[18] [19]
Appendix A. Supplementary data [20]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.06.003.
[21] [22]
References [1] Y.S. Wei, The classification and distribution of the genus Flemingia Roxb. ex Ait. in China, Guihaia 11 (1991) 198–207. [2] M.M. Rahman, S.D. Sarker, M. Byres, A.I. Gray, New salicylic acid and isoflavone derivatives from Flemingia paniculata, J. Nat. Prod. 67 (2004) 402–406. [3] H. Soicke, K. Görler, H. Waring, Flavonol glycosides from Moghania faginea, Planta Med. 56 (1990) 410–412. [4] I. Gumula, J.P. Alao, I.O. Ndiege, P. Sunnerhagen, A. Yenesew, M. Erdélyi, Flemingins G-O, cytotoxic and antioxidant constituents of the leaves of Flemingia grahamiana, J. Nat. Prod. 77 (2014) 2060–2067. [5] H.M. Chiang, H.H. Chiu, S.T. Liao, Y.T. Chen, H.C. Chang, K.C. Wen, Isoflavonoid-rich Flemingia macrophylla extract attenuates UVB-induced skin damage by scavenging
[23] [24] [25] [26]
reactive oxygen species and inhibiting MAP kinase and MMP expression, Evid. Based Complement. Alternat. Med. 2013 (2013) 696879. Y.J. Shiao, C.N. Wang, W.Y. Wang, Y.L. Lin, Neuroprotective flavonoids from Flemingia macrophylla, Planta Med. 71 (2005) 835–840. H.Y. Ho, J.B. Wu, W.C. Lin, Flemingia macrophylla extract ameliorates experimental osteoporosis in ovariectomized rats, Evid. Based Complement. Alternat. Med. 2011 (2011) 752302. Y.L. Lin, H.J. Tsay, Y.F. Liao, M.F. Wu, C.N. Wang, Y.J. Shiao, Components of Flemingia macrophylla attenuate amyloid β-protein accumulation by regulating amyloid βprotein metabolic pathway, Evid. Based Complement. Alternat. Med. 2012 (2012) 795843. G. Xie, B. Lin, X. Qin, G. Wang, Q. Wang, J. Yuan, C. Li, M. Qin, New flavonoids with cytotoxicity from the roots of Flemingia latifolia, Fitoterapia 104 (2015) 97–101. M. Chen, S.Q. Luo, J.H. Chen, Studies on the chemical constituents of Flemingia philippinensis, Acta Pharmacol. Sin. 26 (1990) 42–48. H. Li, F. Zhai, M. Yang, X. Li, P. Wang, X. Ma, A new benzofuran derivative from Flemingia philippinensis Merr. et Rolfe, Molecules 17 (2012) 7637–7644. E.M. Ahn, N. Nakamura, T. Akao, K. Komatsu, M.H. Qiu, M. Hattori, Prenylated flavonoids from Moghania philippinensis, Phytochemistry 64 (2003) 1389–1394. E.M. Ahn, N. Nakamura, T. Akao, T. Nishihara, M. Hattori, Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents, Biol. Pharm. Bull. 27 (2004) 548–553. Y. Wang, M.J. Curtis-Long, B.W. Lee, H.J. Yuk, D.W. Kim, X.F. Tan, K.H. Park, Inhibition of tyrosinase activity by polyphenol compounds from Flemingia philippinensis roots, Bioorg. Med. Chem. 22 (2014) 1115–1120. L. Li, X. Deng, L. Zhang, P. Shu, M. Qin, A new coumestan with immunosuppressive activities from Flemingia philippinensis, Fitoterapia 82 (2011) 615–619. Y. Wang, M.J. Curtis-Long, H.J. Yuk, D.W. Kim, X.F. Tan, K.H. Park, Bacterial neuraminidase inhibitory effects of prenylated isoflavones from roots of Flemingia philippinensis, Bioorg. Med. Chem. 21 (2013) 6398–6404. E.M. Bickoff, A.N. Booth, R.L. Lyman, A.L. Livingston, C.R. Thompson, F. Deeds, Coumestrol, a new estrogen isolated from forage crops, Science 126 (1957) 969–970. S. Tahara, J.L. Ingham, J. Mizutani, New coumaronochromones from white lupin, Lupinus albus L. (Leguminosae), Agric. Biol. Chem. 49 (1985) 1775–1783. J.E. Kinjo, J.I. Furusawa, J. Baba, T. Takeshita, M. Yamasaki, T. Nohara, Studies on the constituents of Pueraria lobata. III. Isoflavonoids and related compounds in the roots and the voluble stems, Chem. Pharm. Bull. 35 (1987) 4846–4850. Y.R. Deng, T. Wang, Y.Z. He, Studies on chemical constituents of Caragana spinifera, China J. Chin. Materia. Med. 33 (2008) 775–777. P.G. Waterman, E.G. Crichton, Xanthones and biflavonoids from Garcinia densivenia stem bark, Phytochemistry 19 (1980) 2723–2726. D.M. Giuliano, S. Rosalba, V. Alberto, B. Bruno, M. Barbara, P. Gabriella, P. Cleofe, C. Enrico, Two isoflavones and a flavone from the fruits of Maclura pomifera, Phytochemistry 37 (1994) 893–898. H. Li, M. Yang, J. Miao, X. Ma, Prenylated isoflavones from Flemingia philippinensis, Magn. Reson. Chem. 46 (2008) 1203–1207. M. Chen, S.Q. Lou, J.H. Chen, Two isoflavones from Flemingia philippinensis, Phytochemistry 30 (1991) 3842–3844. W.G. Ma, N. Fuzzati, Q.S. Li, C.R. Yang, H. Stoeckli-Evans, K. Hostettmann, Polyphenols from Eriosema tuberosum, Phytochemistry 39 (1995) 1049–1061. J. Wu, W.B. Hou, T.J. Zhang, Y.M. Han, Chemical constituents of Eclipta prostrata, Chin. Tradit. Herb. Drugs 39 (2008) 814–816.
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New chalcone and pterocarpoid derivatives from the roots of Flemingia philippinensis with antiproliferative activity and apoptosis-inducing property Wen-Jia Kang 1, Da-Hong Li 1, Tong Han, Lin Sun, Yan-Bin Fu, Chun-Mei Sai, Zhan-Lin Li ⁎, Hui-Ming Hua ⁎ Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e
i n f o
Article history: Received 19 April 2016 Received in revised form 11 June 2016 Accepted 13 June 2016 Available online 14 June 2016 Keywords: Flemingia philippinensis Leguminosae Chalcone Pterocarpoid Apoptosis
a b s t r a c t Investigation of the roots of Flemingia philippinensis resulted in the isolation of two new chalcones, flemiphilippinones B (1) and C (2), and one new pterocarpoid, demethylwedelolactone-11-methyl ether (3), together with 12 known compounds (4-15). The antiproliferative activity against PC-3 cells was evaluated and most compounds showed cytotoxicity, among which, compound 2 exhibited GI50 value of 14.12 μM. The antiproliferative activity of 2 against Bel-7402 and CaEs-17 cells was also measured, with GI50 values of 1.91 and 2.58 μM, respectively. Intensive mechanism study showed that 2 caused cell-cycle arrest at S/G2 phase and induced apoptosis in Bel-7402 cells through mitochondria-related pathway. © 2016 Published by Elsevier B.V.
Introduction The Flemingia (Leguminosae) genus, containing N40 species, is mainly distributed in tropical region of Africa, Asia, and Australia. About 20 species can be found in China, which are widely distributed from the east to the southwest. Several species are used as ethnodrugs for a long history to cure rheumatoid arthritis, lumbocrural pain, lumbar muscle strain, swollen feet, chronic nephritis, menopausal syndrome and so on [1]. Previous chemical investigations have demonstrated that the genus is a rich source of isoflavonoids, chalcones, flavones, terpenoids, and phenolics [2–6], exhibiting various biological activities [5–8]. Among them, isoflavonoids are the major bioactive ingredients and have been obtained from the roots, stems, flowers and seeds of the Flemingia plants, some of which exhibit anti-cancer, antioxidant and chemical protective activities [4,5]. Flemingia philippinensis (Merr. et Rolfe) Li is a medicinal valuable species distributing in the southwest of China and its dried roots have long been used as folk medicine for the treatment of rheumatoid arthritis and swelling of throat. It is also being used to relieve pain and induce sleep. The previous studies on chemical constituents of the roots and dried leaves of F. philippinensis afforded isoflavones, flavanones, flavonoid glycosides, and steroids [9–13]. It has been found that some isolated compounds showed anti-cancer, antioxidant, immunosuppressive, estrogenic and antiestrogenic activities [13–16]. ⁎ Corresponding authors. E-mail addresses: [email protected] (Z.-L. Li), [email protected] (H.-M. Hua). 1 Da-Hong Li and Wen-Jia Kang contributed equally to this work.
http://dx.doi.org/10.1016/j.fitote.2016.06.003 0367-326X/© 2016 Published by Elsevier B.V.
In this paper, the chemical constituents of the roots of F. philippinensis were isolated by various kinds of column chromatography (CC) on silica gel, Sephadex LH-20, and ODS, and also preparative thin layer chromatography (PTLC) to yield fifteen compounds (Fig. 1), including three new ones, flemiphilippinones B (1), C (2), and demethylwedelolactone-11-methyl ether (3). The structures were identified by the physico-chemical characters and the spectroscopic analysis. By comparison with the published data, the known compounds were identified as coumestrol (4) [17], lupinalbin A (5) [18], formononetin (6) [19], biochanin A (7) [20], 5,7-dihydroxy-6,8diprenylchromone (8) [21], eriosematin (9) [21], isoeriosematin (10) [21], auriculasin (11) [22], flemiphilippinin A (12) [23,24], (2R)flemichin D (13) [25], 5,2′,4′-trihydroxy-8,5′-di-(3-methylbut-2enyl)-6,7-(2,2-dimethylpyrano)flavanone (14) [12], and flemiphilippinin D (15) [10]. Herein, we report the isolation and structural elucidation of three new compounds, as well as the antiproliferative activity of the obtained compounds against PC-3 cell line by MTT method. Structure-activity relationship (SAR) was also concluded. Antiproliferative activity against Bel-7402 and CaEs-17 cells and apoptosis related mechanism of new compound 2 in Bel-7402 cells concerning apoptosis-inducing effects and cell cycle arrest were also disclosed.
Materials and methods The UV spectra were recorded with a Shimadzu UV-2201 spectrometer. HR ESIMS was measured on a Bruker Micro-TOFQ-Q mass
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223
Fig. 1. The structures of compounds 1–15 from F. philippinensis.
spectrometer. NMR data were obtained from Bruker AV-600 NMR spectrometer using TMS as an internal standard. Silica gel (200–300 mesh) performed for column chromatography was purchased from Qingdao Ocean Chemical Factory and ODS (50 μm) was purchased from YMC Co. Ltd., Kyoto Japan. The Sephadex LH-20 was purchased from GE Healthcare. A Shimadzu SPD-20A series equipped with an YMC C18 column (250 × 20 mm, 5 μm) was used for HPLC analysis. 5-fluorouracil (5-Fu, purity N 99% by HPLC) was purchased from Aladdin. All the organic solvents were purchased from Yuwang Chemicals Industries, Ltd., China, and used without further purification. PC-3 (human prostate cancer) cell line was obtained from the American Type Culture Collection.
The roots of F. philippinensis were collected from Guangxi province, China, in 2008 and were identified by Prof. Qishi Sun, Shenyang Pharmaceutical University, Shenyang, China. The voucher sample (HHM200807) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China. The dry roots of F. philippinensis (20.6 kg) were extracted with ethanol (3 × 20 L 95:5, v/v) under reflux for three times (2 h, each) and concentrated under reduced pressure to give black-brown gum of 1626 g. The residue was suspended in water and partitioned with petroleum ether (3 × 3 L) and CHCl3 (3 × 3 L), successively. The CHCl3 soluble part (390 g) was chromatographed on silica gel CC (i.d. 10 × 150 cm) eluting
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with a PE/acetone gradient system from 100:0 to 0:100 to give 5 fractions Fr.A–E based on their TLC characteristic. Fr.C (PE/acetone 85:15, 32 g) was performed on silica gel CC (i.d. 4 × 80 cm, PE/acetone gradient system from 100:0 to 0:100) to give 5 fractions Fr.C1–C5. Fr.C1 (317 mg) was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to afford compounds 6 (5 mg) and 13 (35 mg). Fr.C2 was recrystallized with PE/acetone to get 11 (25 mg). Fr.C3 was performed on silica gel CC (i.d. 1 × 50 cm, PE/acetone 8:1) to give 14 (40 mg). Fr.C4 was recrystallized with PE/acetone to get 12 (30 mg). Silica gel CC (i.d. 4 × 80 cm) eluting with PE/acetone gradient system from 100:0 to 0:100 of Fr.D (PE/acetone 85:15, 20 g) gave 5 major fractions Fr.D1–D5. Fr.D1 (127 mg) then submitted to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to give 5 (10 mg). Fr.D2 (116 mg) was chromatographed on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to obtain 7 (10 mg). Fr.D3 was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to give 3 (2 mg), and 4 (10 mg). Fr.D4 (76 mg) was separated on ODS CC (i.d. 1.5 × 60 cm) eluting with MeOH/H2O 75:25 to yield 1 (3 mg). Fr.D5 was applied to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to afford 15 (30 mg). The PE soluble part (33 g) was fractionated by silica gel column chromatography (CC, i.d. 5 × 120 cm) and eluted with a PE/acetone gradient system from 100:0 to 0:100 to give 4 fractions Fr.F–I based on their TLC characteristic. Fr.F (PE/acetone 98:2, 421 mg) was separated on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and followed by PTLC (20 × 20 cm, PE/acetone 7:1) to obtain 2 (18 mg). Fr.G (PE/acetone 97:3, 267 mg) was performed on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and then PTLC (20 × 20 cm, PE/acetone 5:1) to obtain 9 (5 mg). Fr.H (PE/acetone 95:5, 325 mg) was separated on Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) to get 10 (13 mg). Fr.I (PE/acetone 95:5, 265 mg) was subjected to Sephadex LH-20 CC (i.d. 2 × 60 cm, CHCl3/MeOH 1:1) and PTLC (20 × 20 cm, cyclohexane/acetone 5:1) to afford 8 (5 mg). PC-3 cells were plated in a 96-well plate and the test samples were dissolved in DMSO to obtain stock solution. After 24 h spreading, different dilutions of compound 2 were added into each well at 37 °C incubating for 4 days. The medium was removed and cells were then treated with MTT solution for an additional 4 h. After discarding culture
Table 2 NMR dataa and HMBC correlations of compound 3 (in DMSO-d6). 1
No
H
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 5,7,12-OH 11-OCH3 a
6.37 (1H, s) 6.40 (1H, s) 7.30 (1H, s)
7.21 (1H, s)
10.82 (1H, s), 10.44 (1H, s), 9.50 (1H, s) 3.88 (3H, s)
13
C
158.0 101.2 159.7 95.5 155.2 95.0 161.3 99.2 155.5 101.7 146.9 146.3 99.2 149.4 113.8
HMBC
C-5, C-8 C-4, C-6, C-7 C-11, C-12, C-15
C-11, C-12, C-14
56.2
Chemical shifts (δ) were expressed in ppm and J in Hz.
medium, the cells were dissolved in 200 μL DMSO and the optical density (OD) at 570 nm was measured by a microplate reader spectrophotometer. The concentration of a compound (purity N 98% by HPLC) inhibiting half of the cell growth was calculated. The results were obtained from three independent experiments. Bel-7402 cells (5.0 × 103 cells/well) were plated in 6-well plates and incubated with 2 at 37 °C for 24 h, and stained with PI. Cellular DNA content, for cell cycle distribution analysis, was measured using a flow cytometer (FACS Calibur Becton-Dickinson). Cells were incubated with 2 for 72 h and apoptosis was analyzed using Annexin-V and PI double staining by flow cytometry according to the manufacturer's instructions in order to detect apoptotic cells. Bel-7402 cells were cultured in six-well plates after treatment with 2 or vehicle for 48 h. The cells were stained with the lipophilic cationic dye JC-1, according to the manufacturer's instruction (Keygen, KGA601). The percentage of cells
Table 1 NMR dataa of compounds 1 and 2. 1 (in CDCl3) No 1 2 3 4 5 6 C=O α β 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴ 2′–OH OCH3 a
2 (in CDCl3) 1
H
7.15 (1H, d, J = 8.6 Hz) 6.82 (1H, d, J = 8.6 Hz) 6.82 (1H, d, J = 8.6 Hz) 7.15 (1H, d, J = 8.6 Hz) 3.33 (1H, m), 3.25 (1H, m) 2.96 (2H, t, J = 7.6 Hz)
2.98 (1H, m) 3.08 (1H, dd, J = 15.1, 9.5 Hz) 4.73 (1H, d, J = 9.6 Hz) 1.22 (3H, s) 1.33 (3H, s) 3.04 (2H, d, J = 9.2 Hz) 4.76 (1H, d, J = 9.2 Hz) 1.31 (3H, s) 1.22 (3H, s) 13.30 (1H, s) 3.78 (3H, s)
Chemical shifts (δ) were expressed in ppm and J in Hz.
13
C
133.0 129.3 114.0 157.9 114.0 129.3 212.8 44.4 29.7 99.5 159.6 105.7 161.8 96.5 163.7 27.2 92.5 72.1 24.0 26.0 26.8 92.0 71.8 26.0 24.0 55.4
No 1 2 3 4 5 6 C=O α β 1′ 2′ 3′ 4′ 5′ 6′ 1″, 1‴ 2″, 2‴ 3″, 3‴ 4″, 4‴ 5″, 5‴ 1″″ 2″″ 3″″ 4″″ 5″″ OH OCH3
1
H
7.20 (1H, d, J = 8.6 Hz) 6.83 (1H, d, J = 8.6 Hz) 6.83 (1H, d, J = 8.6 Hz) 7.20 (1H, d, J = 8.6 Hz) 3.30 (2H, t, J = 7.8 Hz) 2.87 (2H, t, J = 7.8 Hz)
2.70 (1H, dd, J = 13.8, 7.5 Hz) 2.50 (1H, dd, J = 13.8, 7.5 Hz) 4.78 (2H, d, J = 7.5 Hz) 1.57 (3H, s) 1.57 (3H, s) 6.46 (1H, d, J = 10.1 Hz) 5.35 (1H, d, J = 10.1 Hz) 1.44 (3H, s) 1.44 (3H, s) 18.88 (1H, s) 3.78 (3H, s)
13
C
133.6 129.6 113.9 158.0 113.9 129.6 202.1 42.1 30.4 108.0 195.4 57.2 172.0 106.2 186.3 37.8 118.2 134.9 25.9 18.2 114.6 123.4 81.3 28.9 28.9 55.4
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Fig. 2. Key HMBC correlations of compounds 1 and 2.
Table 3 GI50a values of fifteen compounds isolated from F. philippinensis against PC-3 cells. Compound
GI50 ± SD (μM)
Compound
GI50 ± SD (μM)
1 2 3 4 5 6 7 8
NTb 14.12 ± 0.64 NT N100 42.76 ± 2.64 32.84 ± 0.33 64.25 ± 2.32 N100
9 10 11 12 13 14 15 5-Fuc
N100 N100 8.33 ± 0.12 10.13 ± 0.30 22.73 ± 0.85 27.45 ± 1.03 10.88 ± 0.34 39.37 ± 2.76
a GI50 is the concentration of a compound inhibiting half of the cell growth. The results given are means ± SD of three independent experiments carried out in duplicate. b NT = not test. c Positive control.
with healthy or collapsed mitochondrial membrane potentials was monitored by flow cytometry analysis. Flemiphilippinone B (1): yellow oil; UV (MeOH) λmax: 291 and 222 nm; 1H and 13C NMR data (measured in CDCl3) see Table 1; ESIMS: m/z = 457.3 [M + H]+, 479.2 [M + Na]+, 455.1 [M-H]−; HR ESIMS: m/z = 457.2240 (calcd. for C H32O7H+: 457.2226). Flemiphilippinone C (2): yellow oil; UV (MeOH) λmax: 245, 271, 221, and 202 nm; 1H and 13C NMR data (measured in CDCl3) see Table 1; ESIMS: m/z = 491.3 [M + H]+, 513.3 [M + Na]+, 489.1 [M-H]−; HR ESIMS: m/z = 513.2616 (calcd. for C31H38O5Na+: 513.2617). Demethylwedelolactone-11-methyl ether (3): white amorphous powder; UV (MeOH) λmax: 348 and 254 nm; 1H and 13C NMR data (measured in DMSO-d6) see Table 2; HR ESIMS: m/z = 315.0501 [M + H]+ (calcd. for C16H10O7H+, 315.0499).
Fig. 3. Cell cycle effects of compound 2 in Bel-7402 cells.
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Results and discussion Compound 1 was obtained as yellow oil and designated with a molecular formula of C26H32O7 based on quasi-molecular ion at m/z 457.2240 [M + H]+ (cacld. 457.2226) in its HRESIMS. The UV absorptions at 291 and 222 nm indicated that 1 established the presence of an o-hydroxyl aromatic carbonyl moiety [14]. The 1H NMR spectrum (Table 1) displayed 4 methyl singlets at δ 1.33 (3H, s), 1.31 (3H, s), and 1.22 (6H, s), two oxygenated methine doublets at δ 4.73 (1H, d, J = 9.6 Hz) and 4.76 (1H, d, J = 9.2 Hz), two aromatic proton doublets at δ 7.15 (2H, d, J = 8.6 Hz) and 6.82 (2H, d, J = 8.6 Hz) for a para-disubstituted benzene ring (ring B), two methylenes at δ 3.33 (1H, m), 3.25 (1H, m), and 2.96 (2H, t, J = 7.8 Hz) attributed to -CO-CH2-CH2-Ar and a methoxy signal at δ 3.78 (3H, s), as well as a downfield signal of a chelated hydroxyl group at δ 13.30 (1H, s). The 13C NMR spectrum (Table 1) of 1 showed 26 signals which disclosed the presence of a fully substituted aromatic ring (δ 96.5, 99.5, 105.7, 159.6, 161.8 and 163.7), a para-disubstituted benzene and a carbonyl carbon at δ 212.8. Base on the above data, compound 1 was inferred to be a dihydrochalcone with prenyl-related functionalities. The 1 H and 13 C NMR data were assigned by the HSQC and HMBC spectra (Fig. 2). The HMBC correlations of H-1″ with C-2′, C-3′, C4′, C-2″, and C-3″, and of H3–4″ and H3–5″ with C-2″ and C-3″ confirmed the presence of a furan ring substituted with an isopropanol, which was fused to C-3′ and C-4′. Similar method ensured that the other furan ring was attached to C-5′ and C-6′. The phenolic hydroxyl of 2′–OH at δ 13.30 showed correlations with C-1′, C-2′ and C-3′. The above data established the tricyclic structure in the left hand. The methoxy proton signal at δ 3.78 correlated with C-4 suggesting the
methoxy located at C-4. Therefore, 1 was determined as shown and named flemiphilippinone B. Compound 2 was isolated as yellow oil. Its molecular formula was deduced to be C31H38O5 by HR ESIMS (m/z 513.2616 [M + Na]+). The UV spectrum showed absorption maximum at 245, 271, 221, and 202 nm. The 1H NMR spectrum of 2 (Table 1) exhibited signals for a para-disubstituted aromatic ring at δ 7.20 (2H, d, J = 8.6 Hz), and 6.83 (2H, d, J = 8.6 Hz), a dimethylchromene moiety at δ 6.46 (1H, d, J = 10.1 Hz), 5.35 (1H, d, J = 10.1 Hz), and 1.44 (6H, s), two prenyl groups at δ 4.78 (2H, t, J = 7.5 Hz), 2.70 (2H, dd, J = 13.8, 7.5 Hz), 2.50 (2H, dd, J = 13.8, 7.5 Hz) and δ 1.57 (12H, s, Me × 4), a methoxy group at δ 3.78 and an enol hydroxyl at δ 18.88. The 13C NMR spectrum exhibited 31 carbon signals, including two ketone carbonyl carbons (δ 202.1 and 195.4). Comparison of 1H and 13C NMR data of 2 with 1 indicated that they possess the same 4-methoxy hydrocinnamoyl. The structure of 2 was deduced from detailed analysis of 1H and 13C NMR data (Table 1) together with 2D-NMR experiments (Fig. 2). The left moiety was established by the HMBC correlations from the proton at δ 6.46 of a dimethylchromene to C-4′ (δ 172.0), C-5′ (δ 106.2) and C-6′ (δ 186.3), from H-1″ and H-1‴ of two prenyls at δ 2.70, 2.50 to C-2′ (δ 195.4), C3′ (δ 57.2), and C-4′ (δ 172.0). The long-range correlations of H-β at δ 2.87 (2H, t, J = 7.8 Hz) to a carbonyl carbon at δ 202.1, and of H-α at δ 3.30 (2H, t, J = 7.8 Hz) to the carbonyl carbon (δ 202.1) and C-1 (δ 133.6) determined the linkage of two moieties via C-1′ and the carbonyl carbon. Thus, the structure of compound 2 was assigned as shown and named flemiphilippinone C. Compound 3 was obtained as white amorphous powder with the molecular formula C16H10O7 determined by a quasi-molecular ion
Fig. 4. Apoptotic property of compound 2 in Bel-7402 cells.
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peak at m/z 315.0501 [M + H]+ in its HR ESIMS. It exhibited UV absorption bands at 348 and 254 nm. The 1H NMR spectrum of 3 (Table 2) exhibited signals for three phenolic hydroxyl protons at δ 10.82, 10.44, and 9.50, and four aromatic proton singlets at δ 7.30, 7.21, 6.40, and 6.37, as well as a methoxy signal at δ 3.88. The 13C NMR spectrum (Table 2) revealed 16 carbon resonances, including a methoxyl group at δ 56.2 and 15 sp2 carbons. The 1H and 13C NMR chemical shifts were closely similar to those of the known compound wedelolactone [26], with the difference of H-6 and H-8 appearing at a higher field and H-10 appearing at a lower field, indicating the methoxy located at C-11 instead of C-7. The above deduction was supported by the HMBC correlations from H-8 (δ 6.40) to C-4 (δ 95.5), C-6 (δ 95.0) and C-7 (δ 161.3), from H-6 (δ 6.37) to C-5 (δ 155.2), and C-8 (δ 99.2), from H-10 (δ 7.30) to C-11 (δ 146.9), C-12 (δ 146.3) and C-15 (δ 113.8), and from H-13 (δ 7.21) to C-11, C-12 and C-14 (δ 149.4), as well as between the methoxy signal δ 3.88 and C-11. Thus, compound 3 was established as demethylwedelolactone-11-methyl ether. All the isolated compounds were tested for the growth inhibitory effects against PC-3 cell line using the MTT assay; the results were listed in Table 3. Among them, 11 exhibited the most potent in vitro antiproliferative effect with a GI50 value of 8.33 μM. The flavonoids (2, 5–7, and 11– 15) showed moderate inhibitory effects, while the chromone derivatives (8–10) showed no activity. Compared with 9, B ring with 3′,4′-dihydroxy group in the isoflavonone structure (11) would play an important role in inhibiting the growth of PC-3 cells. Isoflavonone 12 processing 3′,4′-dihydroxy group also showed strong antiproliferative activity with a GI50 value of 10.13 μM. Flavanones 13, 14, and 15 demonstrated good antiproliferative effects with GI50 values ranging from 10.88 to 27.45 μM, of which 15 with two prenyls is more active than the related compound 13 with dimethylchromene formed by a prenyl group. It was noteworthy that novel compound 2 also exhibited potent effect with a GI50 value of
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14.12 μM. Furthermore, the cytotoxicity of 2 against Bel-7402 (human hepatoma) and CaEs-17 (human gastric cancer) cell lines was evaluated and the GI50 values were 1.91 and 2.58 μM, respectively. Intensive mechanism study of 2 was carried out in Bel-7402 cells. Bel-7402 cells were treated with compound 2 at concentrations of 0.5, 1.0, and 2.0 μM, which resulted in accumulation of 34.03, 45.08, and 48.61% of cells at the S phase, respectively, compared with the untreated cells 22.30% (Fig. 3). There were almost no changes of G1 phase cells with 42.34, 38.14, and 41.86% cells, and the decline of G2 phase cells were observed of 23.63, 16.78 and 9.53%, respectively. So, compound 2 influenced cell-cycle progression of Bel-7402 cells at low micromolar concentrations and arrested at S/G2 phase. In order to examine the involvement of apoptosis in the loss of cancer cell viability of compound 2 in Bel-7402 cells, an annexin V-FITC/ propidium iodide (PI) binding assay was carried out. Bel-7402 cells were treated with different concentrations of 2 and percentages of apoptotic cells were determined by flow cytometry. As shown in Figs. 4, 2 exhibited potent dose-dependent activity in the induction of apoptosis. Treatment of Bel-7402 cells with 2 at 0.5, 1.0, and 1.5 μM, apoptotic cell rates (early and late) were 18.86, 35.45, and 59.79%, as compared with 8.09% in an untreated vehicle control, indicating that 2 was able to induce apoptotic cell death in Bel-7402 cells. Apoptosis could result in the loss of mitochondrial membrane potential. To determine plausible pathways that compound 2 triggered cell apoptosis, Bel-7402 cells were incubated with 0, 0.5, 1.5, and 2.0 μM of 2 prior to staining with the lipophilic mitochondrial probe JC-1. The number of cells with collapsed mitochondrial membrane potentials in different groups was determined by flow cytometry analysis (Fig. 5), yielding 2.87, 9.01, 19.53, and 28.49% apoptotic cells, respectively. These results showed that incubation with 2 increased the number of cells with collapsed mitochondrial membrane potentials.
Fig. 5. Effect of mitochondrial membrane potentials by compound 2 in Bel-7402 cells.
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Conclusions The chemical investigation of the roots of F. philippinensis led to the isolation of three new (1–3) and twelve known compounds (4–15). The structures of 1–3 were elucidated on the basis of their spectroscopic data. The results of in vitro antiproliferative activity showed that 2 and 11–15 exhibited strong growth inhibitory effects against PC-3 cells with GI50 values b 30 μM. The GI50 values of 2 against Bel-7402 and CaEs-17 cells were 1.91 and 2.58 μM, respectively. Intensive mechanism study of 2 was also carried out, and Bel-7402 cells were treated with compound 2 at concentrations of 0, 0.5, 1.0, and 2.0 μM, which revealed that 2 caused cell cycle arrest at S/G2 phase in a dose-dependent manner at low micromolar concentrations. The annexin V-FITC/PI binding assay and mitochondrial probe JC-1 assay revealed that the loss of cancer cell viability by 2 was involved of apoptosis. Conflict of interest
[6] [7]
[8]
[9] [10] [11] [12] [13]
[14]
The authors declare no conflict of interest. [15]
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31570350), General Scientific Research Projects of Department of Education in Liaoning Province (L2014382), and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University.
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Appendix A. Supplementary data [20]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.06.003.
[21] [22]
References [1] Y.S. Wei, The classification and distribution of the genus Flemingia Roxb. ex Ait. in China, Guihaia 11 (1991) 198–207. [2] M.M. Rahman, S.D. Sarker, M. Byres, A.I. Gray, New salicylic acid and isoflavone derivatives from Flemingia paniculata, J. Nat. Prod. 67 (2004) 402–406. [3] H. Soicke, K. Görler, H. Waring, Flavonol glycosides from Moghania faginea, Planta Med. 56 (1990) 410–412. [4] I. Gumula, J.P. Alao, I.O. Ndiege, P. Sunnerhagen, A. Yenesew, M. Erdélyi, Flemingins G-O, cytotoxic and antioxidant constituents of the leaves of Flemingia grahamiana, J. Nat. Prod. 77 (2014) 2060–2067. [5] H.M. Chiang, H.H. Chiu, S.T. Liao, Y.T. Chen, H.C. Chang, K.C. Wen, Isoflavonoid-rich Flemingia macrophylla extract attenuates UVB-induced skin damage by scavenging
[23] [24] [25] [26]
reactive oxygen species and inhibiting MAP kinase and MMP expression, Evid. Based Complement. Alternat. Med. 2013 (2013) 696879. Y.J. Shiao, C.N. Wang, W.Y. Wang, Y.L. Lin, Neuroprotective flavonoids from Flemingia macrophylla, Planta Med. 71 (2005) 835–840. H.Y. Ho, J.B. Wu, W.C. Lin, Flemingia macrophylla extract ameliorates experimental osteoporosis in ovariectomized rats, Evid. Based Complement. Alternat. Med. 2011 (2011) 752302. Y.L. Lin, H.J. Tsay, Y.F. Liao, M.F. Wu, C.N. Wang, Y.J. Shiao, Components of Flemingia macrophylla attenuate amyloid β-protein accumulation by regulating amyloid βprotein metabolic pathway, Evid. Based Complement. Alternat. Med. 2012 (2012) 795843. G. Xie, B. Lin, X. Qin, G. Wang, Q. Wang, J. Yuan, C. Li, M. Qin, New flavonoids with cytotoxicity from the roots of Flemingia latifolia, Fitoterapia 104 (2015) 97–101. M. Chen, S.Q. Luo, J.H. Chen, Studies on the chemical constituents of Flemingia philippinensis, Acta Pharmacol. Sin. 26 (1990) 42–48. H. Li, F. Zhai, M. Yang, X. Li, P. Wang, X. Ma, A new benzofuran derivative from Flemingia philippinensis Merr. et Rolfe, Molecules 17 (2012) 7637–7644. E.M. Ahn, N. Nakamura, T. Akao, K. Komatsu, M.H. Qiu, M. Hattori, Prenylated flavonoids from Moghania philippinensis, Phytochemistry 64 (2003) 1389–1394. E.M. Ahn, N. Nakamura, T. Akao, T. Nishihara, M. Hattori, Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents, Biol. Pharm. Bull. 27 (2004) 548–553. Y. Wang, M.J. Curtis-Long, B.W. Lee, H.J. Yuk, D.W. Kim, X.F. Tan, K.H. Park, Inhibition of tyrosinase activity by polyphenol compounds from Flemingia philippinensis roots, Bioorg. Med. Chem. 22 (2014) 1115–1120. L. Li, X. Deng, L. Zhang, P. Shu, M. Qin, A new coumestan with immunosuppressive activities from Flemingia philippinensis, Fitoterapia 82 (2011) 615–619. Y. Wang, M.J. Curtis-Long, H.J. Yuk, D.W. Kim, X.F. Tan, K.H. Park, Bacterial neuraminidase inhibitory effects of prenylated isoflavones from roots of Flemingia philippinensis, Bioorg. Med. Chem. 21 (2013) 6398–6404. E.M. Bickoff, A.N. Booth, R.L. Lyman, A.L. Livingston, C.R. Thompson, F. Deeds, Coumestrol, a new estrogen isolated from forage crops, Science 126 (1957) 969–970. S. Tahara, J.L. Ingham, J. Mizutani, New coumaronochromones from white lupin, Lupinus albus L. (Leguminosae), Agric. Biol. Chem. 49 (1985) 1775–1783. J.E. Kinjo, J.I. Furusawa, J. Baba, T. Takeshita, M. Yamasaki, T. Nohara, Studies on the constituents of Pueraria lobata. III. Isoflavonoids and related compounds in the roots and the voluble stems, Chem. Pharm. Bull. 35 (1987) 4846–4850. Y.R. Deng, T. Wang, Y.Z. He, Studies on chemical constituents of Caragana spinifera, China J. Chin. Materia. Med. 33 (2008) 775–777. P.G. Waterman, E.G. Crichton, Xanthones and biflavonoids from Garcinia densivenia stem bark, Phytochemistry 19 (1980) 2723–2726. D.M. Giuliano, S. Rosalba, V. Alberto, B. Bruno, M. Barbara, P. Gabriella, P. Cleofe, C. Enrico, Two isoflavones and a flavone from the fruits of Maclura pomifera, Phytochemistry 37 (1994) 893–898. H. Li, M. Yang, J. Miao, X. Ma, Prenylated isoflavones from Flemingia philippinensis, Magn. Reson. Chem. 46 (2008) 1203–1207. M. Chen, S.Q. Lou, J.H. Chen, Two isoflavones from Flemingia philippinensis, Phytochemistry 30 (1991) 3842–3844. W.G. Ma, N. Fuzzati, Q.S. Li, C.R. Yang, H. Stoeckli-Evans, K. Hostettmann, Polyphenols from Eriosema tuberosum, Phytochemistry 39 (1995) 1049–1061. J. Wu, W.B. Hou, T.J. Zhang, Y.M. Han, Chemical constituents of Eclipta prostrata, Chin. Tradit. Herb. Drugs 39 (2008) 814–816.