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New amino butenolides from the bulbs of Fritillaria unibracteata
Fitoterapia 98 (2014) 53–58
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New amino butenolides from the bulbs of Fritillaria unibracteata Juan Liu a,b, Cheng Peng a,b,⁎, Cheng-Jun He a,b, Jian-Lin Liu a, Ya-Cong He a,b, Li Guo a,b, Qin-Mei Zhou a,b, Huai Yang a,b, Liang Xiong a,b,⁎⁎ a State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China b School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China
a r t i c l e
i n f o
Article history: Received 14 May 2014 Accepted in revised form 8 July 2014 Available online 23 July 2014 Keywords: Fritillaria unibracteata Liliaceae Amino butenolides Amide alkaloids
a b s t r a c t Five new amino γ-butenolides, fritenolide A (1), B (2), C (3), D (4), and E (5), along with four known compounds, were isolated from the bulbs of Fritillaria unibracteata. Their structures were determined by spectroscopic analysis, including 1D NMR, 2D NMR, HRESIMS, HRESIMS/MS, IR, and CD techniques. All isolates were evaluated for the protective activity on injured hepatocytes and cytotoxic activity on human cancer cells in vitro. The unusual amino butenolides were isolated from the Liliaceae family for the first time. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Fritillaria (Liliaceae) is approximately composed of 130 species in the whole world, and mainly distributed in temperate regions of the Northern Hemisphere [1]. The bulbs of several Fritillaria plants have been used as a traditional Chinese medicine to relieve coughing for nearly 2000 years in China [2]. Previous phytochemical and pharmacological studies on the genus have led to the isolation of a lot of bioactive secondary metabolites, such as alkaloids, steroids, saponins, and terpenoids [3–7]. In order to search for more active ingredients, we carried out an investigation on the ethanolic extract of the bulbs of Fritillaria unibracteata Hsiao et K. C. Hsia, which is one of the sources for “Chuanbeimu” in Pharmacopoeia Commission of the
⁎ Correspondence to: C. Peng, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel.: +86 028 61800018. ⁎⁎ Correspondence to: L. Xiong, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel.:+86 028 62135056. E-mail addresses: [email protected] (C. Peng), [email protected] (L. Xiong).
http://dx.doi.org/10.1016/j.fitote.2014.07.009 0367-326X/© 2014 Elsevier B.V. All rights reserved.
People's Republic of China [2]. This paper describes the isolation, structure elucidation, and bioassays of the isolates. The new compounds 1–5 (Fig. 1) are α,β-unsaturated γ-butenolides with an amide unit. It is of interest to note that the unusual amide butenolides have been only reported from marine organisms and fungus before [8–11]. 2. Experimental 2.1. General NMR spectra were recorded on a Bruker-AVIIIHD-600 spectrometer. HRESIMS and HRESIMS/MS were measured with a Waters Synapt G2 Q-TOF HDMS spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument. Optical rotations were measured with a Perkin-Elmer 341 plus. CD spectra were recorded on a JASCO J-815 CD spectrometer. Column chromatography was performed with silica gel (200–300 mesh, Yantai Institute of Chemical Technology, Yantai, China) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Preparative TLC (0.4–0.5 mm) was conducted with glass plates precoated silica gel GF254 (Yantai). HPLC separation was performed on an instrument
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J. Liu et al. / Fitoterapia 98 (2014) 53–58
Fig. 1. Chemical structures of compounds 1–5.
consisting of a Cometro 6000LDS pump and a Cometro 6000PVW UV/VIS detector with an Ultimate (250 × 10 mm) preparative column packed with C18 (5 μm). 2.2. Plant material The bulbs of F. unibracteata were purchased from Sichuan Neautus Traditional Chinese Medicine Inc., and were originally collected from Sichuan province, China. Plant identity was verified by Prof. Min Li (Chengdu University of Traditional Chinese Medicine, Chengdu, China). A voucher specimen (FU20111202) was deposited at the School of Pharmacy, Chengdu University of Traditional Chinese Medicine. 2.3. Extraction and isolation The air-dried bulbs of F. unibracteata (5 kg) were extracted three times with 95% EtOH (3 × 30 L, total amount 90 L) for 2 h under reflux. The EtOH extract was concentrated in vacuo to yield a semi-solid (275 g), which was suspended in H2O (2 L) and then partitioned sequentially with chloroform (6 × 1.5 L) and n-BuOH (6 × 1.5 L). The chloroform extract (28 g) was chromatographed on silica gel with petroleum ether–ethyl acetate (100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:2, v/v) to afford 27 fractions (Fr.1–Fr.27) based on their TLC patterns. Fr.15 (2.1 g) was applied to Sephadex LH-20 using petroleum ether–CHCl3– MeOH (5:5:1, v/v) as the mobile phase to yield five subfractions (Fr.15-1–Fr.15-5). Eluting with a gradient of petroleum ether– acetone (100:1–4:1, v/v), Fr.15-1 (0.5 g) was separated by column chromatography over alkaline silica gel, to give six subfractions (Fr.15-1a–Fr.15-1f). Fr.15-1a (68 mg) was further purified by preparative TLC (petroleum ether–acetone, 4:1) followed by reversed-phase semi-preparative HPLC (98% MeOH in H2O) to yield 1 (6.8 mg), 2 (4.6 mg), 3 (2.1 mg), 4 (2.4 mg), and 5 (1.7 mg). Fritenolide A (1): white amorphous powder; [α]20 D − 7.3 (c 0.05, MeOH); CD (MeOH) 246.5 (Δε + 1.12) nm; IR νmax = 3330, 2954, 2921, 2851, 1744, 1694, 1654, 1548, 1468, 1347, 1072, 965, 786, 721 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 526.4232 [M + Na]+ (calcd for C32H57NO3Na, 526.4236); HRESIMS/MS m/z: 482.4340 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2253 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2354 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide B (2): white amorphous powder; [α]20 D − 7.7 (c 0.04, MeOH); CD (MeOH) 243.5 (Δε + 1.11) nm; IR νmax =
3326, 2955, 2921, 2852, 1743, 1692, 1652, 1546, 1465, 1354, 1071, 877, 787, 719 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 526.4242 [M + Na]+ (calcd for C32H57NO3Na, 526.4236); HRESIMS/MS m/z: 482.4339 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2254 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2355 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide C (3): white amorphous powder; [α]20 D − 6.9 (c 0.04, MeOH); CD (MeOH) 244 (Δε + 0.94) nm; IR νmax = 3330, 2955, 2923, 2853, 1743, 1693, 1654, 1548, 1454, 1053, 965, 798 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z:498.3926 [M + Na]+ (calcd for C30H53NO3Na, 498.3923). HRESIMS/MS m/z: 454.4022 [M − CO2 + Na]+ (calcd for C29H53NONa, 454.4025), 316.2249 [M − C12H22O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2352 [M − CO2 − C12H22O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide D (4): white amorphous powder; [α]20 D − 6.4 (c 0.05, MeOH); CD (MeOH) 244 (Δε + 0.99) nm; IR νmax =
Table 1 13 C NMR data (150 MHz) for compounds 1–5 in CDCl3a (δ in ppm). no.
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11–15 16 17 18 1′ 2′ 3′ 4′–9′ 10′ 11′ 12′ 13′ 14′
170.0 125.4 129.5 82.2 33.4 24.9 32.3 129.0 131.8 32.7 29.3–29.8b 32.1 22.8 14.3 172.1 37.0 25.3
169.9 125.4 129.4 82.2 33.5 25.1 26.9 128.5 131.2 27.4 29.3–29.8 32.1 22.8 14.3 172.1 37.0 25.3
170.0 125.4 129.6 82.3 34.0 25.1
170.0 125.4 129.6 82.3 34.0 25.1
29.3–29.8
29.3–29.9
32.1 22.8 14.3 172.1 37.0 25.3
29.3–29.8
29.3–29.8
32.1 22.8 14.3
32.1 22.8 14.3
170.0 125.4 129.5 82.2 33.4 24.9 32.3 129.0 131.8 32.7 29.3–29.8 32.1 22.8 14.3 172.1 37.0 25.3 29.3–29.8 32.1 22.8 14.3
32.1 22.9 14.3 172.1 37.0 25.4 29.3–29.9 32.1 22.9 14.3
a
29.3–29.8 32.1 22.8 14.3
The assignments were based on 1H–1H COSY, HSQC, and HMBC experiments. The 13C NMR signals for methylenes in the aliphatic chains were overlapped between 29.3–29.8 ppm (C-11–C-15 and C-4′–C-11′ in 1 and 2, C-11–C-15 and C-4′–C-9′ in 3, C-7–C-15 and C-4′–C-11′ in 4, C-7–C-15 and C-4′–C-9′ in 5). b
J. Liu et al. / Fitoterapia 98 (2014) 53–58
55
Table 2 1 H NMR data (600 MHz) for compounds 1–5 in CDCl3a (δ in ppm, J in Hz). no.
1
2
3
4
5
3 4 5 6 7 8 9 10 11–15 16 17 18 2′ 3′ 4′–9′ 10′ 11′ 12′ 13′ 14′ NH
7.42 d (1.8) 5.05 td (6.0, 1.8) 1.74 m, 1.66 m 1.52 m 2.03 q (6.6) 5.33 dt (15.6, 6.6) 5.41 dt (15.6, 6.6) 1.96 q (6.6) 1.25–1.35 mb 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.2) 1.69 m
7.42 d (1.2) 5.05 td (5.4, 1.2) 1.77 m, 1.68 m 1.50 m 2.08 q (7.2) 5.30 dt (10.2, 7.2) 5.40 dt (10.2, 7.2) 1.99 q (7.2) 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m
7.42 d (1.8) 5.04 td (5.4, 1.8) 1.70 m 1.44 m
7.43 d (1.2) 5.04 td (6.0, 1.2) 1.70 m 1.45 m
1.25–1.35 m
1.25–1.35 m
1.25 m 1.28 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m
1.25–1.35 m
1.25–1.35 m
1.25 m 1.29 m 0.88 t (7.2) 7.42 s
1.25 m 1.29 m 0.88 t (7.2) 7.42 s
7.42 d (1.8) 5.05 td (5.4, 1.8) 1.75 m, 1.66 m 1.52 m 2.03 q (6.6) 5.33 dt (15.6, 6.6) 5.41 dt (15.6, 6.6) 1.96 q (6.6) 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.8) 1.68 m 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2)
1.25 m 1.28 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m 1.25–1.35 m 1.25 m 1.28 m 0.88 t (7.2)
a
1
7.41 s
1.25–1.35 m 1.25 m 1.28 m 0.88 t (7.2) 7.41 s
7.41 s
1
The assignments were based on H– H COSY, HSQC, and HMBC experiments. The 1H NMR signals for methylenes in the aliphatic chains were overlapped between 1.25–1.35 ppm (H2-11–H2-15 and H2-4′–H2-11′ in 1 and 2, H2-11–H2-15 and H2-4′–H2-9′ in 3, H2-7–H2-15 and H2-4′–H2-11′ in 4, H2-7–H2-15 and H2-4′–H2-9′ in 5). b
3324, 2957, 2920, 2851, 1742, 1690, 1654, 1547, 1466, 1356, 1033, 785, 721 cm− 1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z:528.4391 [M + Na]+ (calcd for C32H59NO3Na, 528.4393); HRESIMS/MS m/z: 484.4497 [M − CO2 + Na]+ (calcd for C31H59NONa, 484.4494), 318.2411 [M − C14H26O + Na]+ (calcd for C18H33NO2Na, 314.2409), and 274.2513 [M − CO2 − C14H26O + Na]+ (calcd for C17H33NNa, 274.2511). Fritenolide E (5): white amorphous powder; [α]20 D − 6.6 (c 0.05, MeOH); CD (MeOH) 247 (Δε + 0.87) nm; IR νmax = 3338, 2955, 2921, 2853, 1742, 1693, 1645, 1559, 1464, 1380, 1035, 786, 722 cm− 1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 500.4081 [M + Na]+ (calcd for C30H55NO3Na, 500.4080); HRESIMS/MS m/z: 456.4185 [M − CO2 + Na]+ (calcd for C29H55NONa, 456.4181), 318.2413 [M − C12H22O + Na]+ (calcd for C18H33NO2Na, 318.2409), and 274.2512 [M − CO2 − C12H22O + Na]+ (calcd for C17H33NNa, 274.2511).
2.5. Cytotoxic activity assay The human lung cancer cell lines H1975, H358 and A549, human hepatocellular carcinoma cell lines HepG-2 and SMMC7721, human colorectal carcinoma cell line HCT116, human mammary carcinoma cell lines MDA-MB-231 and MCF7, human melanoma cell line A2058, human pancreatic cancer cell line PANC-1 and human acute myeloid leukemia cell line MV4-11 were obtained from the American Type Culture Collection (ATCC) and grown in RPMI1640, DMEM or IMDM containing 10% fetal bovine serum (v/v) in 5% CO2 at 37 °C. Cells (2 × 103–10 × 103) were seeded in 96-well plates and cultured for 24 h, followed by the test compounds treatment at concentrations of 10 μM, 1 μM, and 0.1 μM for 72 h, respectively. After the culture period, 20 μL of MTT (5 mg/mL) was added per well and incubated for 4 h at 37 °C, then 50 μL of 20% acidified SDS was used to lyse the cells. Finally, absorbance was measured at 570 nm using a microplate reader. Each assay was replicated three times. The effect of the compounds on tumor cells viability was calculated and expressed by IC50 of each cell line.
2.4. Hepatoprotective activity assay The hepatoprotective effects were determined by a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in LO-2 cells. Each cell suspension of 1 × 105 cells/mL in DMEM containing 10% fetal bovine serum (v/v) was seeded in 96-well plates and cultured for 12 h in 5% CO2 at 37 °C. After fresh medium (100 μL) containing test sample was added, the cells were cultured for 24 h. Then, the cultured cells were exposed to 1 μM H2O2 for 1 h. The medium was changed into a fresh one containing 5 mg/mL MTT. After 4 h incubation at 37 °C, the supernatant was removed and 150 μL of DMSO was added to dissolve formazan crystals. The optical density (OD) of the formazan solution was measured on a microplate reader at 490 nm.
3. Results and discussion Compound 1 was obtained as a white amorphous powder, and the presence of ester (1744 cm−1), amide (3330, 1694 and 1548 cm−1), and olefinic (1654 cm−1) groups were indicated by its IR spectrum. The molecular formula C32H57NO3 of 1, with five hydrogen deficiencies, was indicated by HRESIMS at m/z 526.4232 (calcd for C32H57NO3Na, 526.4236). The 1H NMR spectrum of 1 (Table 2) displayed resonances attributable to two terminal methyls [δH 0.88 (6H, t, J = 7.2 Hz, H3-18 and H3-14′)], several aliphatic methylenes [δH 2.35 (t, J = 7.2 Hz, H2-2′), 2.03 (q, J = 6.6 Hz, H2-7), 1.96 (q, J = 6.6 Hz, H2-10), 1.74 (m, H-5a), 1.69 (m, H2-3′), 1.66 (m, H-5b), 1.52 (m, H2-6),
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J. Liu et al. / Fitoterapia 98 (2014) 53–58
1.25–1.35 (m, H2-11–17 and H2-4′–13′)], an oxymethine [δH 5.05 (td, J = 6.0, 1.8 Hz, H-4)], a trans-olefinic bond [δH 5.41 (dt, J = 15.6, 6.6 Hz, H-9), 5.33 (dt, J = 15.6, 6.6 Hz, H-8)], and an olefinic methine group [δH 7.42 (d, J = 1.8 Hz, H-3)]. The 13C NMR and DEPT spectrum of 1 revealed carbon resonances (Table 1) corresponding to the above protonated units and three quaternary carbons [two carbonylic (δC 172.1 and 170.0) and an olefinic (δC 125.4)]. The chemical shifts of the two carbonyl groups (δC b 175 ppm) in the 13C NMR spectrum, in combination with the molecular composition and the IR signals, suggested that there were an amide group and an ester group in 1. All these spectroscopic data indicated that compound 1 was an α-amino α,β-unsaturated γ-lactone with substitutions of an aliphatic acyl group and an aliphatic alkyl group containing one trans double bond [8,12]. This conjecture was proved by 2D NMR data analysis. In the HMBC spectrum, two- and three-bond correlations of H-3/C-1, C-2 and C-4; H-4/C-1, C-2 and C-3, further verified the α-amino α,βunsaturated γ-lactone unit in 1 (Fig. 2). The HMBC correlations of H-4/C-5 and C-6, H-8/C-7 and C-10, H-9/C-7 and C-10, in combination with the homonuclear coupling correlations of H-4/H2-5/H2-6/H2-7/H-8/H-9/H2-10 in the 1H–1H gCOSY spectrum, indicated that the aliphatic alkyl group was substituted at C-4 and the double bond was located at C-8 and C-9. The HMBC correlations from H2-2′ and H2-3′ to C-1′ confirmed the presence of the aliphatic acyl group. The length of two aliphatic chains was determined by HRESI-QTOF-MS/MS. The positively charged HRESI-QTOFMS/MS of 1 (Fig. 3) showed sodiated molecular ion peak at m/z 526.4237 [M + Na]+ (calcd for C32H57NO3Na, 526.4236) and its fragment ions at m/z 482.4340 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2253 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2354 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). These MS/MS spectroscopic data suggested that both of the two aliphatic chains were C14-long chains corresponding to a tetradecanoyl and a tetradecenyl group, respectively. In the CD spectrum, a positive Cotton effect at 246.5 nm for a π–π* transition of the α-amino α,β-unsaturated γ-lactone moiety suggested 4S configuration for 1 [12], which was supported by the negative optical rotation {[α]20 D −7.3 (c 0.05, MeOH)} [13]. Thus, compound 1 was determined to be (−)-(4S)-2tetradecanoylamino-4-[4(E)-tetradecenyl]but-2-enolide and named as fritenolide A. Compound 2 was an isomer of 1, as indicated by the HRESIMS, NMR and IR spectroscopic data. Comparison of the
13
C NMR data between 1 and 2 displayed that the resonances for C-7 and C-10 were shielded from δ 32.3 and 32.7 ppm in 1 to δ 26.9 and 27.4 ppm in 2, respectively (Table 1). This suggested Z configuration for the double bond in the unsaturated long chain in 2, on the basis of the 13C NMR chemical shifts of the methylene carbons adjacent to the olefinic carbons (δ ≈ 27 ppm for Z, δ ≈ 32 ppm for E) [14]. Meanwhile, the geometry (Z) of the double bond was confirmed by the coupling constant J8, 9 = 10.2 Hz in the 1H NMR spectrum of 2. The positive mode HRESIMS/MS of 2 showed the same sodiated fragment ions (C31H57NONa+, C18H31NO2Na+, and C17H31NNa+) as those of 1, which indicated that 2 also had the tetradecanoyl and tetradecenyl groups. In the 2D NMR spectrum, the 1H–1H gCOSY correlations of H-4/H2-5/H2-6/H2-7/H-8/H-9/H2-10 and the HMBC correlations of H-4/C-5 and C-6, H-8 and H-9/C-7 and C-10 verified the location of the double bond (C-8–C-9). In addition, the optical rotation ([α]20 D –7.7º) of 2 was consistent with that of 1. Therefore, 2 was assigned as (−)-(4S)-2tetradecanoylamino-4-[4(Z)-tetradecenyl]but-2-enolide and named as fritenolide B. Compound 3 was also obtained as a white amorphous powder. The 1D NMR, 2D NMR, IR, and [α]20 D data of 3 were very similar to those of 1, except that the HRESIMS data of 3 indicated the molecular formula C30H53NO3 for 3 with two fewer CH2 units than 1. This suggested the only difference between 3 and 1 was the length of the aliphatic chain. In the comparison of their HRESIMS/MS data, the different sodiated fragment ion C29H53NONa+ and the same fragment ions C18H31NO2Na+ and C17H31NNa+ revealed that the tetradecanoyl unit in 1 was substituted by a dodecanoyl unit in 3. Thus, 3 was determined to be (−)-(4S)-2-dodecanoylamino-4[4(E)-tetradecenyl]but-2-enolide and named as fritenolide C. The spectroscopic data of 4 showed that it was another analog of 1. The HRESIMS data indicated that it had the molecular formula C32H59NO3 with two more hydrogen atom than 1. Comparison of the NMR data of 1 and 4 displayed the resonances for the double bond in the tetradecenyl unit disappeared in 4, which indicated replacement of the tetradecenyl group in 1 by a tetradecyl group in 4. This was proved by the 2D NMR and HRESIMS/MS analysis. Specifically, the MS/MS fragment ions C31H59NONa+ ([M − CO2 + Na]+), C18H33NO2Na+ ([M − C14H26O + Na]+), and C17H33NNa+ ([M − CO2 − C14H26O + Na]+) confirmed the presence of the tetradecyl and tetradecanoyl groups. Accordingly, compound 4 was assigned as (−)-(4S)-2-tetradecanoylamino4-tetradecyl but-2-enolide named as fritenolide D.
NH O
5
1
O
O
1
H, 1H-COSY HMBC (H C)
Fig. 2. Key 1H, 1H-COSY and HMBC correlations of compound 1.
7
J. Liu et al. / Fitoterapia 98 (2014) 53–58
57
Fig. 3. HRESI-QTOF-MS/MS fragmentation pattern of compound 1.
Compound 5 possessed the molecular formula C30H55NO3, with two fewer CH2 units than 4, as indicated by HRESIMS spectra. The NMR and HRESIMS/MS data analysis of 5 suggested that it was an analog of 4 except for substitution of the tetradecanoyl unit by a dodecanoyl unit. Thus, compound 5 was determined as (−)-(4S)-2-dodecanoylamino-4-tetradecylbut2-enolide and named as fritenolide E. The known compounds were identified as β-sitosterol [15], 7-ketositosterol [16], 3-methoxy-4-(palmitoyloxy)benzaldehyde [17], and methyl octadecanoate [18] by comparing their spectroscopic data with those reported in the corresponding literature. The protective activities on hepatocyte injury induced by H2O2 were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay in LO-2 cells [19]. Compounds 1–5 showed weak hepatoprotective activity at a concentration of 10 μM, with the survival rate of 27.0%, 24.9%, 27.8%, 30.1%, and 29.4%, respectively (H2O2 model group: 22.5%). In addition, all compounds were evaluated for the cytotoxic activities against 11 kinds of human tumor cells, including H1975, H358, A549, HepG-2, SMMC7721, HCT116, MDA-MB-231, MCF-7, A2058, PANC-1, and MV4-11 [20]. However no obvious effect was observed at 10 μM. Fritillaria plants are known to contain alkaloids, especially steroidal alkaloids, such as cevanine-type, veratramine-type, jervine-type, solanidine-type, and verazine-type [21–23]. However, the amide alkaloids have not been reported before. Thus, the isolation of new compounds 1–5 enriched the types of alkaloids in the genus. In addition, compounds 1–5 were unusual α-amino butenolides, which were very probably biosynthesized from unsaturated long-chain aliphatic α-amino acids in the natural plants. In the previous study, although a lot of butenolides derived from fatty acids metabolism have been found from the natural products [24–26], the α-amino butenolides from amino acids metabolism were rather rare in the terrestrial plants. To the best of our knowledge, there were only six natural α-amino butenolides obtained from the marine bacterium Pseudoalteromonas sp. F-420 [8], marine ascomycete Leptosphaeria oraemaris [9], marine sponge Anthosigmella aft. raromicrosclera [10], and fungus Penicillium sp. No. 13 [11]. Thus, the α-amino butenolides, especially those with an amide unit, could be used as a primary chemical character of Fritillaria unibracteata.
Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was financially supported by grants from the National Technology R&D Program for the “Eleventh Five-Year” Plan of China (grant No. 2009BAI84B04) is acknowledged. The authors are grateful to Prof. Min Li (Chengdu University of Traditional Chinese Medicine) for the plant identification. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.07.009. References [1] Xiao PG, Jiang Y, Li P, Luo YB, Liu Y. The botanical origin and pharmacophylogenetic treatment of Chinese materia medica Beimu. Acta Phytotaxon Sin 2007;45:473–87. [2] Commission of Chinese Pharmacopoeia. Pharmacopoeia of the People's Republic of China, vol. 1. Beijing: Chemical Industry Press; 2010. p. 34–5. [3] Wang DD, Wang S, Chen X, Xu XL, Zhu JY, Nie LH, et al. Antitussive, expectorant and anti-inflammatory activities of four alkaloids isolated from bulbus of Fritillaria wabuensis. J Ethnopharmacol 2012;139:189–93. [4] Akhtar MN, Atta-ur-Rahman, Choudhary MI, Sener B, Erdogan I, Tsuda Y. New class of steroidal alkaloids from Fritillaria imperialis. Phytochemistry 2003;63:115–22. [5] Ori K, Mimaki Y, Sashida Y, Nikaido T, Ohmoto T. Steroidal alkaloids from the bulbs of Fritillaria persica. Phytochemistry 1992;31:4337–41. [6] Shou QY, Tan Q, Shen ZW. A novel sulfur-containing diterpenoid from Fritillaria anhuiensis. Tetrahedron Lett 2009;50:4185–7. [7] Shen S, Li GY, Huang J, Chen CJ, Ren B, Lu G, et al. Steroidal saponins from Fritillaria pallidiflora Schrenk. Fitoterapia 2012;83:785–94. [8] Yoshikawa K, Takadera T, Adachi K, Nishijima M, Sano H. Korormicin, a novel antibiotic specifically active against marine gram-negative bacteria, produced by a marine bacterium. J Antibiot 1997;50:949–53. [9] White JD, Badger RA, Kezar HS, Pallenberg AJ, Schiehser GA. Structure, synthesis and absolute configuration of leptosphaerin, a metabolite of the marine ascomycete Leptosphaeria oraemaris. Tetrahedron 1989;45:6631–44. [10] Tsukamoto S, Kato H, Hirota H, Fusetani N. Pipecolate derivatives, anthosamines A and B, inducers of larval metamorphosis in ascidians, from a marine aponge Anthosigmella aft. raromicrosclera. Tetrahedron 1995;51:6687–94. [11] Kimura Y, Mizuno T, Kawano T, Okada K, Shimada A. Peniamidienone and penidilamine, plant growth regulators produced by the fungus Penicillium sp. No. 13. Phytochemistry 2000;53:829–31.
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[12] Berova N, Breinholt J, Jensen GW, Kjaer A, Lo LC, Nakanishi K, et al. Malonofungin: an antifungal aminomalonic acid from Phaeoramularia fusimaculans. Acta Chem Scand 1994;48:240–51. [13] Hamada Y, Kawai A, Matsui T, Hara O, Shioiri T. 4-alkoxycarbonyloxazoles as β-hydroxy-α-amino acid synthons: efficient stereoselective syntheses of 3-amino-2,3,6-trideoxyhexoses and a hydroxy amino acid moiety of Al-77-B. Tetrahedron 1990;46:4823–46. [14] Kumar N, Singh B, Gupta AP, Kaul VK. Lonijaposides, novel cerebrosides from Lonicera japonica. Tetrahedron 2006;62:4317–22. [15] Wei XH, Yang SJ, Liang N, Hu DY, Jin LH, Xue W, et al. Chemical constituents of Caesalpinia decapetala (Roth) Alston. Molecules 2013;18:1325–36. [16] Zhang X, Geoffroy P, Miesch M, Julien-David D, Raul F, Aoudé-Werner D, et al. Gram-scale chromatographic purification of β-sitosterol: synthesis and characterization of β-sitosterol oxides. Steroids 2005;70:886–95. [17] Fiorentino A, D'Abrosca B, Pacifico S, Mastellone C, Piscopo V, Caputo R, et al. Isolation and structure elucidation of antioxidant polyphenols from quince (Cydonia vulgaris) peels. J Agric Food Chem 2008;56:2660–7. [18] Minakawa M, Baek H, Yamada YMA, Han JW, Uozumi Y. Direct dehydrative esterification of alcohols and carboxylic acids with a macroporous polymeric acid catalyst. Org Lett 2013;15:5798–801.
[19] Ding X, Wang MY, Yao YX, Li GY, Cai BC. Protective effect of 5hydroxymethylfurfural derived from processed Fructus Corni on human hepatocyte LO2 injured by hydrogen peroxide and its mechanism. J Ethnopharmacol 2010;128:373–6. [20] Xiong L, Cao ZX, Peng C, Li XH, Xie XF, Zhang TM, et al. Phenolic glucosides from Dendrobium aurantiacum var. denneanum and their bioactivities. Molecules 2013;18:6153–60. [21] Jiang Y, Li P, Li HJ, Yu H. New steroidal alkaloids from the bulbs of Fritillaria puqiensis. Steroids 2006;71:843–8. [22] Qian ZZ, Nohara T. Steroidal alkaloids of Fritillaria maximowiczii. Phytochemistry 1995;40:979–81. [23] Xu DM, Xu ML, Wang SQ, Hung EX, Wen XG. Two new steroidal alkaloids from Fritillaria ussuriensis. J Nat Prod 1990;53:549–52. [24] Kim MR, Jung HJ, Min BS, Oh SR, Kim CS, Ahn KS, et al. Constituents from the stems of Actinodaphne lancifolia. Phytochemistry 2002; 59:861–5. [25] Chen IS, Lai-Yaun IL, Duh CY, Tsai IL. Cytotoxic butanolides from Litsea akoensis. Phytochemistry 1998;49:745–50. [26] Tanaka H, Nakamura T, Ichino K, Ito K, Tanaka T. Butanolides from Litsea japonica. Phytochemistry 1990;29:857–9.
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New amino butenolides from the bulbs of Fritillaria unibracteata Juan Liu a,b, Cheng Peng a,b,⁎, Cheng-Jun He a,b, Jian-Lin Liu a, Ya-Cong He a,b, Li Guo a,b, Qin-Mei Zhou a,b, Huai Yang a,b, Liang Xiong a,b,⁎⁎ a State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China b School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China
a r t i c l e
i n f o
Article history: Received 14 May 2014 Accepted in revised form 8 July 2014 Available online 23 July 2014 Keywords: Fritillaria unibracteata Liliaceae Amino butenolides Amide alkaloids
a b s t r a c t Five new amino γ-butenolides, fritenolide A (1), B (2), C (3), D (4), and E (5), along with four known compounds, were isolated from the bulbs of Fritillaria unibracteata. Their structures were determined by spectroscopic analysis, including 1D NMR, 2D NMR, HRESIMS, HRESIMS/MS, IR, and CD techniques. All isolates were evaluated for the protective activity on injured hepatocytes and cytotoxic activity on human cancer cells in vitro. The unusual amino butenolides were isolated from the Liliaceae family for the first time. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The genus Fritillaria (Liliaceae) is approximately composed of 130 species in the whole world, and mainly distributed in temperate regions of the Northern Hemisphere [1]. The bulbs of several Fritillaria plants have been used as a traditional Chinese medicine to relieve coughing for nearly 2000 years in China [2]. Previous phytochemical and pharmacological studies on the genus have led to the isolation of a lot of bioactive secondary metabolites, such as alkaloids, steroids, saponins, and terpenoids [3–7]. In order to search for more active ingredients, we carried out an investigation on the ethanolic extract of the bulbs of Fritillaria unibracteata Hsiao et K. C. Hsia, which is one of the sources for “Chuanbeimu” in Pharmacopoeia Commission of the
⁎ Correspondence to: C. Peng, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel.: +86 028 61800018. ⁎⁎ Correspondence to: L. Xiong, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, PR China. Tel.:+86 028 62135056. E-mail addresses: [email protected] (C. Peng), [email protected] (L. Xiong).
http://dx.doi.org/10.1016/j.fitote.2014.07.009 0367-326X/© 2014 Elsevier B.V. All rights reserved.
People's Republic of China [2]. This paper describes the isolation, structure elucidation, and bioassays of the isolates. The new compounds 1–5 (Fig. 1) are α,β-unsaturated γ-butenolides with an amide unit. It is of interest to note that the unusual amide butenolides have been only reported from marine organisms and fungus before [8–11]. 2. Experimental 2.1. General NMR spectra were recorded on a Bruker-AVIIIHD-600 spectrometer. HRESIMS and HRESIMS/MS were measured with a Waters Synapt G2 Q-TOF HDMS spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument. Optical rotations were measured with a Perkin-Elmer 341 plus. CD spectra were recorded on a JASCO J-815 CD spectrometer. Column chromatography was performed with silica gel (200–300 mesh, Yantai Institute of Chemical Technology, Yantai, China) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Preparative TLC (0.4–0.5 mm) was conducted with glass plates precoated silica gel GF254 (Yantai). HPLC separation was performed on an instrument
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Fig. 1. Chemical structures of compounds 1–5.
consisting of a Cometro 6000LDS pump and a Cometro 6000PVW UV/VIS detector with an Ultimate (250 × 10 mm) preparative column packed with C18 (5 μm). 2.2. Plant material The bulbs of F. unibracteata were purchased from Sichuan Neautus Traditional Chinese Medicine Inc., and were originally collected from Sichuan province, China. Plant identity was verified by Prof. Min Li (Chengdu University of Traditional Chinese Medicine, Chengdu, China). A voucher specimen (FU20111202) was deposited at the School of Pharmacy, Chengdu University of Traditional Chinese Medicine. 2.3. Extraction and isolation The air-dried bulbs of F. unibracteata (5 kg) were extracted three times with 95% EtOH (3 × 30 L, total amount 90 L) for 2 h under reflux. The EtOH extract was concentrated in vacuo to yield a semi-solid (275 g), which was suspended in H2O (2 L) and then partitioned sequentially with chloroform (6 × 1.5 L) and n-BuOH (6 × 1.5 L). The chloroform extract (28 g) was chromatographed on silica gel with petroleum ether–ethyl acetate (100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:2, v/v) to afford 27 fractions (Fr.1–Fr.27) based on their TLC patterns. Fr.15 (2.1 g) was applied to Sephadex LH-20 using petroleum ether–CHCl3– MeOH (5:5:1, v/v) as the mobile phase to yield five subfractions (Fr.15-1–Fr.15-5). Eluting with a gradient of petroleum ether– acetone (100:1–4:1, v/v), Fr.15-1 (0.5 g) was separated by column chromatography over alkaline silica gel, to give six subfractions (Fr.15-1a–Fr.15-1f). Fr.15-1a (68 mg) was further purified by preparative TLC (petroleum ether–acetone, 4:1) followed by reversed-phase semi-preparative HPLC (98% MeOH in H2O) to yield 1 (6.8 mg), 2 (4.6 mg), 3 (2.1 mg), 4 (2.4 mg), and 5 (1.7 mg). Fritenolide A (1): white amorphous powder; [α]20 D − 7.3 (c 0.05, MeOH); CD (MeOH) 246.5 (Δε + 1.12) nm; IR νmax = 3330, 2954, 2921, 2851, 1744, 1694, 1654, 1548, 1468, 1347, 1072, 965, 786, 721 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 526.4232 [M + Na]+ (calcd for C32H57NO3Na, 526.4236); HRESIMS/MS m/z: 482.4340 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2253 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2354 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide B (2): white amorphous powder; [α]20 D − 7.7 (c 0.04, MeOH); CD (MeOH) 243.5 (Δε + 1.11) nm; IR νmax =
3326, 2955, 2921, 2852, 1743, 1692, 1652, 1546, 1465, 1354, 1071, 877, 787, 719 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 526.4242 [M + Na]+ (calcd for C32H57NO3Na, 526.4236); HRESIMS/MS m/z: 482.4339 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2254 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2355 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide C (3): white amorphous powder; [α]20 D − 6.9 (c 0.04, MeOH); CD (MeOH) 244 (Δε + 0.94) nm; IR νmax = 3330, 2955, 2923, 2853, 1743, 1693, 1654, 1548, 1454, 1053, 965, 798 cm−1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z:498.3926 [M + Na]+ (calcd for C30H53NO3Na, 498.3923). HRESIMS/MS m/z: 454.4022 [M − CO2 + Na]+ (calcd for C29H53NONa, 454.4025), 316.2249 [M − C12H22O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2352 [M − CO2 − C12H22O + Na]+ (calcd for C17H31NNa, 272.2354). Fritenolide D (4): white amorphous powder; [α]20 D − 6.4 (c 0.05, MeOH); CD (MeOH) 244 (Δε + 0.99) nm; IR νmax =
Table 1 13 C NMR data (150 MHz) for compounds 1–5 in CDCl3a (δ in ppm). no.
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11–15 16 17 18 1′ 2′ 3′ 4′–9′ 10′ 11′ 12′ 13′ 14′
170.0 125.4 129.5 82.2 33.4 24.9 32.3 129.0 131.8 32.7 29.3–29.8b 32.1 22.8 14.3 172.1 37.0 25.3
169.9 125.4 129.4 82.2 33.5 25.1 26.9 128.5 131.2 27.4 29.3–29.8 32.1 22.8 14.3 172.1 37.0 25.3
170.0 125.4 129.6 82.3 34.0 25.1
170.0 125.4 129.6 82.3 34.0 25.1
29.3–29.8
29.3–29.9
32.1 22.8 14.3 172.1 37.0 25.3
29.3–29.8
29.3–29.8
32.1 22.8 14.3
32.1 22.8 14.3
170.0 125.4 129.5 82.2 33.4 24.9 32.3 129.0 131.8 32.7 29.3–29.8 32.1 22.8 14.3 172.1 37.0 25.3 29.3–29.8 32.1 22.8 14.3
32.1 22.9 14.3 172.1 37.0 25.4 29.3–29.9 32.1 22.9 14.3
a
29.3–29.8 32.1 22.8 14.3
The assignments were based on 1H–1H COSY, HSQC, and HMBC experiments. The 13C NMR signals for methylenes in the aliphatic chains were overlapped between 29.3–29.8 ppm (C-11–C-15 and C-4′–C-11′ in 1 and 2, C-11–C-15 and C-4′–C-9′ in 3, C-7–C-15 and C-4′–C-11′ in 4, C-7–C-15 and C-4′–C-9′ in 5). b
J. Liu et al. / Fitoterapia 98 (2014) 53–58
55
Table 2 1 H NMR data (600 MHz) for compounds 1–5 in CDCl3a (δ in ppm, J in Hz). no.
1
2
3
4
5
3 4 5 6 7 8 9 10 11–15 16 17 18 2′ 3′ 4′–9′ 10′ 11′ 12′ 13′ 14′ NH
7.42 d (1.8) 5.05 td (6.0, 1.8) 1.74 m, 1.66 m 1.52 m 2.03 q (6.6) 5.33 dt (15.6, 6.6) 5.41 dt (15.6, 6.6) 1.96 q (6.6) 1.25–1.35 mb 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.2) 1.69 m
7.42 d (1.2) 5.05 td (5.4, 1.2) 1.77 m, 1.68 m 1.50 m 2.08 q (7.2) 5.30 dt (10.2, 7.2) 5.40 dt (10.2, 7.2) 1.99 q (7.2) 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m
7.42 d (1.8) 5.04 td (5.4, 1.8) 1.70 m 1.44 m
7.43 d (1.2) 5.04 td (6.0, 1.2) 1.70 m 1.45 m
1.25–1.35 m
1.25–1.35 m
1.25 m 1.28 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m
1.25–1.35 m
1.25–1.35 m
1.25 m 1.29 m 0.88 t (7.2) 7.42 s
1.25 m 1.29 m 0.88 t (7.2) 7.42 s
7.42 d (1.8) 5.05 td (5.4, 1.8) 1.75 m, 1.66 m 1.52 m 2.03 q (6.6) 5.33 dt (15.6, 6.6) 5.41 dt (15.6, 6.6) 1.96 q (6.6) 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2) 2.35 t (7.8) 1.68 m 1.25–1.35 m 1.25 m 1.29 m 0.88 t (7.2)
1.25 m 1.28 m 0.88 t (7.2) 2.35 t (7.2) 1.68 m 1.25–1.35 m 1.25 m 1.28 m 0.88 t (7.2)
a
1
7.41 s
1.25–1.35 m 1.25 m 1.28 m 0.88 t (7.2) 7.41 s
7.41 s
1
The assignments were based on H– H COSY, HSQC, and HMBC experiments. The 1H NMR signals for methylenes in the aliphatic chains were overlapped between 1.25–1.35 ppm (H2-11–H2-15 and H2-4′–H2-11′ in 1 and 2, H2-11–H2-15 and H2-4′–H2-9′ in 3, H2-7–H2-15 and H2-4′–H2-11′ in 4, H2-7–H2-15 and H2-4′–H2-9′ in 5). b
3324, 2957, 2920, 2851, 1742, 1690, 1654, 1547, 1466, 1356, 1033, 785, 721 cm− 1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z:528.4391 [M + Na]+ (calcd for C32H59NO3Na, 528.4393); HRESIMS/MS m/z: 484.4497 [M − CO2 + Na]+ (calcd for C31H59NONa, 484.4494), 318.2411 [M − C14H26O + Na]+ (calcd for C18H33NO2Na, 314.2409), and 274.2513 [M − CO2 − C14H26O + Na]+ (calcd for C17H33NNa, 274.2511). Fritenolide E (5): white amorphous powder; [α]20 D − 6.6 (c 0.05, MeOH); CD (MeOH) 247 (Δε + 0.87) nm; IR νmax = 3338, 2955, 2921, 2853, 1742, 1693, 1645, 1559, 1464, 1380, 1035, 786, 722 cm− 1; 1H NMR and 13C NMR data see Tables 1 and 2; HRESIMS m/z: 500.4081 [M + Na]+ (calcd for C30H55NO3Na, 500.4080); HRESIMS/MS m/z: 456.4185 [M − CO2 + Na]+ (calcd for C29H55NONa, 456.4181), 318.2413 [M − C12H22O + Na]+ (calcd for C18H33NO2Na, 318.2409), and 274.2512 [M − CO2 − C12H22O + Na]+ (calcd for C17H33NNa, 274.2511).
2.5. Cytotoxic activity assay The human lung cancer cell lines H1975, H358 and A549, human hepatocellular carcinoma cell lines HepG-2 and SMMC7721, human colorectal carcinoma cell line HCT116, human mammary carcinoma cell lines MDA-MB-231 and MCF7, human melanoma cell line A2058, human pancreatic cancer cell line PANC-1 and human acute myeloid leukemia cell line MV4-11 were obtained from the American Type Culture Collection (ATCC) and grown in RPMI1640, DMEM or IMDM containing 10% fetal bovine serum (v/v) in 5% CO2 at 37 °C. Cells (2 × 103–10 × 103) were seeded in 96-well plates and cultured for 24 h, followed by the test compounds treatment at concentrations of 10 μM, 1 μM, and 0.1 μM for 72 h, respectively. After the culture period, 20 μL of MTT (5 mg/mL) was added per well and incubated for 4 h at 37 °C, then 50 μL of 20% acidified SDS was used to lyse the cells. Finally, absorbance was measured at 570 nm using a microplate reader. Each assay was replicated three times. The effect of the compounds on tumor cells viability was calculated and expressed by IC50 of each cell line.
2.4. Hepatoprotective activity assay The hepatoprotective effects were determined by a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in LO-2 cells. Each cell suspension of 1 × 105 cells/mL in DMEM containing 10% fetal bovine serum (v/v) was seeded in 96-well plates and cultured for 12 h in 5% CO2 at 37 °C. After fresh medium (100 μL) containing test sample was added, the cells were cultured for 24 h. Then, the cultured cells were exposed to 1 μM H2O2 for 1 h. The medium was changed into a fresh one containing 5 mg/mL MTT. After 4 h incubation at 37 °C, the supernatant was removed and 150 μL of DMSO was added to dissolve formazan crystals. The optical density (OD) of the formazan solution was measured on a microplate reader at 490 nm.
3. Results and discussion Compound 1 was obtained as a white amorphous powder, and the presence of ester (1744 cm−1), amide (3330, 1694 and 1548 cm−1), and olefinic (1654 cm−1) groups were indicated by its IR spectrum. The molecular formula C32H57NO3 of 1, with five hydrogen deficiencies, was indicated by HRESIMS at m/z 526.4232 (calcd for C32H57NO3Na, 526.4236). The 1H NMR spectrum of 1 (Table 2) displayed resonances attributable to two terminal methyls [δH 0.88 (6H, t, J = 7.2 Hz, H3-18 and H3-14′)], several aliphatic methylenes [δH 2.35 (t, J = 7.2 Hz, H2-2′), 2.03 (q, J = 6.6 Hz, H2-7), 1.96 (q, J = 6.6 Hz, H2-10), 1.74 (m, H-5a), 1.69 (m, H2-3′), 1.66 (m, H-5b), 1.52 (m, H2-6),
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1.25–1.35 (m, H2-11–17 and H2-4′–13′)], an oxymethine [δH 5.05 (td, J = 6.0, 1.8 Hz, H-4)], a trans-olefinic bond [δH 5.41 (dt, J = 15.6, 6.6 Hz, H-9), 5.33 (dt, J = 15.6, 6.6 Hz, H-8)], and an olefinic methine group [δH 7.42 (d, J = 1.8 Hz, H-3)]. The 13C NMR and DEPT spectrum of 1 revealed carbon resonances (Table 1) corresponding to the above protonated units and three quaternary carbons [two carbonylic (δC 172.1 and 170.0) and an olefinic (δC 125.4)]. The chemical shifts of the two carbonyl groups (δC b 175 ppm) in the 13C NMR spectrum, in combination with the molecular composition and the IR signals, suggested that there were an amide group and an ester group in 1. All these spectroscopic data indicated that compound 1 was an α-amino α,β-unsaturated γ-lactone with substitutions of an aliphatic acyl group and an aliphatic alkyl group containing one trans double bond [8,12]. This conjecture was proved by 2D NMR data analysis. In the HMBC spectrum, two- and three-bond correlations of H-3/C-1, C-2 and C-4; H-4/C-1, C-2 and C-3, further verified the α-amino α,βunsaturated γ-lactone unit in 1 (Fig. 2). The HMBC correlations of H-4/C-5 and C-6, H-8/C-7 and C-10, H-9/C-7 and C-10, in combination with the homonuclear coupling correlations of H-4/H2-5/H2-6/H2-7/H-8/H-9/H2-10 in the 1H–1H gCOSY spectrum, indicated that the aliphatic alkyl group was substituted at C-4 and the double bond was located at C-8 and C-9. The HMBC correlations from H2-2′ and H2-3′ to C-1′ confirmed the presence of the aliphatic acyl group. The length of two aliphatic chains was determined by HRESI-QTOF-MS/MS. The positively charged HRESI-QTOFMS/MS of 1 (Fig. 3) showed sodiated molecular ion peak at m/z 526.4237 [M + Na]+ (calcd for C32H57NO3Na, 526.4236) and its fragment ions at m/z 482.4340 [M − CO2 + Na]+ (calcd for C31H57NONa, 482.4338), 316.2253 [M − C14H26O + Na]+ (calcd for C18H31NO2Na, 316.2252), and 272.2354 [M − CO2 − C14H26O + Na]+ (calcd for C17H31NNa, 272.2354). These MS/MS spectroscopic data suggested that both of the two aliphatic chains were C14-long chains corresponding to a tetradecanoyl and a tetradecenyl group, respectively. In the CD spectrum, a positive Cotton effect at 246.5 nm for a π–π* transition of the α-amino α,β-unsaturated γ-lactone moiety suggested 4S configuration for 1 [12], which was supported by the negative optical rotation {[α]20 D −7.3 (c 0.05, MeOH)} [13]. Thus, compound 1 was determined to be (−)-(4S)-2tetradecanoylamino-4-[4(E)-tetradecenyl]but-2-enolide and named as fritenolide A. Compound 2 was an isomer of 1, as indicated by the HRESIMS, NMR and IR spectroscopic data. Comparison of the
13
C NMR data between 1 and 2 displayed that the resonances for C-7 and C-10 were shielded from δ 32.3 and 32.7 ppm in 1 to δ 26.9 and 27.4 ppm in 2, respectively (Table 1). This suggested Z configuration for the double bond in the unsaturated long chain in 2, on the basis of the 13C NMR chemical shifts of the methylene carbons adjacent to the olefinic carbons (δ ≈ 27 ppm for Z, δ ≈ 32 ppm for E) [14]. Meanwhile, the geometry (Z) of the double bond was confirmed by the coupling constant J8, 9 = 10.2 Hz in the 1H NMR spectrum of 2. The positive mode HRESIMS/MS of 2 showed the same sodiated fragment ions (C31H57NONa+, C18H31NO2Na+, and C17H31NNa+) as those of 1, which indicated that 2 also had the tetradecanoyl and tetradecenyl groups. In the 2D NMR spectrum, the 1H–1H gCOSY correlations of H-4/H2-5/H2-6/H2-7/H-8/H-9/H2-10 and the HMBC correlations of H-4/C-5 and C-6, H-8 and H-9/C-7 and C-10 verified the location of the double bond (C-8–C-9). In addition, the optical rotation ([α]20 D –7.7º) of 2 was consistent with that of 1. Therefore, 2 was assigned as (−)-(4S)-2tetradecanoylamino-4-[4(Z)-tetradecenyl]but-2-enolide and named as fritenolide B. Compound 3 was also obtained as a white amorphous powder. The 1D NMR, 2D NMR, IR, and [α]20 D data of 3 were very similar to those of 1, except that the HRESIMS data of 3 indicated the molecular formula C30H53NO3 for 3 with two fewer CH2 units than 1. This suggested the only difference between 3 and 1 was the length of the aliphatic chain. In the comparison of their HRESIMS/MS data, the different sodiated fragment ion C29H53NONa+ and the same fragment ions C18H31NO2Na+ and C17H31NNa+ revealed that the tetradecanoyl unit in 1 was substituted by a dodecanoyl unit in 3. Thus, 3 was determined to be (−)-(4S)-2-dodecanoylamino-4[4(E)-tetradecenyl]but-2-enolide and named as fritenolide C. The spectroscopic data of 4 showed that it was another analog of 1. The HRESIMS data indicated that it had the molecular formula C32H59NO3 with two more hydrogen atom than 1. Comparison of the NMR data of 1 and 4 displayed the resonances for the double bond in the tetradecenyl unit disappeared in 4, which indicated replacement of the tetradecenyl group in 1 by a tetradecyl group in 4. This was proved by the 2D NMR and HRESIMS/MS analysis. Specifically, the MS/MS fragment ions C31H59NONa+ ([M − CO2 + Na]+), C18H33NO2Na+ ([M − C14H26O + Na]+), and C17H33NNa+ ([M − CO2 − C14H26O + Na]+) confirmed the presence of the tetradecyl and tetradecanoyl groups. Accordingly, compound 4 was assigned as (−)-(4S)-2-tetradecanoylamino4-tetradecyl but-2-enolide named as fritenolide D.
NH O
5
1
O
O
1
H, 1H-COSY HMBC (H C)
Fig. 2. Key 1H, 1H-COSY and HMBC correlations of compound 1.
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57
Fig. 3. HRESI-QTOF-MS/MS fragmentation pattern of compound 1.
Compound 5 possessed the molecular formula C30H55NO3, with two fewer CH2 units than 4, as indicated by HRESIMS spectra. The NMR and HRESIMS/MS data analysis of 5 suggested that it was an analog of 4 except for substitution of the tetradecanoyl unit by a dodecanoyl unit. Thus, compound 5 was determined as (−)-(4S)-2-dodecanoylamino-4-tetradecylbut2-enolide and named as fritenolide E. The known compounds were identified as β-sitosterol [15], 7-ketositosterol [16], 3-methoxy-4-(palmitoyloxy)benzaldehyde [17], and methyl octadecanoate [18] by comparing their spectroscopic data with those reported in the corresponding literature. The protective activities on hepatocyte injury induced by H2O2 were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay in LO-2 cells [19]. Compounds 1–5 showed weak hepatoprotective activity at a concentration of 10 μM, with the survival rate of 27.0%, 24.9%, 27.8%, 30.1%, and 29.4%, respectively (H2O2 model group: 22.5%). In addition, all compounds were evaluated for the cytotoxic activities against 11 kinds of human tumor cells, including H1975, H358, A549, HepG-2, SMMC7721, HCT116, MDA-MB-231, MCF-7, A2058, PANC-1, and MV4-11 [20]. However no obvious effect was observed at 10 μM. Fritillaria plants are known to contain alkaloids, especially steroidal alkaloids, such as cevanine-type, veratramine-type, jervine-type, solanidine-type, and verazine-type [21–23]. However, the amide alkaloids have not been reported before. Thus, the isolation of new compounds 1–5 enriched the types of alkaloids in the genus. In addition, compounds 1–5 were unusual α-amino butenolides, which were very probably biosynthesized from unsaturated long-chain aliphatic α-amino acids in the natural plants. In the previous study, although a lot of butenolides derived from fatty acids metabolism have been found from the natural products [24–26], the α-amino butenolides from amino acids metabolism were rather rare in the terrestrial plants. To the best of our knowledge, there were only six natural α-amino butenolides obtained from the marine bacterium Pseudoalteromonas sp. F-420 [8], marine ascomycete Leptosphaeria oraemaris [9], marine sponge Anthosigmella aft. raromicrosclera [10], and fungus Penicillium sp. No. 13 [11]. Thus, the α-amino butenolides, especially those with an amide unit, could be used as a primary chemical character of Fritillaria unibracteata.
Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was financially supported by grants from the National Technology R&D Program for the “Eleventh Five-Year” Plan of China (grant No. 2009BAI84B04) is acknowledged. The authors are grateful to Prof. Min Li (Chengdu University of Traditional Chinese Medicine) for the plant identification. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.07.009. References [1] Xiao PG, Jiang Y, Li P, Luo YB, Liu Y. The botanical origin and pharmacophylogenetic treatment of Chinese materia medica Beimu. Acta Phytotaxon Sin 2007;45:473–87. [2] Commission of Chinese Pharmacopoeia. Pharmacopoeia of the People's Republic of China, vol. 1. Beijing: Chemical Industry Press; 2010. p. 34–5. [3] Wang DD, Wang S, Chen X, Xu XL, Zhu JY, Nie LH, et al. Antitussive, expectorant and anti-inflammatory activities of four alkaloids isolated from bulbus of Fritillaria wabuensis. J Ethnopharmacol 2012;139:189–93. [4] Akhtar MN, Atta-ur-Rahman, Choudhary MI, Sener B, Erdogan I, Tsuda Y. New class of steroidal alkaloids from Fritillaria imperialis. Phytochemistry 2003;63:115–22. [5] Ori K, Mimaki Y, Sashida Y, Nikaido T, Ohmoto T. Steroidal alkaloids from the bulbs of Fritillaria persica. Phytochemistry 1992;31:4337–41. [6] Shou QY, Tan Q, Shen ZW. A novel sulfur-containing diterpenoid from Fritillaria anhuiensis. Tetrahedron Lett 2009;50:4185–7. [7] Shen S, Li GY, Huang J, Chen CJ, Ren B, Lu G, et al. Steroidal saponins from Fritillaria pallidiflora Schrenk. Fitoterapia 2012;83:785–94. [8] Yoshikawa K, Takadera T, Adachi K, Nishijima M, Sano H. Korormicin, a novel antibiotic specifically active against marine gram-negative bacteria, produced by a marine bacterium. J Antibiot 1997;50:949–53. [9] White JD, Badger RA, Kezar HS, Pallenberg AJ, Schiehser GA. Structure, synthesis and absolute configuration of leptosphaerin, a metabolite of the marine ascomycete Leptosphaeria oraemaris. Tetrahedron 1989;45:6631–44. [10] Tsukamoto S, Kato H, Hirota H, Fusetani N. Pipecolate derivatives, anthosamines A and B, inducers of larval metamorphosis in ascidians, from a marine aponge Anthosigmella aft. raromicrosclera. Tetrahedron 1995;51:6687–94. [11] Kimura Y, Mizuno T, Kawano T, Okada K, Shimada A. Peniamidienone and penidilamine, plant growth regulators produced by the fungus Penicillium sp. No. 13. Phytochemistry 2000;53:829–31.
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