Four new compounds from the roots of Euphorbia ebracteolata and their inhibitory effect on LPS-induced NO production

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Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Four new compounds from the roots of Euphorbia ebracteolata and their inhibitory effect on LPS-induced NO production Jiao Baia,b, Xue-Yan Huanga,b, Zhi-Guo Liua,b, Chi Gonga,b, Xin-Yu Lia,b, Da-Hong Lia,b, ⁎ ⁎ Hui-Ming Huaa,b, , Zhan-Lin Lia,b, a b

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Euphorbia ebracteolata Diterpenoids Sesquiterpenes Anti-inflammation

Three new diterpenoids, ebractenoids O~Q (1–3), and a new phenolic glucoside, γ-pyrone-3-O-β-D-(6-galloyl)glucopyranoside (4), together with 6 known compounds, were isolated from the 95% ethanol extract of the roots of Euphorbia ebracteolata, and their structures were elucidated on the basis of spectroscopic data. The absolute configurations of 1–3 were determined by electronic circular dichroism (ECD) calculations. The inhibitory effects of all the isolates with exception of compounds 8 and 10 on the NO production in lipopolysaccharide (LPS)induced macrophages were evaluated. All of them exhibited significant inhibitory activity.

1. Introduction The root of Euphorbia ebracteolata Hayata (Euphorbiaceae) is one of the two origins of the traditional Chinese medicine “Langdu”, which is employed for the treatment of scrofula and ringworm by external application [1]. Previous phytochemical investigation on E. ebracteolata led to the isolation of diverse scaffolds including diterpenoids [2–4], triterpenoids [5–7], steroids [8], acetophenones [9–11], and organic acids [12,13]. Diterpenoids are the most abundant metabolites in E. ebracteolata, which showed a wide spectrum of pharmacological activities such as anti-inflammatory [14–16], antitumor [17,18], and antifungal [19] effects. During our exploring for the structurally diverse natural anti-inflammatory terpenoids, three new diterpenes (1–3) and one new γ-pyrone glycoside (4), together with six known terpenoids (5–10, Fig. 1), were isolated from the roots of E. ebracteolata after the reports on an array of diterpenes [14,15]. Additionally, their anti-inflammatory activities against the NO production in LPS-induced macrophages were evaluated. 2. Experimental 2.1. General Optical rotations were obtained on a JASCO-2000 polarimeter. The UV spectra were acquired on a Shimadzu UV-2201 spectrometer. The FT-IR spectra were obtained on a Bruker IFS-55 spectrometer (by the

⁎

KBr disk method). ECD spectra were recorded on a BioLogic MOS-450 spectrometer. HRESIMS were recorded on an Agilent 6210 TOF mass spectrometer. NMR spectra were recorded on Bruker ARX-300 and AV600 NMR spectrometers using TMS as an internal standard. Chromatographic silica gel (200–300 mesh) was purchased from Qingdao Marine Chemical Factory (Qingdao, China), and ODS (50 μm) was obtained from YMC Co. Ltd., Kyoto, Japan. Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. RPHPLC separations were conducted using a Shimadzu LC-6AD liquid chromatograph with a YMC-PACK ODS-A column (250 × 10 mm, 5 μm) and a Shimadzu SPD-10A VP UV/vis detector. TLC spots were visualized under UV light and/or with 10% H2SO4 in EtOH followed by heating. HPLC-ORD was run on a Shodex Asahipak NH2P-50 4E (250 × 4.6 mm I. D., 5 μm) equipped with a LC-NetII/ADC pump and a JASCO OR-4090 detector (JASCO Co. Ltd., Japan). 2.2. Plant material Plant material was purchased from Traditional Chinese Medicinal Materials Trading Center, Bozhou City, Anhui Province, China, and identified by Professor Jin-Cai Lu, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (LD-20110530) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China.

Corresponding authors at: Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China. E-mail addresses: [email protected] (H.-M. Hua), [email protected] (Z.-L. Li).

https://doi.org/10.1016/j.fitote.2017.12.006 Received 15 August 2017; Received in revised form 30 November 2017; Accepted 4 December 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Bai, J., Fitoterapia (2017), https://doi.org/10.1016/j.fitote.2017.12.006

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Fig. 1. Structures of compounds 1–10.

gradient system of increasing polarity with petroleum ether-acetone (100:0–0:100, v/v) to afford 6 fractions (Fr. 2A–Fr. 2F). Fr. 2B (10 g) was separated by ODS CC eluting with MeOH-H2O (from 20 to 80%), followed by a Sephadex LH-20 column (MeOH) to afford 4 (10.8 mg). Fr. 2E (4.0 g) was subjected to ODS CC eluting with MeOH-H2O (from 20 to 80%) to afford two subfractions (Fr. 2E1 and Fr. 2E2). Fr. 2E1 (0.5 g) was subjected to Sephadex LH-20 CC eluting with MeOH to afford 9 (10.9 mg). Compound 6 (4.2 mg) was obtained from Fr. 2E2 by preparative TLC using petroleum ether-EtOAc (2:1) as the developing solvent.

2.3. Extraction and isolation The dried roots of E. ebracteolata (18 kg) were extracted with 95% EtOH (3 × 60 L) three times (3 h, 2 h, and 1 h, respectively) under reflux. The EtOH extract was concentrated to afford a crude extract (1.2 kg), which was suspended in H2O (20 L) and partitioned successively with petroleum ether (3 × 20 L) and CH2Cl2 (3 × 20 L). The petroleum ether extract (380 g) was subjected to silica gel column chromatography (CC) using a gradient system of increasing polarity with petroleum ether-acetone (100:0–0:100, v/v) to afford 10 fractions (Fr. 1A–Fr. 1 J). Fr. 1F (20 g) was subjected to ODS CC eluting with MeOH-H2O (from 40 to 80%), followed by preparative TLC using petroleum ether-acetone (2:1) to yield 3 (20.6 mg). Fr. 1G (25 g) was separated by ODS CC eluting with a MeOH-H2O gradient from 40:60 to 80:20, followed by a Sephadex LH-20 column (MeOH) and further purified by semi-preparative HPLC (MeOH-H2O, 65:35) to afford 1 (2.1 mg) and 2 (17.4 mg). Fr. 1H (17 g) was subjected to an ODS column eluting with MeOH-H2O (from 20 to 80%), followed by preparative TLC using CH2Cl2-MeOH (15:1) to yield 7 (15.2 mg) and 10 (20.2 mg). Fr. 1I was repeatedly recrystallized in MeOH to give 5 (36.2 mg). Compound 8 (5.6 mg) was obtained from the remaining part of Fr. 1I by preparative TLC using petroleum ether-acetone (2:1) as the developing solvent. The CH2Cl2 extract (100 g) was subjected to silica gel CC using a

2.3.1. Ebractenoid O (1) White amorphous powder; [α]D20 – 118.0 (c 0.10, MeOH); HRESIMS m/z 343.2244 [M + Na]+ (calcd. for C20H32O3Na, 343.2244); 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data see Table 1. 2.3.2. Ebractenoid P (2) White amorphous powder; [α]D20 – 99.1 (c 0.11, MeOH); HRESIMS m/z 325.2134 [M + Na]+ (calcd. for C20H30O2Na, 325.2138); 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data see Table 1. 2.3.3. Ebractenoid Q (3) White amorphous powder; [α]D20 – 161.0 (c 0.11, MeOH); HRESIMS m/z 499.3055 [M + H]+ (calcd. for C30H43O6, 499.3054); 1H NMR 2

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Table 1 1 H NMR (300 MHz) and Position

13

1

2 δC

δH (mult. J in Hz)

δC

1

5.58 (dd, 6.0, 2.4)

117.8

39.1

2

4.05 (d, 6.0)

78.2

2.08 1.70 2.63 2.37

3 4 5 6

3.81 (br s)

79.7 48.5 46.8 19.5

7 8 9 10 11 12 13 14 15 16 17 18 19 20

(m) (m) (m) (m) (m) (m)

25.0 31.9 36.9 154.9 35.0

1.58 1.36 1.50 1.22

(m) (m) (m) (m)

1.18 1.09 5.77 4.89 4.83 0.96 1.13 3.93 3.36 0.95

(m) (m) (dd, 17.4, 10.8) (dd, 17.4, 1.2) (dd, 10.8, 1.2) (s) (s) (d, 9.0) (d, 9.0) (s)

Table 2 1 H NMR (300 MHz) and

1.99 1.74 1.59 1.71 1.23 2.29

13

36.6 39.4

1.40 1.02 5.77 4.90 4.84 0.92 1.18 4.02 3.56 1.24

151.3 109.1 22.7 15.6 70.3 20.9

δC

Position

1

1.93 1.20 1.53 1.48 1.58 1.46

(m) (m) (m) (m) (m) (m)

41.7

1.05 1.57 1.38 2.11 1.68

(brd, 12.0) (m) (m) (m) (m)

15 16 17 18 19 20 1′ 2′ 3′ 4′

3 4 5 6 7 8 9 10 11 12 13 14

a

35.9

(m) (m) (m) (m) (m) (m)

27.1 28.9 148.7 37.6 117.7

1″ 2″, 6″ 4″ 3″, 5″ 7″

37.7

(ddd, 12.7, 5.2, 2.1) (t, 11.4) (dd, 17.4, 10.8) (dd, 17.4, 1.2) (dd, 10.8, 1.2) (s) (s) (d, 10.8) (d, 10.8) (s)

35.0 41.7

18.9 41.6 33.5 55.1 20.4 39.9

2.07 (brs) 4.58 (d, 3.6) 5.13 (dq, 3.6, 1.8) 5.61 (s)

74.6 62.2 38.1 68.8 79.0 152.0 73.7

5′ 6′ 7′ 8′ 9′ 10′ 8-OH 11-OH

δH (mult. J in Hz)

2.01 0.90 0.87 1.19

(d, 1.8) (s) (s) (s)

6.78 2.49 1.15 2.13 2.43 2.36 2.77

(m) (m) (d, 9.1) (m) (m) (m) (td, 5.6, 1.3)

1.30 0.77 5.37 5.19

(s) (s) (s)a (br s)a

150.1 109.5

13

δH (mult., J in Hz)

δC

8.13 (d, 0.6)

155.7 145.7 172.4 116.1 144.1 100.1 73.1 76.3 69.5 73.9 63.1

6.37 8.05 4.88 3.27 3.27 3.27 3.63 4.43 4.24

(d, 5.4) (dd, 5.4, 0.6) (d, 7.2) (m, overlapped) (m, overlapped) (m, overlapped) (m) (d, 11.7) (d, 11.7)

6.94, s

119.3 108.7 138.6 145.6 165.7

2.5. Inhibitory assay for NO production

22.6 21.3 66.2

The nitrite concentration in the medium was measured according to the Griess reaction as an indicator of NO production. Briefly, RAW 264.7 cells were seeded into 96-well plates at a density of 1 × 105 cells/well and stimulated with 1 μg/mL LPS in the presence or absence of test compounds. After incubation at 37 °C for 24 h, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.2% (w/v) N-(1-naphthyl)-ethylenediamine solution to determine nitrite production. The absorbance was measured in a microplate reader at 540 nm and compared with a calibration curve prepared using NaNO2 standards. The experiments were performed in triplicate, and the data are expressed as the means ± SD of three independent experiments.

24.7

δC 130.1 174.4 9.1 34.3 22.2 18.3 165.5 139.8 138.3 31.4

3. Results and discussion Ebractenoid O (1) was obtained as white amorphous powder. Its molecular formula was determined to be C20H32O3 based on the 13C NMR data and the HRESIMS ion peak at m/z 343.2244 [M + Na]+ (calcd. for C20H32O3Na, 343.2244). The 1H NMR data (Table 1) indicated the presence of a vinyl group (δ 5.77, dd, J = 17.4, 10.8 Hz; 4.89, dd, J = 17.4, 1.2 Hz; 4.83, dd, J = 10.8, 1.2 Hz), a trisubstituted olefinic moiety (δ 5.58, dd, J = 6.0, 2.4 Hz), a hydroxymethyl group (δ 3.93, 3.36, each d, J = 9.0 Hz), two oxymethine groups (δ 4.05, d, J = 6.0 Hz; 3.81, br s), and three tertiary methyl groups (δ 1.13, 0.96, and 0.95). The 13C NMR and HSQC (Table 1) data showed 20 carbon resonances, including three methyl carbons, seven methylene carbons (including one sp2 carbon), six methine carbons (containing two sp2 carbons), and four quaternary carbons. The aforementioned spectroscopic data suggested that compound 1 was probably a rosane-type diterpenoid similar to that of hugorosediol [20], with the difference of one more hydroxyl group. The Δ1(10) and Δ15 double bonds were confirmed based on the HMBC correlations (Fig. 2a) from H-1 to C-3, C-5, and C-9 and from H-15 to C-12, C-14, and C-17. In the HMBC spectrum, the cross peaks from H-3 to C-5, C-18, and C-19, from H-19 to C-3, C-5, and C-18, and from H-2 to C-1, C-4, C-10 allowed the assignment of three hydroxyl groups to C-2, C-3, and C-19, respectively. The NOESY correlations (Fig. 2b) of CH3-18/H-3, H-5 and H-6α, H-5/H-3 and H-8, H-8/CH3-17, H-15/H-12β, and CH3-20/H-6β, H-12β, and CH2-19,

40.4 32.6 41.8 37.8 25.9 21.1

Recorded in DMSO-d6.

(300 MHz, CDCl3) and

4

reflux for 1 h. The reaction solution was analyzed by HPLC-ORD on a Shodex Asahipak NH2P-50 4E column.

C NMR (75 MHz) data for compound 3 in CDCl3.

δH (mult. J in Hz)

C NMR (75 MHz) data for compounds 4 in DMSO-d6.

2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′

217.4 52.9 48.0 18.8

2.01 (m) 1.73 (m)

Position

2

(m) (m) (ddd, 15.4, 13.0, 6.5) (ddd, 15.4, 5.6, 3.0)

5.42 (dd, 5.4, 2.4)

32.8

13

Position

δH (mult. J in Hz)

2.61 1.68 1.40 1.56 1.28 1.73

Table 3 1 H NMR (300 MHz) and

C NMR (75 MHz) data for compounds 1 and 2 in CDCl3.

C NMR (75 MHz, CDCl3) data see Table 2.

2.3.4. γ-Pyrone-3-O-β-D-(6-galloyl)-glucopyranoside (4) Yellow amorphous powder; [α]D20 –119.3 (c 0.14, MeOH); λmax nm: 215, 266, 360; HRESIMS m/z 449.0686 [M + Na]+ (calcd. for C18H18O12Na, 449.0690); 1H NMR (300 MHz, DMSO-d6) and 13C NMR (75 MHz, DMSO-d6) data see Table 3. 2.4. Acid hydrolysis A total of 1.5 mg of 4 was hydrolyzed with 10% HCl (4 mL) under 3

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b Fig. 2. Key HMBC (a) and NOESY (b) correlations of 1.

indicated that H-3, H-5, H-8, CH3-17, and CH3-18 were in α-orientation, while 3-OH, HOCH2-19, and CH3-20 were β-oriented. Due to the small coupling constant between H-2 and H-3, 2-OH and 3-OH possessed the same β-orientation [17]. The absolute configuration was determined by comparing its experimental ECD spectrum with the calculated ones of (2S,3R,4S,5R,8R,9R,13R)-1a and (2R,3S,4R,5S,8S,9S,13S)-1b (Supplementary Information, Fig. S23). The ECD spectrum of 1 showed positive Cotton effects at 250 nm, and negative Cotton effect at 212 nm, respectively, which was in agreement with calculated ECD spectrum of 1a. Therefore, the structure of compound 1 was defined as (2S,3R,4S,5R,8R,9R,13R)-2,3,19-trihydroxy-1(10),15-rosadiene. Ebractenoid P (2) was obtained as white amorphous powder. Its molecular formula was determined to be C20H30O2 based on the 13C NMR data and the HRESIMS ion at m/z 325.2134 [M + Na]+ (calcd. for C20H30O2Na, 325.2138). The 1H NMR data (Table 1) indicated the presence of a vinyl group (δ 5.77, dd, J = 17.4, 10.8 Hz; 4.90, dd, J = 17.4, 1.2 Hz; 4.84, dd, J = 10.8, 1.2 Hz), a trisubstituted olefinic moiety (δ 5.42, dd, J = 5.4, 2.4 Hz), a hydroxymethyl group (δ 4.02, 3.56, each d, J = 10.8 Hz), and three tertiary methyl groups (δ 1.24, 1.18, and 0.92). The 13C NMR and HSQC (Table 1) data showed 20 carbon resonances, including three methyl carbons, eight methylene carbons (including one sp2 carbon), four methine carbons (containing two sp2 carbons), and five quaternary carbons (including one carbonyl carbon), suggesting that compound 2 was likely a isopimarane-type diterpenoid. The 1D and 2D NMR data showed that the structure of 2 was similar to that of 19-hydroxy-9(11),15-isopimaradien-3-one [21]. In the HMBC spectrum (Fig. 3a), the correlations from H-18 to C-3, C-4, C-5, and C-19, and from H-20 to C-1, C-5, and C-9, and from H-11 to C8, C-10, and C-13 confirmed the positions of the functional groups of compound 2. The relative configuration of 2 was assigned on the basis of the NOESY experiment. The NOE correlations (Fig. 3b) of CH3-18/H5, H-5/H-8, and H-8/CH3-17 indicated their cofacial arrangement and allowed tentative assignment of the α-orientation. CH3-20 had a β-orientation based on the NOE correlation between CH3-20 and CH2-19. The ECD spectrum of 2 showed positive Cotton effects at 215 and 295 nm, and negative Cotton effect at 200 nm, respectively, which was in good agreement with calculated ECD spectrum of 2a (Supplementary Information, Fig. S24). Therefore, the structure of 2 was elucidated as (4S,5R,8R,10S,13R)-19-hydroxy- 9(11),15-pimaradien-3-one. Ebractenoid Q (3) was obtained as white amorphous powder. Its molecular formula was determined to be C30H42O6 based on the 13C NMR data and the HRESIMS ion at m/z 499.3055 [M + H]+ (calcd. for

a

Fig. 4. Key HMBC correlations of 3.

C30H43O6, 499.3054). The 1H NMR data (Table 2) indicated the presence of a trisubstituted olefinic proton at δ 6.78 (1H, m), three oxymethine protons (δ 5.61, s; 5.13, dq, J = 3.6, 1.8 Hz; 4.58, br d, J = 3.6 Hz), one olefinic methyl doublet at δ 2.01 (3H, d, J = 1.8 Hz), and five methyl singlets at δ 1.30, 1.19, 0.90, 0.87, and 0.77. The 13C NMR and HSQC (Table 2) data showed 30 carbon resonances, including six methyl carbons, seven sp3 methylene carbons, seven sp3 methine carbons, four sp3 quaternary carbons, four olefinic carbons, and two ester carbonyl carbons. Comparison of its 1D NMR data with those of yuexiandajisu E [17] and myrtenic acid [22], indicated that 3 was composed of yuexiandajisu E and myrtenic acid moieties. In the HMBC spectrum (Fig. 4a), the correlations from CH3-20 to C-1, C-9, and C-10, from H-11 to C-8, C-10, C-12, and C-13, from H-14 to C-9, C-12, C-13, and C-15, and from CH3-17 to C-13, C-15, and C-16 confirmed the planar structure of abietane lactone part of 3 as yuexiandajisu E. The similar 1H and 13C NMR data of abietane lactone part of 3 to those of yuexiandajisu E [17] indicated that 3 possessed the same relative configuration as yuexiandajisu E, which was partially supported by the NOE correlations of CH3-18/H-5, H-5/H-9, CH3-19/CH3-20, CH3-20/H11 and H-12. And the cross peaks of H-3′ to C-1′, C-5′, and C-7′, of H-4′ to C-2′, and C-6′ and of H-9′ and H-10′ with C-5′, C-7′, and C-8′ in HMBC spectrum, confirmed the presence of a myrtenic acid moiety. These two parts were connected by an ester bond at C-14 on the basis of the cross peak from H-14 to C-1′. Considered the biogenetic relationship, the absolute configuration of diterpene part was same as yuexiandajisu E from E. ebracteolata [17]. Therefore, the structure of 3 was defined as (5R,8R,9R,10R,11R,12S,14R)-8,11-dihydroxy-14-myrtenoyloxy-entabieta-13(15)-ene-16,12-lactone. Compound 3 is the first hybrid of myrtenic acid with diterpene, and definitely, can be employed as the chemotaxonomic marker for Euphorbia ebracteolata. Compound 4 was obtained as a yellow amorphous powder, with the molecular formula determined to be C18H18O12 based on the 13C NMR data and the HRESIMS ion at m/z 449.0686 [M + Na]+ (calcd. for C18H18O12Na, 449.0690). The 1H NMR spectrum of 4 demonstrated the resonances of one set of ABX coupling system (δ 8.13, d, J = 0.6 Hz; 8.05, dd, J = 5.4, 0.6 Hz; 6.37, d, J = 5.4 Hz) assigned to a γ-pyrone, a two-proton aromatic singlet (δ 6.94, s) due to 1″,3″,4″,5″-tetrasubstituted aromatic ring, and a β-anomeric proton signal (δ 4.88, d, J = 7.2 Hz). The 13C NMR and HSQC (Table 3) data showed 18 carbon resonances, which were assigned to a γ-pyrone (δ 172.4, 155.7, 145.7, 144.1, and 116.1), a β-glucose (δ 100.1, 76.3, 73.9, 73.1, 69.5, and 63.1), and a galloyl (δ 165.7, 145.6, 145.6, 138.6, 119.3, 108.7, and 108.7), respectively. In the HMBC spectrum, the cross peaks from H-1′ to C-3 (δ 145.7), and from H-2″ and H-6′ to the carbonyl C-7″ (δ 165.7) of the galloyl confirmed that the glucose was connected to C-3 of γpyrone, and the galloyl was connected to C-6′ of the glucose moiety. The absolute configuration of the glucose was assigned by comparing

b

Fig. 3. Key HMBC (a) and NOESY (b) correlations of 2.

4

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Table 4 Inhibitory effect of isolated compounds on LPS-induced NO production in macrophages. [3] Compound

IC50 ± SD (μM)

Compound

IC50 ± SD (μM)

1 2 3 4 5

6.04 ± 0.55 10.23 ± 0.73 1.97 ± 0.96 42.49 ± 3.0827 13.21 ± 1.10

6 7 9 Hydrocortisone Indomethacin

6.25 ± 0.47 6.73 ± 0.52 18.50 ± 1.04 54.23 ± 4.12 16.67 ± 1.24

[4] [5] [6] [7] [8]

the hydrolysis product of 4 with D-glucose by HPLC with optical rotation detector. Based on the above analyses, the structure of 4 was defined as γ-pyrone-3-O-β-D-(6-galloyl)-glucopyranoside. The known compounds were identified as tricyclohumuladiol (5) [23], ingenol (6) [24], ingenol-20-acetate (7) [25], ingenol-20-palmitate (8) [26], langduin A4 (9) [27], and jolkinol A (10) [28] by comparison of the observed and reported spectroscopic data. The anti-inflammatory activities of the isolated compounds were evaluated by examining their abilities to inhibit LPS-induced NO production in vitro in macrophages. Indomethacin and hydrocortisone were selected as positive controls. As shown in Table 4, all the test terpenoids (1–3, 5–7, and 9) exhibited potent inhibitory activity, of which compound 3 was the most active one with an IC50 value of 1.97 μM, while the γ-pyrone-3-O-β-D-(6-galloyl)-glucopyranoside (4) showed weaker activity.

[9]

[10] [11]

[12] [13]

[14]

[15]

Conflict of interest

[16] [17]

The authors declare no conflict of interest.

[18]

Acknowledgements

[19]

This work was supported by the National Natural Science Foundation of China (No. 81172958) and the National Key Technology R&D Program (2012BAI30B02). The authors wish to thank Mr. Y. Sha and Mrs. W. Li (Analytical Test Center, Shenyang Pharmaceutical University, Shenyang, People's Republic of China) for acquiring the NMR data.

[20]

[21] [22]

[23]

Appendix A. Supplementary data

[24]

The UV, IR, HR-ESI-MS, 1D NMR, 2D NMR, and ECD spectra for compounds 1–4, were available in the Supplementary data. Supplementary data to this article can be found online at https://doi. org/10.1016/j.fitote.2017.12.006.

[25] [26] [27]

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