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Oct 14, 2016 - ABSTRACT: Medicago sativa L. is the most important cultivated herbage, known as “the king of forage” and “feed queen”, in the w...
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Characterization of Chalcones from Medicago sativa L. and Their Hypolipidemic and Antiangiogenic Activities Qin-Ge Ma,*,† Ting Li,† Rong-Rui Wei,*,‡ Wen-Min Liu,† Zhi-Pei Sang,† and Zhong-Wen Song† †

College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang, Henan 473061, People’s Republic of China ‡ Department of Pharmacology, College of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, People’s Republic of China ABSTRACT: Medicago sativa L. is the most important cultivated herbage, known as “the king of forage” and “feed queen”, in the world. A total of 8 new chalcones (1−8), and 12 known chalcones (9−20) were isolated from the aerial parts of M. sativa for the first time. Their structures were identified by extensive spectral data and references. The hypolipidemic and antiangiogenic activities of compounds (1−20) were evaluated for the first time. Compounds 3, 4, 11, 12, and 20 (10 μM) exhibited significant hypolipidemic activities by measuring the triglyceride content in HepG2 cells, with simvastatin as the positive control. Moreover, compounds 6, 8, 18, and 19 exhibited moderate antiangiogenic activities, which inhibited vascular-endothelial-growth-factorinduced human umbilical vein endothelial cell proliferation in vitro, with IC50 values of 13.86 ± 0.43, 15.53 ± 0.19, 39.52 ± 0.24, and 45.04 ± 0.51 μM, respectively. These research results may guide the search for new natural products with hypolipidemic and antiangiogenic attributes. KEYWORDS: Medicago sativa L., chalcone, hypolipidemic, antiangiogenic

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INTRODUCTION

General Experimental Procedures. The infrared (IR) spectra were recorded on a Nicolet 5700 FTIR spectrometer with KBr pellets (Shanghai Xiangrun Industry Co., Ltd., Shanghai, China). The ultraviolet (UV) spectra were measured with an Australia GBC UV916 spectrophotometer (GBC Scientific Equipment Pty Ltd, Braeside, Australia). The one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectral data were determined by a Bruker-400 spectrometer, with tetramethylsilane (TMS) as the internal standard (Bruker Corporation, Madison, WI, U.S.A.). The high-resolution electrospray ionization mass spectrometry (HR-ESI− MS) data were measured with an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA, U.S.A.), and ESI−MS spectra were recorded on a LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.).20 Preparative high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm, Shimadzu Corporation, Japan). The HPLC data were measured using Agilent 1200 series with a DIKMA (4.6 × 250 mm) analytical column packed with C18 (5 μm, Agilent Technologies, Santa Clara, CA, U.S.A.). The column chromatography (CC) was subjected to silica gel (100−200 or 200−300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia Biotech Co., Ltd., Tokyo, Japan). Thin-layer chromatography (TLC) was applyed to silica gel GF254 plates (Qingdao Marine Chemical, Inc., Qingdao, China), and the spots were observed under UV light (254/ 365 nm) or spraying with 10% H2SO4 in 95% EtOH, followed by heating.21 Plant Material. The aerial parts of M. sativa were collected from Xihe County, Gansu Province, in October 2014. This plant was

Medicago sativa L., belonging to the Leguminosae family, is called “the king of forage” and “feed queen” of the world.1 It contains a variety of nutritional components and is used as an industrial plant for herdsmen. M. sativa is rich in natural resources, which is widely introduced and cultivated in the world.2 M. sativa is an excellent pasture for animals as a result of its digestibility, protein content, and other nutrients.3 Moreover, M. sativa is also served as a medicinal plant for diseases in many countries. Previous phytochemical investigations of M. sativa contained flavonoids,4 isoflavones,5 volatiles,6 organic acids,7 saponins,8 polysaccharides,9 tannins,10 etc. Modern pharmacology showed that M. sativa possessed a range of biological activities, such as antibacterial,11 antioxidant,12 anticancer,2 O-methyltransferase,13 antihyperlipidemic,14 and anxiolytic15 activities. On the basis of previous pharmacological investigations of M. sativa, it was found that there had been few reports on hypolipidemic and antiangiogenic activities of M. sativa, which prompted us to study its further chemical constituents and bioactivities. In this work, we carried out a bioactivity-guided investigation of M. sativa to evaluate its further pharmacological activities. As a result, 8 new chalcones (1−8) and 12 known chalcones (9−20) were isolated from the aerial parts of M. sativa for the first time. Modern pharmacology showed that chalcones showed a range of pharmacological effects, such as anticancer,16 anti-HIV,17 antihypertensive,18 insect antifeedant,19 etc. The structures of compounds (1−20) were identified by their spectral data and references. Moreover, all of the compounds were evaluated for their hypolipidemic and antiangiogenic activities for the first time. © 2016 American Chemical Society

MATERIALS AND METHODS

Received: Revised: Accepted: Published: 8138

August 31, 2016 October 7, 2016 October 14, 2016 October 14, 2016 DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

Article

Journal of Agricultural and Food Chemistry identified by Dr. Zhanwen Feng (pharmacist of traditional Chinese medicine, Henan Fusen Pharmaceutical Co., Ltd.). The voucher specimen (MXC-2014010) has been deposited at the Nanyang Normal University, Nanyang, Henan, China. Extraction and Isolation. The aerial parts of M. sativa (18.0 kg) was extracted with 54 L of 95% EtOH heating under reflux for 3 h each time. The extract was concentrated by a rotary evaporator at 45 °C under reduced pressure, which obtained a dark black residue (2.1 kg). The combined extracts were successively partitioned with petroleum ether, ethyl acetate (EtOAc), and n-butanol to yield three fractions:22 petroleum-ether-soluble fraction (132.5 g), EtOAc-soluble fraction (226.7 g), and n-butanol-soluble fraction (308.3 g). According to the screening results of the bioactivity-guided investigation, the EtOAc-soluble fraction was identified as the active site according to the hypolipidemic activity of 0.2208 ± 0.0031 (10−5 M) and the IC50 value of antiangiogenic activity of 21.62 ± 0.37 μM. The active extract of the EtOAc-soluble fraction was performed on silica gel (100−200 mesh) eluted with a gradient elution (petroleum ether/EtOAc = 15:1 → 8:1 → 6:1 → 3:1 → 1:1), and yielded five fractions: A (22.8 g), B (50.7 g), C (87.3 g), D (31.6 g), and E (12.6 g). Fraction B was subjected to silica gel (200−300 mesh) eluted with petroleum ether/EtOAc gradients (10:1 → 8:1 → 6:1) to give three subfractions, B1−B3. Subfraction B2 (19.3 g) with antiangiogenic activity (28.39 ± 0.46 μM) was separated by Sephadex LH-20 and silica gel (100−200 or 200−300 mesh) repeatedly, yielding compounds 7 (10.15 mg), 8 (11.58 mg), 14 (10.25 mg), and 18 (12.32 mg). In a similar way, fraction C was chromatographed over silica gel (200−300 mesh) eluted with petroleum ether/EtOAc gradients (8:1 → 6:1 → 3:1) to give three subfractions, C1−C3. Subfraction C2 (35.7 g) with hypolipidemic activity (0.2018 ± 0.0072, 10−5 M) was separated by silica gel (100−200 or 200−300 mesh) and Sephadex LH-20 successively, yielding compounds 1 (8.42 mg), 2 (8.69 mg), 10 (10.74 mg), 12 (11.36 mg), 17 (12.16 mg), and 20 (13.45 mg). Fraction D was applied to the Sephadex LH-20 column (eluted with MeOH in H2O, 85, 95, and 100% in a step gradient manner) to afford three subfractions, D1−D3. Subfraction D2 (15.4 g) with antiangiogenic activity (21.68 ± 0.65 μM) was purified by medium-pressure liquid chromatography (MPLC) (70−100% MeOH−H2O), followed by preparative HPLC (detection at 210− 400 nm, 6 mL/min), to yield compounds 5 (8.68 mg), 6 (9.26 mg), 15 (10.24 mg), 16 (12.28 mg), and 19 (11.79 mg). In the same way, subfraction D3 (8.55 g) with hypolipidemic activity (0.2119 ± 0.0037, 10−5 M) was subjected to MPLC (85−100% MeOH−H2O) successively, using preparative HPLC (detection at 220−390 nm, 6 mL/min), to yield compounds 3 (9.06 mg), 4 (8.63 mg), 9 (10.96 mg), 11 (13.26 mg), and 13 (12.09 mg). The structures of these compounds (1−20) were shown in Figure 1. Hypolipidemic Activity Assay. The compounds (1−20) were assayed for their hypolipidemic activities by measuring the triglyceride content in HepG2 cells in vitro, with 10 μM simvastatin as the positive control.23 All of the compounds were dissolved in dimethyl sulfoxide (DMSO) with a concentration of 10−2 M and then diluted to 10−5 M. The HepG2 cells were cultured in Ham’s F-12/Leibovitz L-15 (1:1, v/ v) supplemented with 7% newborn calf serum, 50 units of penicillin/ mL, and 50 μg of streptomycin/mL.24 After the HepG2 cells were detached with trypsin, they were inoculated in 96-well plates with a density of inoculation of 5 × 104 cells/mL. These cells were starved for 12 h in Dulbecco’s modified Eagle’s medium (DMEM) with 1% bovine serum albumin (BSA), where the cell growth reached 80−90% confluency. After stimulation for 24 h in DMEM with fatty acids (1.2 mM), the culture medium was discarded, washed 3 times with phosphate-buffered saline (PBS), and then fixed with paraformaldehyde (4%) at 4 °C for 12 h. Each hole was filled with Oil Red O (20 μL) for 15 min. After the Oil Red O dye was washed, DMSO (100 μL) was added to each well to dissolve the dye attached to the lipid. Lastly, the optical density (OD) values were measured at 358 nm.25 Antiangiogenic Activity Assay. We had evaluated the compounds (1−20) for their antiangiogenic activities in vitro, with axitinib (Pfizer, New York, U.S.A.) as the positive control.26 Compounds (1−20) were evaluated by the 3-(4,5-dimethylthiazol-2-

Figure 1. Structures of compounds 1−20.

yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine whether they decreased vascular endothelial growth factor (VEGF)mediated cell proliferation in human umbilical vein endothelial cells (HUVECs). The HUVECs were cultured in DMEM with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The HUVECs were seeded in 96-well plates and treated with 10 ng/mL VEGF and test compounds (purity of >98.7% by HPLC). Compounds (1−20) were dissolved in DMSO and diluted with the culture medium to afford a final DMSO concentration (0.2%) in the assay. After incubation for 24 h, the MTT reagent was added to these cultured cells and then they were measured at 450 nm. All of the results were typically expressed as IC50 values, and they were calculated using linear interpolation of inhibition curves for five independent experiments.27

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RESULTS AND DISCUSSION Structure Elucidation of New Compounds. Compound 1 was obtained as a yellow powder, and its molecular formula was deduced to be C26H28O4 from the HR-ESI−MS ion peak at m/z 427.3681 [M + Na]+ (calcd for C26H28O4Na, 427.3629), corresponding to 13 degrees of unsaturation. The UV spectrum showed the absorptions at λmax of 225, 264, 356, and 385 nm, which indicated the characteristic absorption peaks of 8isopentenyl chalcone.28 The IR spectrum suggested the presence of hydroxyl (3410.8 cm−1), carbonyl (1637.2 cm−1), and aromatic ring (1610.8 cm−1) functionalities. 8139

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

number

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 1‴ 2‴ 3‴ 4″-CH3 5″-CH3 4‴-CH3 5‴-CH3 2′-OCH3 4′-OCH3

d (8.0) dd (8.0, 2.0) d (7.5) t (7.5, 1.5)

8140

1.74 1.81 1.51 1.51 3.84

s s s s s

6.68 d (10.0) 5.72 d (10.0)

6.91 7.22 3.33 5.39

7.30 d (2.0)

7.91 s

7.79 s

7.62 d (15.6) 7.98 d (15.6)

δH mult (J in Hz) δC

192.7 118.5 145.2 121.3 154.2 118.5 133.2 128.3 131.6 128.4 114.5 148.7 149.2 116.7 123.6 33.6 123.3 132.5 123.2 129.1 79.6 25.6 18.5 28.3 28.3 56.2

1

C-5 C-6

C-4′

C-4′

C-1, C-1″

C-1″, C-1‴

C-1′ C-2′, C-6′

HMBC

d (8.0) d (8.0) d (7.5) t (7.5, 1.5)

1.74 1.80 1.50 1.50

s s s s

6.68 d (10.0) 5.73 d (10.0)

6.91 7.63 3.34 5.39

7.63 d (8.0) 6.91 d (8.0)

7.91 s

7.78 s

7.63 d (15.6) 7.99 d (15.6)

δH mult (J in Hz)

δC 192.7 118.6 145.2 121.5 154.3 118.5 133.5 128.4 131.6 128.5 130.5 116.3 159.6 116.3 130.5 33.5 123.4 132.5 123.3 129.1 79.5 25.6 18.6 28.4 28.4

2

C-5 C-6

C-4′

C-4′

C-1, C-1″

C-1″, C-1‴

C-1′ C-2′, C-6′

HMBC

d (8.0) d (8.0) d (7.5) t (7.5, 1.5)

1.73 1.80 1.51 1.51

s s s s

6.69 d (10.0) 5.72 d (10.0)

6.90 7.64 3.35 5.38

7.64 d (8.0) 6.90 d (8.0)

7.22 s

7.63 d (15.6) 7.98 d (15.6)

δH mult (J in Hz)

δC 192.8 118.5 145.3 120.8 155.2 117.3 163.7 127.6 130.5 128.7 130.4 116.4 149.1 116.4 130.4 32.1 123.2 132.4 122.8 129.0 79.5 25.5 18.4 28.3 28.3

3

Table 1. 1H NMR (400 MHz, CD3COCD3), 13C NMR (100 MHz, CD3COCD3), and Key HMBCs of Compounds 1−4

C-5 C-6

C-4′

C-4′

C-1, C-1″

C-1′ C-2′, C-6′

HMBC

1.73 1.80 1.51 1.51 3.85 3.85

s s s s s s

6.69 d (10.0) 5.73 d (10.0)

7.01 s 3.35 d (7.5) 5.38 t (7.5, 1.5)

7.01 s

7.23 s

7.64 d (15.6) 7.99 d (15.6)

δH mult (J in Hz)

δC 192.8 118.6 145.3 120.7 155.3 117.4 163.7 127.5 130.5 128.1 109.5 148.3 138.4 148.3 109.5 32.2 123.3 132.4 122.8 129.1 79.6 25.5 18.5 28.4 28.4 56.3 56.3

4

C-5 C-6

C-4′

C-4′

C-1, C-1″

C-1′ C-2′, C-6′

HMBC

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

Article

Journal of Agricultural and Food Chemistry

Figure 2. Key HMBC (H → C), 2D NOESY, and 1H−1H COSY correlations of compounds 1−8.

The 1H NMR spectrum of compound 1 displayed the presence of an isopentenyl (δH 3.33, 2H, d, J = 7.5 Hz, H-1″; δH 5.39, 1H, t, J = 7.5 and 1.5 Hz, H-2″; δH 1.74, 3H, s, 4″-CH3; δH 1.81, 3H, s, 5″-CH3) (Table 1). The signals of compound 1 for a trans-configured double bond group (δH 7.62, 1H, d, J = 15.6 Hz, H-2; δH 7.98, 1H, d, J = 15.6 Hz, H-3) and a typical carbonyl carbon (δC 192.7, C-1) confirmed the existence of the chalcone skeleton.28 In addition, an unsaturated hexatomic ring (δH 6.68, 1H, d, J = 10.0 Hz, H-1‴; δH 5.72, 1H, d, J = 10.0 Hz, H-2‴; δH 1.51, 6H, s, 4‴/5‴-CH3) was assigned to H-5 and H6 according to the heteronuclear multiple-bond correlations (HMBCs) of H-7/C-1‴, H-1‴/C-5, and H-2‴/C-6 (Figure 2) and the 2D nuclear Overhauser effect spectroscopy (NOESY) correlations of H-7/H-1″, H-7/H-1‴, H-2‴/4‴-CH3, and H2‴/5‴-CH3 (Figure 2). Typical ABX system aromatic proton signals (δH 7.30, 1H, d, J = 2.0 Hz, H-2′; δH 6.91, 1H, d, J = 8.0 Hz, H-5′; δH 7.22, 1H, dd, J = 8.0, 2.0 Hz, H-6′) (Table 1) indicated that a 1,3,4-trisubstituted phenyl moiety was in compound 1. The structure of compound 1 was determined by the HMBCs of H-2/C-1′, H-3/C-2′, H-3/C-6′, H-7/C-1″, H9/C-1, H-9/C-1″, H-2′/C-4′, and H-6′/C-4′ (Figure 2) and the 1H−1H correlation spectroscopy (COSY) correlations of H-2/H-3, H-5′/H-6′, and H-1‴/H-2‴ (Figure 2). Therefore, compound 1 was identified as (E)-1-[6-(3‴-methylbut-2‴enyl)-8-isopent-2″-enyl]-3-(4′-hydroxy-3′-methoxyphenyl)propenone. Compound 2, a yellow powder, was designated with a molecular formula of C25H26O3 on the basis of HR-ESI−MS

and produced a molecular ion, [M + Na]+, at m/z 397.3521 (calcd for C25H26O3Na, 397.3518), indicating 13 degrees of unsaturation. Its UV spectrum showed absorbance at λmax of 226, 264, 355, and 385 nm, and its IR spectrum displayed characteristic absorption peaks (3411.7, 1636.8, 1610.1, and 845.6 cm−1). The UV and IR spectral data of compound 2 were similar to those of compound 1. Therefore, compound 2 was concluded to be an analogue of compound 1. Moreover, the spectral data of 1H and 13C NMR (Table 1) were closely similar to those of compound 1. The only difference between compounds 2 and 1 was the absence of a set of the AA′BB′ system (δH 7.63, 2H, d, J = 8.0 Hz, H-2′/6′; δH 6.91, 2H, d, J = 8.0 Hz, H-3′/5′) in compound 2. The structure of compound 2 was determined by the HMBCs of H-2/C-1′, H-3/C-2′, H-3/ C-6′, H-7/C-1″, H-7/C-1‴, H-9/C-1, H-9/C-1″, H-2′/C-4′, H-6′/C-4′, H-1‴/C-5, and H-2‴/C-6 (Figure 2), the 2D NOESY correlations of H-7/H-1″, H-7/H-1‴, H-2‴/4‴-CH3, and H-2‴/5‴-CH3 (Figure 2), and the 1H−1H COSY correlations of H-2/H-3, H-2′/H-3′, H-5′/H-6′, and H-1‴/ H-2‴ (Figure 2). Thus, compound 2 was elucidated as (E)-1[6-(3‴-methylbut-2‴-enyl)-8-isopent-2″-enyl]-3-(4′hydroxyphenyl)propenone. Compound 3 was obtained as a yellow powder, and its molecular formula was determined as C25H26O4 from HR-ESI− MS (m/z 413.0563 [M + Na]+, calcd for C25H26O4Na, 413.0512), corresponding to 13 degrees of unsaturation. The UV spectrum of compound 3 showed the absorptions at λmax of 225, 265, and 385 nm, and its IR absorptions indicated the 8141

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

number

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 1‴ 2‴ 3‴ 4‴-CH3 5‴-CH3 5-OCH3 7-OCH3 5′-OCH3

8142

d (8.0) dd (8.0, 2.0) s q (5.0) d (5.0) d (7.5) t (7.5, 1.5)

1.72 s 1.81 s

6.95 7.11 6.04 6.23 1.83 3.30 5.40

7.28 d (2.0)

7.67 d (15.6) 7.93 d (15.6)

δH mult (J in Hz)

5 δC

191.8 119.6 143.6 103.2 149.1 112.9 150.8 126.5 147.7 129.1 115.4 149.6 148.9 117.0 124.3 101.3 108.9 22.3 20.8 124.2 131.9 25.4 18.3 C-5, C-7 C-6

C-3′, C-4′ C-8, C-9

C-1′ C-2′, C-6′

HMBC

d (8.0) dd (8.0, 2.0) s q (5.0) d (5.0) d (7.5) t (7.5, 1.5)

3.86 s

1.72 s 1.80 s

6.95 7.10 6.04 6.22 1.82 3.31 5.41

7.28 d (2.0)

7.66 d (15.6) 7.93 d (15.6)

δH mult (J in Hz)

6 δC

58.6

191.7 119.8 143.7 103.1 149.0 111.2 150.7 126.4 147.6 129.0 115.5 149.5 148.9 117.1 124.4 101.3 108.9 22.4 21.2 124.0 131.9 25.5 18.4 C-5, C-7 C-6

C-3′, C-4′ C-8, C-9

C-1′ C-2′, C-6′

HMBC

s s q (5.0) d (5.0) d (7.5) t (7.5, 1.5)

3.85 s

1.73 s 1.82 s

6.80 6.05 6.25 1.84 3.34 5.42

6.80 s

7.23 s

7.12 s

7.65 d (15.6) 7.97 d (15.6)

δH mult (J in Hz)

δC

56.2

191.4 118.5 143.3 119.3 121.6 128.8 118.4 146.5 147.2 128.5 107.6 148.3 149.0 149.8 107.6 102.2 108.2 22.2 28.5 126.1 132.1 25.6 18.4

7

Table 2. 1H NMR (400 MHz, CD3COCD3), 13C NMR (100 MHz, CD3COCD3), and Key HMBCs of Compounds 5−8

C-5, C-7 C-6

C-3′, C-4′ C-8, C-9

C-1‴

C-1

C-1′ C-2′, C-6′

HMBC

1.72 1.81 3.86 3.86 3.84

6.80 6.05 6.24 1.84 3.31 5.41

s s s s s

s s q (5.0) d (5.0) d (7.5) t (7.5, 1.5)

6.80 s

7.66 d (15.6) 7.94 d (15.6)

δH mult (J in Hz)

δC 191.7 119.8 143.6 103.3 149.2 111.4 150.6 126.5 147.6 129.0 107.8 148.2 148.9 149.9 107.8 102.2 108.8 22.4 21.0 124.1 132.0 25.5 18.4 58.5 58.5 56.3

8

C-5, C-7 C-6

C-3′, C-4′ C-8, C-9

C-1′ C-2′, C-6′

HMBC

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

Article

Journal of Agricultural and Food Chemistry existence of hydroxyl (3409.9 cm−1), carbonyl (1637.5 cm−1) and aromatic ring (1610.5 and 1526.8 cm−1) functional groups. The 1H and 13C NMR spectroscopic data (Table 1) were very similar to those of compound 2, except for a 7-hydroxyl group in compound 3. Furthermore, the observed correlations of H2/C-1′, H-3/C-2′, H-3/C-6′, H-9/C-1, H-9/C-1″, H-2′/C-4′, H-6′/C-4′, H-1‴/C-5, and H-2‴/C-6 (Figure 2), the 2D NOESY correlations of 7-OH/H-1″, 7-OH/H-1‴, H-2‴/4‴CH3, and H-2‴/5‴-CH3 (Figure 2), and the 1H−1H COSY correlations of H-2/H-3, H-2′/H-3′, H-5′/H-6′, and H-1‴/H2‴ (Figure 2). According to the above data, compound 3 was elucidated as (E)-1-[6-(3‴-methylbut-2‴-enyl)-7-hydroxy-8isopent-2″-enyl]-3-(4′-hydroxyphenyl)propenone. Compound 4 was isolated as a yellow powder, and its molecular formula was determined as C27H30O6 from the HRESI−MS ion peak at m/z 473.6529 [M + Na]+ (calcd for C27H30O6Na, 473.6507), corresponding to 13 degrees of unsaturation. According to its UV spectrum, showing absorbance at λmax of 225, 266, and 385 nm, and its IR spectrum, displaying characteristic absorption peaks (3412.0, 1637.4, 1611.8, 1345.6 cm−1), we concluded that compound 4 was an analogue of compound 3. The data from 1H and 13C NMR (Table 1) spectra of compound 4 were similar to compound 4, except for 3′-OCH3 and 5′-OCH3 in compound 4. The structure of compound 4 was confirmed by the key HMBC of H-2/C-1′, H-3/C-2′, H-3/C-6′, H-9/C-1, H-9/C-1″, H-2′/C-4′, H-6′/C-4′, H-1‴/C-5, and H-2‴/C-6 (Figure 2), the 2D NOESY correlations of 7-OH/H-1″, 7-OH/H-1‴, H2‴/4‴-CH3, and H-2‴/5‴-CH3 (Figure 2), and the 1H−1H COSY correlations of H-2/H-3, H-5′/H-6′, and H-1‴/H-2‴ (Figure 2). Therefore, compound 4 was determined to be (E)1-[6-(3‴-methylbut-2‴-enyl)-7-hydroxy-8-isopent-2″-enyl]-3(4′-hydroxy-3′,5′-dimethoxyphenyl)propenone. Compound 5 was obtained as a yellow powder. Its molecular formula was deduced as C23H22O7 from HR-ESI−MS at m/z 433.4834 [M + Na]+ (calcd for C23H22O7Na, 433.4820), indicating 13 degrees of unsaturation. The UV spectrum showed the absorptions at λmax of 225, 240, 335, and 385 nm, and its IR spectrum suggested the presence of hydroxyl (3443.6 cm−1), carbonyl (1650.8 cm−1), and aromatic ring (1600.6 and 1523.7 cm−1) functionalities. The 1H NMR spectrum of compound 5 showed the presence of the typical ethylidenedioxy group [−OCH(CH3)O−, δH 6.23, 1H, q, J = 5.0 Hz, H-1″; δH 1.83, 3H, d, J = 5.0 Hz, H-2″] and two trans-coupled olefinic protons (δH 7.67, 1H, d, J = 15.6 Hz, H-2; δH 7.93, 1H, d, J = 15.6 Hz, H-3), and a typical carbonyl carbon (δC 191.8, C-1) in the 13C NMR spectrum confirmed the existence of the chalcone skeleton.29 Moreover, an isopentenyl (δH 3.30, 2H, d, J = 7.5 Hz, H-1‴; δH 5.40, 1H, t, J = 7.5 and 1.5 Hz, H-2‴; δH 1.72, 3H, s, 4‴-CH3; δH 1.81, 3H, s, 5‴-CH3) (Table 2) was assigned to H-6 according to the HMBCs of H-1‴/C-5, H-1‴/C-7, and H-2‴/C-6 (Figure 2). In the 1H and 13C NMR spectra, a typical singlet at δH 6.04 (2H, s, H-7′) and δC 101.3 (Table 2) revealed that the fragment of −O−CH2−O− was assigned to H-3′ and H-4′ according to the HMBCs of H-7′/C-3′ and H-7′/C-4′ (Figure 2). Typical ABX system aromatic proton signals (δH 7.28, 1H, d J = 2.0 Hz, H-2′; δH 6.95, 1H, d, J = 8.0 Hz, H-5′; δH 7.11, 1H, dd, J = 8.0 and 2.0 Hz, H-6′) (Table 2) revealed that a 1,3,4trisubstituted phenyl moiety was in compound 5. The structure of compound 5 was determined by the HMBCs of H-2/C-1′, H-3/C-2′, H-3/C-6′, H-7′/C-3′, H-7′/4′, H-1″/C-8, H-1″/C9, H-1‴/C-5, H-1‴/C-7, and H-2‴/C-6 (Figure 2), the 2D

NOESY correlation of 7-OH/H-1‴ (Figure 2), and the 1H−1H COSY correlations of H-2/H-3 and H-5′/H-6′ (Figure 2). Consequently, compound 5 was identified as (E)-3-(benzo[1,3]dioxol-5′-yl)-1-(5,7-dihydroxy-6-isopentenyl-benzo[1,3]dioxol-4-yl)propenone. Compound 6 was isolated a yellow power, and the molecular formula, C24H24O7, was established by HR-ESI−MS data at m/ z 447.6123 [M + Na]+ (calcd for C24H24O7Na, 447.6163), implying 13 degrees of unsaturation. The UV spectrum showed the absorptions at λmax of 226, 265, and 385 nm, and its IR spectrum suggested that characteristic absorption peaks (3442.9, 1651.0, 1602.3, and 1524.1 cm−1) were closely similar to those of compound 5. It was concluded that compound 6 was an analogue of compound 5. The only difference between compounds 6 and 5 was the appearance of 7-OCH3 (δH 3.86) in compound 6 and the appearance of 7-OH in compound 5 (Table 2). The HMBCs of H-2/C-1′, H-3/C-2′, H-3/C-6′, H7′/C-3′, H-7′/4′, H-1″/C-8, H-1″/C-9, H-1‴/C-5, H-1‴/C-7, and H-2‴/C-6 (Figure 2), the 2D NOESY correlation of 7OCH3/H-1‴ (Figure 2), and the 1H−1H COSY correlations of H-2/H-3 and H-5′/H-6′ (Figure 2) confirmed the structure of compound 6. Analysis of the spectroscopic data established the structure of compound 6 as (E)-3-(benzo[1,3]dioxol-5′-yl)-1(5-methoxy-7-hydroxy-6-isopentenylbenzo[1,3]dioxol-4-yl)proenone. Compound 7 was obtained as a yellow powder, with the molecular formula C24H24O6 determined by HR-ESI−MS at m/z 431.2860 [M + Na]+ (calcd for C24H24O6Na, 431.2816), corresponding to 13 degrees of unsaturation. The UV spectrum of compound 7 exhibited absorption maxima at 226, 280, 350, and 384 nm, and its IR spectrum showed absorptions of hydroxyl (3443.0 cm−1), carbonyl (1652.3 cm−1), and aromatic ring (1601.3 cm−1) functional groups. The presence of two single peaks (δH 7.12, 1H, s, H-5; δH 7.23, 1H, s, H-7) and a methoxyl group (δH 3.85, 3H, s, 5′-OCH3) were observed in the 1H NMR spectrum (Table 2). The structure of compound 7 was determined by the HMBCs of H-2/C-1′, H-3/C-2′, H-3/ C-6′, H-5/C-1, H-7/C-1‴, H-7′/C-3′, H-7′/4′, H-1″/C-8, H1″/C-9, H-1‴/C-5, H-1‴/C-7, and H-2‴/C-6 (Figure 2), the 2D NOESY correlation of H-7/H-1‴ (Figure 2), and the 1 H−1H COSY correlations of H-2/H-3 (Figure 2). Consequently, compound 7 was identified as (E)-3-(7′methoxybenzo[1,3]dioxol-5′-yl)-1-(6-isopentenylbenzo[1,3]dioxol-4-yl)propenone. Compound 8 was obtained as a yellow powder, with the molecular formula C26H28O8 determined by HR-ESI−MS at m/z 491.2703 [M + Na]+ (calcd for C26H28O8Na, 491.2716), corresponding to 13 degrees of unsaturation. Its UV spectrum showed the absorptions at λmax of 225, 266, and 385 nm, and its IR spectrum displayed absorption peaks (3443.2, 1653.1, and 1601.8 cm−1) very similar to those of compound 7, which concluded that compound 8 was an analogue of compound 7. Detailed analysis of the NMR data (Table 2) suggested that the structure of compound 8 was similar to that of compound 7, except for two methoxyl groups (δH 3.86, 3H, s, 5-OCH3; δH 3.86, 3H, s, 7-OCH3) in compound 8. The structure of compound 8 was established on the HMBCs of H-2/C-1′, H3/C-2′, H-3/C-6′, H-7′/C-3′, H-7′/4′, H-1″/C-8, H-1″/C-9, H-1‴/C-5, H-1‴/C-7, and H-2‴/C-6 (Figure 2), the 2D NOESY correlation of H-1‴/5-OCH3 and H-1‴/7-OCH3 (Figure 2), and the 1H−1H COSY correlations of H-2/H-3 (Figure 2). Therefore, compound 8 was identified as (E)-3-(7′8143

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

Article

Journal of Agricultural and Food Chemistry

values of 39.52 ± 0.24 and 45.04 ± 0.51 μM, respectively. These results indicated that the presence of 7-OCH3 and 2methylbenzo[1,3]dioxole moiety in the structure were critical for the antiangiogenic activities of these compounds. The antiangiogenic activities would decrease if these moieties did not contain these compounds. The study of the structure− activity relationship of the active compounds from M. sativa needed further research. In conclusion, in this work, 8 new compounds (1−8) and 12 known compounds (9−20) were isolated from the aerial parts of M. sativa. Their structures were elucidated as chalcones on the basis of spectroscopic analyses and references. The hypolipidemic and antiangiogenic activities of compounds (1−20) were evaluated for the first time. The pharmacological screening results of compounds (1−20) were shown in Tables 3 and 4. Among them, compounds 3, 4, 11, 12, and 20 (10 μM) showed moderate hypolipidemic activity, and their p values less than 0.05 or 0.01 were considered as significantly different. Moreover, compounds 6, 8, 18, and 19 exhibited significant antiangiogenic activities, which inhibited VEGFinduced HUVEC proliferation in vitro, with IC50 values of 13.86 ± 0.43, 15.53 ± 0.19, 39.52 ± 0.24, and 45.04 ± 0.51 μM, respectively. The known compounds (9−20) were isolated from this plant for the first time.

methoxybenzo[1,3]dioxol-5′-yl)-1-(5,7-dimethoxy-6isopentenylbenzo[1,3]dioxol-4-yl)propenone. In addition, other known compounds (9−20) were readily determined by comparison of their spectroscopic data to those reported in the literature. Their structures were identified as broussochalcone A (9),28 broussochalcone B (10),28 7,9,2′,4′tetrahydroxy-8-isopentenyl-5-methoxychalcon (11),30 xanthohumol (12),31 desmethylxanthohumol (13),31 6′-hydroxy2′,3′,4′-trimethoxy-chalcone (14),29 flavokawin B (15),29 2,4,4′-trihydroxychalcone (16),32 2′-hydroxy-4′,6′-dimethoxychalcone (17),33 litseaone B (18),29 xanthohumol M (19),34 and flemiculosin (20). 35 Moreover, all of the known compounds (9−20) were obtained from this plant for the first time. Statistical Analysis of Hypolipidemic and Antiangiogenic Activitivies. The hypolipidemic activities of the compounds (1−20) were evaluated by measuring the triglyceride content in HepG2 cells, with simvastatin as the positive control. The screening results of compounds (1−20) were shown in Table 3. In comparison to the control group, Table 3. Hypolipemic Effects of Selective Compounds on the Triglyceride Content in HepG2 (10 μM) group

concentration (M)

model 3 4 11 12 20 simvastatin

10−5 10−5 10−5 10−5 10−5 10−5

■

resultsa 0.3412 0.2013 0.2210 0.2138 0.2270 0.1986 0.1797

± ± ± ± ± ± ±

0.0115 0.0042b 0.0036c 0.0132c 0.0029c 0.0058c 0.0106b

*Telephone/Fax: +86-10-6352-5153. E-mail: maqinge2006@ 163.com. *E-mail: [email protected]. Funding

This work was supported by the Key Scientific Research Project of Colleges and Universities in Henan Province (17A350011), the Scientific and Technological Project of Henan Provincial Department of Science and Technology (2017, The Protective Effect and Mechanism from D-Galactosamine Induced Liver Injury of Salvia plebaia), the Key Scientific Research Project of Colleges and Universities in Henan Province (15A350009), the Special Project of Nanyang Normal University (ZX2014044), and the Scientific and Technological Project of Nanyang (KJGG23).

Data were expressed as the mean ± standard error (SE) (n = 5). p < 0.01 versus the control group. cp < 0.05.

a

b

compounds 3, 4, 11, 12, and 20 (10 μM) showed moderate hypolipidemic activities. The p values less than 0.05 or 0.01 were considered as significantly different. These pharmacological screening results may suggest that the chemical groups of 7-OH and 8-isopentenyl played positive roles in mediating their hypolipidemic activities, and further investigations have been undertaken in our laboratory. Moreover, compounds (1− 20) were evaluated for their antiangiogenic activities by the MTT assay to determine whether they decreased VEGFmediated cell proliferation in HUVECs. As shown in Table 4,

Notes

The authors declare no competing financial interest.

■

Table 4. HUVEC Proliferation Inhibitory Activities of Selective Compoundsa compound 6 8 18 19 axitinib a

± ± ± ± ±

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IC50 (μM) 13.86 15.53 39.52 45.04 14.56

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Corresponding Authors

0.43 0.19 0.24 0.51 0.91

The values are the mean ± standard deviation (SD) (n = 5).

compounds 6 and 8 showed significantly inhibited VEGFinduced HUVEC proliferation in vitro, with IC50 values of 13.86 ± 0.43 and 15.53 ± 0.19 μM, respectively. Compound 8 was close to the positive control, and compound 6 was lower than the control (IC50 = 14.56 ± 0.91 μM). In contrast, compounds 18 and 19 showed moderately inhibited proliferation, with IC50 8144

DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145

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DOI: 10.1021/acs.jafc.6b03883 J. Agric. Food Chem. 2016, 64, 8138−8145