Article pubs.acs.org/jnp
Inhibition of Proliferation of Vascular Smooth Muscle Cells by Cucurbitanes from Momordica charantia Nguyen Quoc Tuan,†,‡,# Do-Hyung Lee,§,# Joonseok Oh,⊥,∥,# Chung Sub Kim,⊥,∥ Kyung-Sun Heo,§,¶ Chang-Seon Myung,*,§,¶ and MinKyun Na*,†,¶ †
Department of Pharmacognosy and §Department of Pharmacology, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea ‡ Phutho College of Pharmacy, Viettri City, Phutho Province, Vietnam ⊥ Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States ∥ Chemical Biology Institute, Yale University, New Haven, Connecticut 06516, United States ¶ Institute of Drug Research & Development, Chungnam National University, Daejeon 34134, Republic of Korea S Supporting Information *
ABSTRACT: The cucurbitaceous plant Momordica charantia L., named “bitter melon”, inhabits Asia, Africa, and South America and has been used as a traditional medicine. The atypical proliferation of vascular smooth muscle cells (VSMCs) plays an important role in triggering the pathogenesis of cardiovascular diseases. Platelet-derived growth factor (PDGF) is regarded as the most powerful growth factor in promoting the intimal accumulation of VSMCs. The current study features the identification of six new cucurbitane-type triterpenoids (1−6) from the fruits of M. charantia, utilizing diverse chromatographic and spectroscopic techniques. In particular, the 2D structure of 1 was confirmed utilizing the long-range HSQMBC NMR pulse, capable of measuring heteronuclear long-range correlations (4−6JCH). The cucurbitanes were also assessed for their inhibitory activity against PDGF-induced VSMC proliferation. This current study may constitute a basis for developing those chemotypes into sensible pharmacophores alleviating cardiovascular disorders.
T
of vascular smooth muscle cells (VSMCs) is one of the key features that exacerbate plaque formation.11 Generally, VSMCs regulate vasoconstriction and extensions, but they switch the phenotype into a proliferative form when they are exposed to stimuli, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and interleukin-1 (IL-1).12 Among these stimuli, PDGF is the most potent mitogen for provoking intimal accumulation, proliferation, migration, and deposition of an extracellular matrix of VSMCs, which ultimately causes the pathogenesis of cardiovascular diseases.13 Thus, the inhibition of PDGF-induced VSMC proliferation may be a sensible therapeutic strategy for the remedy/prevention of heart diseases and their complications. In continuing attempts to identify plant-derived drug prototypes for the treatment of cardiovascular dysfunctions and complications,14−17 the current study elaborates the identification of six new cucurbitane-type triterpenoids from the fruits of M. charantia. These cucurbitanes were also evaluated for their inhibitory activity against PDGF-induced VSMC proliferation to determine whether these major constituents can be developed into practical chemotypes alleviating cardiovascular disorders and their complications.
he slender-stemmed tendril climber Momordica charantia L. (Cucurbitaceae), commonly known as “bitter melon”, populates tropical areas in the world1 and has been consumed as a vegetable and medicine.2 Extensive investigations of this plant have identified diverse biological activities such as antidiabetic, antibacterial, antiviral, and anticancer activities.3 In particular, the fruits were beneficial in preventing and alleviating heart disease and diabetes mellitus and their complications.4 Such biological activities were partly attributed to its major constituents such as alkaloids, insulin-like peptides, and steroidal saponins.5 Nevertheless, many other ingredients contributing to the reported biological virtues are elusive, attracting attention in the identification of relevant bioactive ingredients. Cucurbitane-type triterpenoids, possessing a C-9 methyl moiety that originates via a Wagner−Meerwein 1,2methyl shift,6 have been purified predominantly from the fruits of M. charantia.7,8 Investigation of these types of triterpenoids unveiled various biological findings including chemopreventive activity against 7,12-dimethylbenz[a]anthracene- and peroxynitrite-induced mouse skin carcinogenesis9 and insulin secretion enhancing activity in MIN6β-cells.7 Atherosclerosis is a vascular disease caused by the formation of atherosclerotic plaques in intima, and such abnormal plaque development is aggravated via hyperlipidemia and lipid oxidation.10 In the atherosclerotic lesion, aberrant proliferation © 2017 American Chemical Society and American Society of Pharmacognosy
Received: February 20, 2017 Published: June 16, 2017 2018
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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Table 1. 1H NMR Data of Compounds 1−6 [δH, mult. (J in Hz)] 1b
position 1 2 3 4 5 6 7 8 9 10
2a
3a
4a
5a
6a
2.00, m, 1.31, m 1.78, d (8.58) 4.74, (brs)
1.52, m 1.38, m 1.82, m 4.88, s
1.69, m, 1.41, m 1.87, m, 1.26, m 4.83, (brs)
1.40, m 1.30, m, 1.93, m 4.90, m
1.61, m, 1.24, m 1.88, m, 1.78, m 4.80, brt (2.6)
1.73, m, 1.40, m 1.90, m, 1.73, m 4.84, (brs)
6.18, d (9.8) 5.51, dd (9.8, 3.6) 2.34, m
5.98, dd (9.7, 2.1) 5.64, dd (9.7, 3.4)
5.90, s 4.09, d (4.5)
5.93, br d (4.0) 3.50, d (6.8)
6.19, dd (9.8, 2.2) 5.71, dd (9.8, 3.3)
5.91, d (4.5) 4.11, d (5.2)
2.92, s
2.02, s
2.06, s
2.53, m
2.01, s
2.37, m
2.56, d (12.2)
2.53, dd (13.7, 3.9)
2.66, dd (12.5, 5.7)
2.56, dd, (12.8, 4.1)
2.40, td (14.5, 5.6), 1.41, m 1.63, dd (4.1, 13.8)
2.26, m, 1.70, m
2.32, td (14.6, 5.6), 1.47, m 1.63, m
1.32, 1.90, 1.50, 0.89, 9.77, 1.35,
1.35, 1.96, 1.49, 0.93,
11
1.73, m, 1.66, m
2.42, dd (12.5, 5.5) 1.63, d (2.7)
12 13 14 15 16 17 18 19 20
1.66, d (8.6)
1.58, s
2.30, td (14.4, 9.5), 1.51, m 1.64, m
1.37, m 2.01, m, 1.44, m 1.54, m 0.94, s 4.40, s 1.59, dd (6.7, 3.2) 0.96, d (6.5) 2.23, m, 1.86, m
1.38, 1.96, 1.45, 0.87, 4.63, 1.55,
1.39, 1.89, 1.49, 0.87, 9.72, 1.54,
0.84, d (6.5) 2.19, m, 1.79, m
0.90, d (6.3) 2.18, d (10.5), 1.77, m
0.92, d (5.9) 1.51, t (10.0), 1.34, m
0.92, d (6.9) 2.20, d, 1.81, m
5.60, m 5.41, d (15.8)
5.52, m 5.40, d (15.8)
5.49, m 5.38, d (15.7)
3.92, td (3.9, 9.4) 4.92, d (br s)
5.52, dd (15.9, 8.6, 5.6) 5.41, d (15.9)
0.89 d (6.4) 2.16 dd (10.7, 6.6), 1.74, m 5.59, m 5.59, m
1.25, 1.25, 0.99, 1.09, 0.91,
1.25, 1.25, 0.95, 1.08, 0.84,
1.24, 1.24, 1.09, 1.17, 0.73,
1.71, 1.78, 1.13, 1.19, 0.77, 3.26,
1.26, 1.26, 1.01, 1.15, 0.85,
1.30, 1.30, 1.11, 1.19, 0.75,
21 22 23 24 25 26 27 28 29 30 7-OCH3 19-OCH3 23-OCH3 25-OCH3 2′ a
s s s s s
m m, 1.38, m m s s d (6.0)
s s s s s
3.36, d (1.4)
3.46, s
3.15, s
3.52, s 3.52, d (2.7)
d (8.3) s s s s m
s s s s s
m m t (10.0) s s m
d (0.9) d (0.9) s s s s
1.70, m 1.57, m
m m, 1.36, m dd (17.9, 9.1) s
1.56, m
s s s s s
1.36, 1.90, 1.47, 0.88, 9.72, 1.53,
m m m s s m
s s s s s
3.22, s 3.13, s 3.33, s
3.38, s
3.15, s 3.40, s
3.32, d (1.8)
Measured in CDCl3. bMeasured in methanol-d4.
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RESULTS AND DISCUSSION Compound 1 was isolated as a pale yellow powder. The molecular formula was established as C34H52O7 on the basis of the sodium-adduct HRESIMS ion at m/z 595.3622 (calcd for C34H52O7Na, 595.3611), along with the 13C NMR data (Table 2). The 1D NMR data (Tables 1 and 2) exhibited methyl moieties (δH 0.91, 0.94, 0.96, 0.99, 1.09, 1.25), two O-methyl groups (δH 3.15, 3.36), Z- and E-configured olefinic moieties (δH 6.18, 5.51 and δH 5.41, 5.60, respectively), one oxymethine functionality (δH 4.74), and an acetal-type methine motif (δH 4.40). These 1D NMR data are reminiscent of those for 5β,19epoxy-19,25-dimethoxycucurbita-6,23-dien-3β-ol from M. foetida.18 The only difference was the presence of an oxalate moiety in 1, which was substantiated by the hydroxy- and estercarbonyl 13C resonances (δC 170.5 and 160.9) and IR absorptions at 2981−1 (O−H stretch) and 1731 cm−1 (CO stretch). This functionality was attached at C-3 based on the HMBC cross-peak from H-3 to the ester carbonyl carbon (Figure 2). The structural assignment was reaffirmed using the
relatively new heteronuclear long-range (LR)-HSQMBC NMR experiment, capable of detecting long-range (4JC,H and 5JC,H) correlations (Figure 3).19 Despite invariable failures in detecting a four-bond correlation from H-3 to C-2′, other four-bond hetero correlations were useful in verifying structural connectivity (Figure 3). The relative configurational analysis of 1 was conducted using NOESY correlations (Figure 4) and comparison of its 13C NMR data with those of the aforementioned cucurbitane. The absolute configuration was assumed on the basis of consistent absolute configurational features of cucurbitane frameworks from natural sources. The structure of compound 1 was consequently established as 3[(5β,19-epoxy-19,25-dimethoxycucurbita-6,23-dien-3-yl)-2oxoacetic acid. Compound 2 was acquired as a white powder. The molecular formula C35H54O7 was established based upon the sodiumadduct ion observed at m/z 609.3765 in the positive mode HRESIMS ([M + Na]+, calcd for C35H54O7Na), in conjunction with the 13C NMR data (Table 2). Inspection of the 1D NMR 2019
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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Table 2. 13C NMR Data of Compounds 1−6 (δC) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 7-OCH3 19-OCH3 23-OCH3 25-OCH3 1′ 2′ 3′ a
1b 18.4, 25.8, 79.5, 38.5, 84.3, 135.4, 130.5, 50.9, 49.9, 39.9, 22.5, 31.7, 46.3, 49.4, 34.6, 28.9, 51.4, 15.4, 114.6, 37.5, 19.2, 40.5, 130.0, 137.7, 76.5, 26.5, 26.2, 25.1, 20.3, 20.5,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 CH CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
56.1, CH3
2a 18.1, 24.9, 79.6, 37.6, 84.4, 131.1, 132.6, 41.8, 48.0, 41.0, 23.3, 30.7, 45.2, 48.1, 33.6, 28.1, 50.2, 14.9, 112.2, 36.3, 18.8, 39.5, 128.3, 136.8, 75.1, 26.3, 25.9, 23.9, 20.0, 20.0,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 CH CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
3a
4a
21.5, 25.7, 79.6, 40.3, 145.2, 122.8, 66.2, 49.0, 49.8, 35.9, 22.7, 28.9, 45.4, 47.7, 34.7, 27.6, 49.8, 14.9, 209.3, 36.0, 18.7, 39.5, 128.5, 136.9, 75.1, 26.2, 25.9, 26.7, 25.1, 18.0,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 C CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
50.3, 166.2, 42.1, 169.3,
CH3 C CH2 C
21.3, 27.8, 80.0, 40.2, 145.7, 121.3, 75.4, 44.5, 49.7, 35.9, 22.5, 29.0, 45.7, 47.5, 34.7, 26.0, 50.8, 14.7, 207.4, 33.1, 19.4, 42.2, 75.9, 126.0, 136.7, 18.5, 25.9, 26.5, 24.9, 18.2, 55.5,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 C CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3 CH3
5a 19.3, 24.2, 77.7, 36.9, 84.1, 131.5, 133.5, 44.4, 50.7, 40.3, 21.7, 29.8, 45.0, 47.7, 33.6, 27.8, 50.2, 15.0, 182.9, 36.5, 19.0, 39.7, 128.6, 137.3, 75.3, 26.5, 26.1, 23.4, 20.3, 19.6,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 C CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
6a 21.5, 25.8, 79.6, 40.3, 145.3, 122.8, 66.3, 49.4, 50.0, 36.1, 22.7, 29.0, 45.6, 47.9, 34.7, 27.6, 49.9, 15.0, 209.8, 36.3, 18.8, 39.2, 125.3, 139.7, 71.3, 30.1, 30.0, 25.8, 25.1, 18.0,
CH2 CH2 CH C C CH CH CH C CH CH2 CH2 C C CH2 CH2 CH CH3 C CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3
58.5, CH3 56.1, CH3
50.6, CH3 169.0, C 170.5, C
50.4, 165.8, 41.2, 166.5,
CH3 C CH2 C
166.8, C 42.2, CH2 167.6, C
50.24, CH3 166.6, C 41.3, CH2 167.4, C
166.1, C 42.1, CH2 169.0, C
Measured in CDCl3, bMeasured in methanol-d4
3-[(5-formyl-7β-hydroxy-25-methoxycucurbita-5,23-dien-3-yl)oxy]-3-oxopropanoic acid. The molecular formula of 4 (white power) was assigned as C35H54O7 based on the sodium-adduct HRESIMS ion ([M + Na]+ at m/z 609.3759, calcd for C35H54O7Na, 609.3767) and 13 C NMR data (Table 2). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 4 shared significant similarities to those of 3 aside from the resonances for a methoxy group (δH 3.50, δC 55.5). The HMBC cross-peaks from the methoxy protons to C-7 (δC 66.2) corroborated that C-7 of compound 4 was substituted with the methoxy functionality (Figure 2). The configurational assignment of 4 was implemented as for the previous compounds. The C-23 configuration was established via comparison of its 13C NMR chemical shift value with that of reported cucurbitanes from the identical material.20 Thus, the structure of compound 4 was established as 3-[(5-formyl-7βmethoxy-7,23S-dimethoxycucurbita-5,23-dien-3-yl)oxy]-3-oxopropanoic acid. Compound 5, purified as a white powder, showed a protonated HRESIMS ion ([M + H]+ at m/z 571.3637, calcd for C34H51O7) and in conjunction with 13C NMR data (Table
data of 2 (Tables 1 and 2) revealed significant similarities to those of 1, except for the presence of the C-3 malonyl moiety deduced from the 13C NMR resonances (δC 165.8 and 166.5) and HMBC cross-peaks [H-3 (δH 4.88) to C-1′ (δC 165.8) and C-3′ (δC 166.5)] (Figure 2). The configurational assignment of 2 was achieved in a similar manner to that for 1. These findings collectively substantiated the structure of 2 as 3-[(5β,19-epoxy19,25-dimethoxycucurbita-6,23-dien-3-yl)oxy]-3-oxopropanoic acid. Compound 3 was purified as a white powder. The sodiumadduct HRESIMS ion ([M + Na]+ at m/z 595.3652, calcd for C34H52O7Na), along with the 13C NMR data (Table 2), evidenced a molecular formula of C34H52O7. The overall 1D NMR data were analogous to those of compound 2 except for the presence of the formyl 13C resonance (δC 209.3). Examination of the COSY and HMBC data indicated that the formyl motif was attached at C-9 (Figure 2). The NOESY correlations were used for the configurational analysis of the formyl moiety and other stereogenic centers in 3 (Figure 4). These observations confirmed the structure of compound 3 as 2020
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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Figure 1. Structures of cucurbitanes 1−6 from the fruits of M. charantia.
Figure 2. Key HMBC (→) cross-peaks and COSY correlations (bold) for compounds 1−6.
presence of an additional ester carbonyl carbon at δC 182.9. The HMBC cross-peaks from H-10 (δH 2.66) to this carbonyl
2) established a molecular formula of C34H50O7. Inspection of the 1D NMR data showed similarity to those of 2 except for the 2021
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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Figure 3. Key LR-HSQMBC (→) cross-peaks in 1. Only long-range correlations (four-bond) are labeled and shown in the portion of the spectrum (numbers in parentheses indicate 1H and 13C chemical shifts).
structure of compound 5 was elucidated as 3-[(25-Omethylkaravilagenin D-3-yl)oxy]-2-oxoacetic acid. Compound 6 was purified as a white powder. The positive sodium-adduct HRESIMS ion observed at m/z 581.3456 and the 13C NMR data (Table 2) were indicative of a molecular formula of C33H50O7 (calcd for C33H50O7Na, 581.3454). The analyses of the 1H and 13C NMR (Tables 1 and 2) data revealed considerable similarities to those of 3, except for the absence of resonances for the methoxy functionality. The 2D NMR and HRESIMS data analyses of 6 confirmed that the 25methoxy group in 3 was replaced by a hydroxy motif (Figure 2). These data collectively verified that the structure of compound 6 was 3-[(5-formyl-7β,25-dihydroxymethoxycucurbita-5,23-dien-3-yl)oxy]-3-oxopropanoic acid. Compound 7 was obtained as a white powder, and its molecular formula C36H56O9 was established considering the sodium-adduct HRESIMS ion at m/z 655.3815 [M + Na]+ (calcd for C36H56O9Na, 655.3822). The NMR data and acid hydrolysis of 7 revealed that the structure was identical to kuguaglycoside I, a cucurbitane obtained from the medicinal food resource.21 It was verified that all these isolates are true natural products by using an HPLC-evaporative light scattering detector (ELSD), showing their presence in a crude M. charantia extract (Figure S2, Supporting Information). To determine the inhibitory activity against VSMC proliferation, cells were pretreated for 2 h with the purified compounds at 5 and 10 μM except for compound 5 (0.5 and 1 μM), given that 5 began to exert cytotoxicity against the examined smooth muscle cells at concentrations higher than 5 μM (Figure S61, Supporting Information). Cells pretreated with these phytochemicals were then treated with PDGF (25
Figure 4. Configurational and conformational analysis of compounds 1 and 3 using the MMFF force field. Yellow dotted lines indicate NOE correlations, and some protons were omitted for a clear presentation of the homonuclear correlations.
carbon confirmed that C-19 constituting the bridge of the bicyclic motif in 2 was oxidized to the corresponding ester carbonyl group, resulting in the formation of the dihydrofuran2(3H)-one functionality in 5 (Figure 2). Consequently, the 2022
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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Figure 5. Effects of compounds purified from M. charantia on PDGF-induced VSMC proliferation and viability. (A) Serum-deprived VSMCs were treated with the indicated concentrations of compounds (μM, shown on the x axis) or paclitaxel (Pac, 1 μM) for 2 h followed by treatment with 25 ng/mL PDGF for 24 h. (B) Serum-deprived VSMCs were treated with control (0.1% DMSO, D), indicated concentrations of compounds (μM, shown on the x axis), or digitonin (Dig, 100 μg/mL) for 24 h. PDGF-induced VSMC proliferation and cell viability levels were determined using the MTT assay, and optical densities at 565 nm are shown. The results are expressed as four independent experiments, each performed in triplicate. (A) ***p < 0.001, **p < 0.01, and *p < 0.05, vs PDGF stimulated,; (B) ***p < 0.001, vs not treated. dark brownish extract (1100 g). The slurry was suspended in H2O and partitioned with n-hexane (3 × 2.5 L), CHCl3 (3 × 2.5 L), EtOAc (3 × 2.5 L), and BuOH (2 × 2.5 L) to obtain the respective extracts (334.0, 38.0, 47.0, and 240.0 g). The CHCl3 extract was subjected to RP-C18 MPLC, eluting with MeOH/H2O (4:6 → 1:0), to yield 10 fractions [Frs. 2-1 (4.3 g), 2-2 (1.5 g), 2-3 (2.8 g), 2-4 (0.7 g), 2-5 (10.6 g), 2-6 (3.3 g), 2-7 (1.6 g), 2-8 (1.3 g), 2-9 (1.2 g), 2-10 (0.9 g)]. An RP-C18 MPLC application with a gradient of MeOH/H2O (6:4 → 1:0) was used to generate 13 subfractions [Frs. 2-5-1 (0.6 g), 2-5-2 (2.5 g), 2-53 (2.1 g), 2-5-4 (1.1 g), 2-5-5 (1.7 g), 2-5-6 (0.6 g), 2-5-7 (0.5 g), 2-5-8 (0.3 g), 2-5-9 (0.3 g), 2-5-10 (0.1 g), 2-5-11 (45.0 mg), 2-5-12 (0.2 g), and 2-5-13 (70.5 mg)]. Fr. 2-5-5 was purified utilizing HPLC eluting with a gradient of MeOH/H2O (0−10 min 0:1, 10−15 min 6:4, 15− 80 min 4:1, 80−145 min 1:0) to furnish 2 (tR: 134 min, 30.0 mg) and 1 (tR: 138 min, 30.0 mg), along with nine subfractions [Frs. 2-5-5-1 (92.0 mg), 2-5-5-2 (433.0 mg), 2-5-5-3 (42.0 mg), 2-5-5-4 (108.0 mg), 2-5-5-5 (102.0 mg), 2-5-5-6 (250.0 mg), 2-5-5-7 (122.0 mg), 2-5-5-8 (82.0 mg), and 2-5-5-9 (98.0 mg)]. From Fr. 2-5-5-2, 3 (tR: 48 min, 20.0 mg) was isolated upon a similar purification strategy to that for the previous fraction. Preparative HPLC applications led to the purification of compound 5 (tR: 62 min, 5.0 mg) from Fr. 2-5-5-3 and 4 (tR: 74 min, 3.2 mg) from Fr. 2-5-5-4. Fr. 2-3 was separated employing RP18 MPLC with a gradient mixture of MeOH/H2O (1:1 → 1:0) to acquire six subfractions [Frs. 2-3-1 (270.0 mg), 2-3-2 (486.0 mg), 2-3-3 (345.0 mg), 2-3-4 (804.0 mg), 2-3-5 (590.0 mg), and 2-3-6 (113.0 mg)]. Fr. 2-3-4 was further purified using a similar protocol to that for the previous fraction to afford four subfractions [Frs. 2-3-4-1 (150.0 mg), 2-3-4-2 (340.0 mg), 2-3-4-3 (200.0 mg), and 2-3-4-4 (38.0 mg)]. A similar HPLC procedure was applied to Fr. 2-3-4-3 to give 6 (tR: 72 min, 20.0 mg). To validate that these isolates were truly derived from Nature, the EtOH extract of the title plant source (14 g) was extracted with EtOH (100 mL) at room temperature (rt) for 3 days without reflux. Compounds 1−6 (0.5 mg of each compound) were dissolved in 1 mL of HPLC-grade MeOH, and each compound and the crude EtOH extract (10 μL) were analyzed using the HPLC-ELSD system [0.3 mL/min; Kinetex C18 5 μm, 150 × 4.6 mm (Phenomenex, Torrance, CA, USA); Gilson prepELSII detector (Gilson Inc., Middleton, WI, USA)] with a gradient program of MeCN/H2O (0−10 min, 60%; 10− 15 min, 80%; 15−80 min, 80−85 min, 90%; 85−95 min, 90%; 95−100 min, 100%; 100−110 min, 100% MeCN) (Figure S2, Supporting Information). 3-[(5β,19-Epoxy-19,25-dimethoxycucurbita-6,23-dien-3-yl)-2oxoacetic acid (1): pale yellow powder; [α]25 D = −99 (MeOH, c 0.2); IR (KBr, νmax) 2946, 1731, 1079 cm−1; UV−vis (MeOH, λmax (log ε)) 272 (2.56) nm; 1H NMR (600 MHz, methanol-d4) see Table 1; 13C NMR (150 MHz, methanol-d4) see Table 2; HRESIMS [M + H]+ at m/z 595.3622 (calcd for C34H52O7Na, 595.3611). 3-[(5β,19-Epoxy-19,25-dimethoxycucurbita-6,23-dien-3-yl)oxy]3-oxopropanoic acid (2): white amorphous powder; [α]25 D = −68
ng/mL) for 24 h to induce aberrant VSMC proliferation. As shown in Figure 5A, compounds 2−4, 6, and 7 inhibited PDGF-induced VSMC proliferation. In particular, 2 and 4 at 10 μM curbed the VSMC proliferation by 72.4% and 67.9%, respectively, demonstrating significant inhibitory activity among the tested phytochemicals. All compounds did not show cytotoxicity on VSMCs at the tested concentrations (Figure 5B). The current study delineates the purification and structural elucidation of new phytochemicals from the edible fruits of M. charantia, possessing ethnopharmacological relevance for the treatment of cardiovascular dysfunctions. Some of the cucurbitanes showed powerful inhibitory potential against PDGF-induced VSMC proliferation. However, triterpenoids were also reportedly recognized as cytotoxic scaffolds,22−24 as seen in the current investigation as well. Thus, it would be imperative to optimize and refine these cucurbitane-based chemotypes for the development of safe and practical pharmacophores targeting the treatment of cardiovascular diseases via inhibition of VSMC proliferation.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured employing a JASCO DIP-1000 polarimeter (Tokyo, Japan), and UV−vis spectra were obtained with a UV mini-1240 spectrophotometer (Kyoto, Japan). FT-IR data were recorded on a Thermo Nicolet 380 (Madison, WI, USA), and HRESIMS results were obtained utilizing a SYNAPT G2 Waters mass spectrometer (Manchester, UK). NMR spectra were recorded using a Bruker Ascend-600 and Fourier 300 spectrometer (Billerica, MA, USA). Medium-pressure liquid chromatography (MPLC) (Biotage-Isolera One, Biotage, Charlotte, NC, USA) was performed using C18 SNAP cartridges (KP-C18-HS; 400 g, 340 g, 120 g, Biotage). MPLC detection was accomplished at 210 and 205 nm wavelengths at a flow rate of 60 mL/min. Semipreparative HPLC (Gilson UV/vis 155, Middleton, WI, USA) was performed utilizing a Phenomenex Luna 5 μ C18 column (250 × 21.20 mm, 5 μm), and identical UV wavelengths for MPLC were used with a flow rate of 6 mL/min. TLC was performed employing precoated silica gel 60 F254 plates (70−230 mesh, EMD Millipore, Darmstadt, Germany). Plant Material. The fruit of M. charantia was purchased from Jungeun Health Care (Gwangju, Korea) and authenticated by one of the authors (M.N.). A voucher specimen was deposited at the Laboratory of Pharmacognosy at the College of Pharmacy, Chungnam National University, Daejeon, Korea (CNU201401). Extraction and Isolation. The dried fruit of M. charantia (7.2 kg) was extracted with 94% EtOH (4 × 72 L) at 50 °C for 48 h to yield a 2023
DOI: 10.1021/acs.jnatprod.7b00151 J. Nat. Prod. 2017, 80, 2018−2025
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(MeOH, c 0.2); IR (KBr, νmax) 2945, 1729 cm−1; UV−vis (MeOH, λmax (log ε)) 273 (2.22) nm; 1H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (150 MHz, CDCl3) see Table 2; HRESIMS [M + Na]+ at m/z 609.3765 (calcd for C35H54O7Na, 609.3767). 3-[(5-Formyl-7β-hydroxy-25-methoxycucurbita-5,23-dien-3-yl)oxy]-3-oxopropanoic acid (3): white, amorphous powder. [α]25 D = +20 (MeOH, c 0.2); IR (KBr, νmax) 3472, 2946, 1736 cm−1; UV−vis (MeOH, λmax (log ε)) 269 (2.50) nm; 1H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (150 MHz, CDCl3) see Table 2; HRESIMS [M + Na]+ at m/z 595.3652 (calcd for C34H52O7Na, 595.3611). 3-[(5-Formyl-7β-methoxy-7,23S-dimethoxycucurbita-5,23-dien3-yl)oxy]-3-oxopropanoic acid (4): white, amorphous powder; [α]25 D = −12 (MeOH, c 0.1); IR (KBr, νmax) 2948, 1716 cm−1; UV−vis 1 (MeOH, λmax (log ε)) 216 (3.14) nm; H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (150 MHz, CDCl3) see Table 2; HRESIMS [M + Na]+ at m/z 609.3759 (calcd for C35H54O7Na, 609.3767). 3-[(25-O-Methylkaravilagenin D-3-yl)oxy]-2-oxoacetic acid (5): white, amorphous powder; [α]25 D = −79 (MeOH, c 0.1); IR (KBr, νmax) 2953, 1747, 1168 cm−1; UV−vis (MeOH, λmax (log ε)) 212 (3.10) nm; 1 H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (150 MHz, CDCl3) see Table 2; HRESIMS [M + H]+ at m/z 571.3637 (calcd for C34H51O7, 571.3635). 3-[(5-Formyl-7β,25-dihydroxymethoxycucurbita-5,23-dien-3-yl)oxy]-3-oxopropanoic acid (6): white, amorphous powder; [α]25 D = −60 (MeOH, c 0.2); IR (KBr, νmax) 3365, 2950, 1713 cm−1; UV−vis (MeOH, λmax (log ε)) 238 (3.07) nm; 1H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (150 MHz, CDCl3) see Table 2; HRESIMS [M + Na]+ at m/z 655.3815 (calcd for C36H56O9Na, 655.3822). Parameters for Performing LR-HSQMBC. The NMR experiment was performed with reference to previous studies.19,25 Briefly, the NMR pulse sequence was implemented utilizing the t1 increments (indirect dimension) of 640 to 1024 to develop long-range heteronuclear correlations with the nJCH value being optimized to 2 Hz (transfer delay of 250 ms).19 The amount of sample required for this experiment was ∼7 mg with our laboratory settings. Computational Analyses. All conformational searches were implemented using the Macromodel (version 9.9, Schrodinger LLC) module in a mixed torsional/low-mode sampling in the MMFF force field. The searches were done in the gas phase with a 50 kJ/mol energy window limit and a maximum of 10 000 steps for complete investigation of stable conformers. The Polak−Ribiere conjugate gradient method was utilized for a minimization process in 10 000 maximum iterations and a 0.001 kJ (mol Å)−1 convergence threshold on the rms gradient. The most stable conformer was drawn utilizing Pymol (1.7.x, Open Source) as shown in Figure 4. Animals and Isolation of VSMCs. The Sprague−Dawley rats were maintained under pathogen-free conditions at the College of Pharmacy at the Chungnam National University. All animal procedures including the isolation of rat smooth muscle cells from the aorta were performed under the approval of the Chungnam National University Animal Care and Use Committee, and the approved protocol number was CNU-00826. Rat aortic VSMCs were isolated using enzymatic dispersion with reference to a previous study.26 VSMC Culture. Rat aortic VSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 IU/mL, and streptomycin 100 μg/ mL at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The VSMC purity was validated using immunocytochemical visualization against β-smooth-muscle actin, and the cells were used at passages 7− 10. Antiproliferation and MTT Assay. Cells were seeded in a 96-well plate at 3.0 × 104 cells/mL and cultured in DMEM containing 10% FBS at 37 °C for 24 h. After incubation in serum-free DMEM for 24 h, cells were pretreated with concentrations (0.5, 1, 5, 10, 15, 20 μM) of the purified compounds for 2 h, followed by treatment with PDGF (25 ng/mL) or paclitaxel (1 μM, as a positive control for antiproliferation) for another 24 h. As a cytotoxicity control, cells were treated with 100 μg/mL digitonin for 24 h. At the end of the stimulation, the media were removed and 200 μL of MTT solution (5 mg/mL) was added to
each well. After 2 h, the MTT solution was replaced with 200 μL of DMSO. PDGF-induced VSMC proliferation or cell viability was analyzed by optical density measurement at 565 nm using a microplate reader (Packard Instrument Co., Downers Grove, IL, USA). Statistical Analyses. All data are presented as mean ± standard error of the mean. Statistical differences were calculated using one-way ANOVA statistical assessment followed by a Dunnett test if p < 0.05, which was deemed significant.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00151. 1D and 2D NMR, IR, and HRESIMS spectra (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +82 42 821 5923. Fax: +82 42 821 8925. E-mail: cm8r@ cnu.ac.kr (C.-S. Myung). *Tel: +82 42 821 5925. Fax: +82 42 823 6566. E-mail: mkna@ cnu.ac.kr (M. Na). ORCID
MinKyun Na: 0000-0002-4865-6506 Author Contributions
# N. Q. Tuan, D.-H. Lee, and J. Oh contributed to this research equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program (NRF-2017R1A2A2A05001340) and Priority Research Centers Program (NRF-2009-0093815) through the National Research Foundation of Korea (NRF) grant funded by the Korean Government. We thank Mr. W. Yoshida (University of Hawaii, HI, USA) and Dr. G. E. Martin (Merck, NJ, USA) for helpful advice on the optimization of LRHSQMBC NMR settings.
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REFERENCES
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