Arborinane Triterpenoids from Rubia philippinensis Inhibit

Article pubs.acs.org/jnp

Arborinane Triterpenoids from Rubia philippinensis Inhibit Proliferation and Migration of Vascular Smooth Muscle Cells Induced by the Platelet-Derived Growth Factor Khong Trong Quan,†,# Hyun-Soo Park,‡ Joonseok Oh,§,∥ Hyun Bong Park,§,∥ Daneel Ferreira,⊥ Chang-Seon Myung,*,‡,∇ and MinKyun Na*,† †

College of Pharmacy and ‡Department of Pharmacology, College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea # Department of Pharmaceutical Analysis and Standardization, National Institute of Medicinal Materials, Hanoi, Vietnam § Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States ∥ Chemical Biology Institute, Yale University, New Haven, Connecticut 06516, United States ⊥ Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States ∇ Institute of Drug Research & Development, Chungnam National University, Daejeon 34134, Republic of Korea S Supporting Information *

ABSTRACT: The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are associated with cardiovascular diseases and related complications. Such deleterious proliferation and migration events are triggered by cytokines and growth factors, and among them, platelet-derived growth factor (PDGF) is recognized as the most potent inducer. Despite the genus Rubia being researched to identify valuable commercial and medicinal virtues, Rubia philippinensis has rarely been investigated. Nine arborinane-type triterpenoids (1−9) were identified from this underutilized plant species. In particular, 4 was identified as the first arborinane derivative carrying a ketocarbonyl motif at C-19. The presence of the cyclopentanone moiety and the associated configurational assignment were determined by utilizing NOE and coupling constant analysis. These compounds were assessed for their inhibitory potential on PDGF-induced proliferation and the migration of VSMCs. Treatment with 5 μM compound 5 (62.6 ± 10.7%) and compound 9 (41.1 ± 4.7%) impeded PDGF-stimulated proliferation without exerting cytotoxicity. Compound 7 exhibited antimigration activity in a dose-dependent manner (38.5 ± 3.0% at 10 μM, 57.6 ± 3.2% at 30 μM). These results suggest that the arborinane-type triterpenoids may be a pertinent starting point for the development of cardiovascular drugs capable of preventing the intimal accumulation of VSMCs.

T

Natural products have provided a multitude of chemical scaffolds capable of inhibiting PDGF-mediated VSMC proliferation and migration. This is exemplified by sesamin,5 murrayafoline A,6 curcumin,7 genistein,8 Ganoderma lucidum polysaccharides,9 and methylallyl thiosulfinate from garlic extracts.10 Triterpenoidal glycosides and their aglycones also exhibited biological activities that ameliorate cardiovascular chronic dysfunctions via preventing abdominal fat accumulation,11 increasing glucose uptake in skeletal muscle cells,12 and inhibiting protein tyrosine phosphatase-1B, a negative insulin regulator.13 Among these bioactive triterpenoidal architectures, arborinane-type triterpenoids have emerged as putative prototype molecules for the remedy of cardiovascular

he plasticity of vascular smooth muscle cells (VSMCs) from adult tissue is an important component of vascular proliferative diseases. Generally, VSMCs show differentiated phenotypes that can be defined by the expression of contractile proteins such as α smooth-muscle actin, calponin, and smoothmuscle myosin heavy chain.1 Several cytokines and growth factors such as platelet-derived growth factor (PDGF) are involved in inducing the abnormal proliferation and migration of VSMCs,2 thereby leading to vascular lesion formation and vascular obstructive diseases.1,3 In particular, PDGF, the most potent mitogen for stimulating deleterious activities of VSMCs, plays a crucial role in the intimal accumulation of smooth muscle cells, inducing the pathogenesis of cardiovascular dysfunctions such as atherosclerosis.4 Thus, it may be a viable therapeutic strategy to alleviate the PDGF-induced proliferation and migration of VSMCs for the treatment of heart disease. © 2016 American Chemical Society and American Society of Pharmacognosy

Received: May 27, 2016 Published: October 5, 2016 2559

DOI: 10.1021/acs.jnatprod.6b00489 J. Nat. Prod. 2016, 79, 2559−2569

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data (600 MHz, CDCl3) for Compounds 1−5a position 1 2 3 5 6 7 8 11 12 15 16 18 19 20 21 22 23 24 25 26 27 28 29 30 OAc-3 OAc-28 a

1.77, 1.53, 1.76, 1.66, 4.47, 0.96, 1.67, 1.44, 1.79, 1.22, 2.02, 5.25, 2.03, 1.71, 1.37, 1.30, 1.68, 1.34, 1.90, 4.44, 1.68, 2.09, 1.41, 1.64, 0.85, 0.88, 1.06, 0.86, 0.98, 3.84, 3.76, 0.95, 0.86, 2.04,

1b

2b

3

4

5

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

m td (12, 3.5) m. ov dd (12.0, 3.6) ov ov td (12.7, 2.9) ov q (13.0, 3.4) ov brd (6.0) ov m m m ov m d (10.2) td (9.6, 3.0) (α) ov (β), dd (9.2, 4.0) q (9.2) m s s s s s d (12.0) d (12.0) d (6.0) d (6.4) s

1.72, 1.47, 1.76, 1.65, 4.45, 0.97, 1.88, 1.55, 3.71,

dt (12.6, 2.8) td (12.6, 3.5) dd (8.6, 3.5) m dd (11.4, 4.2) m dq (12.5, 2.4) m td (10.2, 5.4)

2.04, 5.31, 2.03, 1.76, 2.06, 1.54, 1.77, 1.46, 1.89, 4.35, 2.01, 1.69, 1.43, 1.43, 0.85, 0.87, 1.05, 0.99, 0.89, 4.19, 4.13, 0.92, 0.82, 2.02, 2.04,

ov brd (6.6) m m m m ov ov d (9.3) td (9.3, 2.4) ov m ov ov s s s s s d (12.0) d (12.0) d (5.4) d (5.4) s s

7.16, d (10.8)

1.74, ov 1.39, ov 1.74, ov 1.64, ov 3.19, dd (12.0, 4.2) 0.88, brd (1.8) 1.90,ddd (12.0, 4.8, 1.8) 1.57, ov 3.70, td (10.2, 5.4)

1.74, 1.42, 1.76, 1.66, 3.21, 0.91, 1.90, 1.59, 3.70,

ov ov ov m dd (11.4, 3.6) ov ov ov td (10.2, 4.8)

2.22, brd (9.3) 5.44, br dt (6.6, 2.1) 1.99, ov 1.86, ov 1.91, ov 1.65 (β), ov 1.63, ov 1.51, m 1.63, d (9.3) 4.22, td (9.3, 3.0) 1.87, ov 1.68, ov 1.31, ov 1.43, m 1.14, s 1.04, s 1.29, s 0.98, s 0.96, s 0.81, s

2.05, 5.32, 2.47, 1.71, 2.13, 1.64, 1.78, 1.66, 2.18,

0.88, d (6.6) 0.82, d (6.6)

0.97, ov 0.87, d (6.6)

2.03, 5.36, 1.81, 1.77, 2.21, 1.56, 1.73, 1.73, 1.45, 4.11, 1.89, 1.21, 1.24, 1.71, 0.99, 0.82, 1.06, 0.93, 1.02, 3.73, 3.67, 0.91, 0.88,

m brd (6.0) ov ov m ov ov ov s brd (1.8) ov m ov ov s s s s s d (7.8) d (7.8) d (6.6) d (6.6)

5.94, d (10.8)

1.67, 2.02, 1.78, 3.91,

ov ov m td (9.3, 6.0)

brd (10.2) brd (6.3) dd (17.4, 6.3) ov m ov m ov s

2.36 (α), dd (18.6, 7.8) 1.75, (β) ov 1.41, ov 1.61, ov 0.97, s 0.81, s 1.04, s 0.93, s 0.96, s 0.90, s

ov denotes overlapped resonance. bCoupling constants were acquired using 1D-TOCSY NMR.

relatively rare triterpenoids may serve as prototypes for the development of cardiovascular drug leads.

dysfunctions and related manifestations, as evidenced by several studies revealing their excellent inhibitory potentials against platelet aggregation14 and nitric oxide production.15 The genus Rubia comprises about 70 species and is globally populated. Extensive chemical and biological investigations on Rubia spp. have unearthed valuable commercial and medicinal uses.16 Many Rubia spp. predominantly produce pentacyclic triterpenoids and cyclopeptides, and diverse biological properties such as antitumor, anti-inflammation, anti-HIV, and antiplatelet aggregation activities have been reported.16 One of the members, R. philippinensis Elmer, is a rambling and lowclimbing perennial herb17 and has rarely been investigated for its chemical constituents and their biological activities. As part of a continuous endeavor to probe potential naturederived drug templates for the treatment of cardiovascular disorders and related complications,13,18−20 the current study delineates the isolation of nine new arborinane triterpenoids (1−9) from R. philippinensis in the first chemical investigation of this underutilized plant resource. These compounds were evaluated for their inhibitory potential against PDGF-induced VSMC proliferation and migration to assess whether these

■

RESULTS AND DISCUSSION Compound 1 was obtained as a white, amorphous powder. The molecular formula was established as C32H52O4 on the basis of the HRESIMS sodium adduct ion at m/z 523.3760 (Figure S12, Supporting Information) (calcd [M + Na]+, m/z 523.3763) and 13C NMR data (Table 2). The 1D NMR data of 1 (Tables 1 and 2) were similar to those of rubiarbonol L,21 an arborinane-type triterpenoid from R. yunnanensis, except for NMR resonances of an O-acetyl functionality at δH 2.04 and δC 21.5 and 171.1. The acetoxy moiety was connected to C-3 based on the HMBC cross-peak from H-3 (δH 4.47) to the acetoxy carbonyl carbon resonating at δC 171.1 (Figure 2), and its β-equatorial orientation was determined on the basis of the splitting pattern of H-3 (δH 4.47, dd, J = 12.0 and 3.6 Hz) and the NOE correlation between H-3 and H-5 (δH 0.96) (Figure 3A). The 2D structure was established on the basis of the comparison of the NMR chemical shifts with those of rubiarbonol L21 and the COSY and HMBC cross-peaks shown in Figure 2. The relative configurations of the ring 2560



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Table 2. 13C NMR Spectroscopic Data (150 MHz, CDCl3) for Compounds 1−5 1

2

3

4

5

position

δC, type

δC, type

δC, type

δC, type

δC, type

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 OAc-3

35.7, CH2 24.2, CH2 81.0, CH 38.1, C 52.4, CH 21.4, CH2 26.7, CH2 40.8, CH 148.2, C 39.5, C 114.6, CH 37.0, CH2 37.2, C 38.6, C 30.5, CH2 32.0, CH2 49.4, C 59.6, CH 71.2, CH 42.5, CH2 56.9, CH 30.8, CH 28.3, CH3 16.9, CH3 22.3, CH3 17.3, CH3 16.1, CH3 63.9, CH2 23.0, CH3 23.3, CH3 171.1, C 21.5, CH3

36.1, CH2 24.2, CH2 80.6, CH 37.8, C 48.2, CH 32.9, CH2 72.2, CH 49.0, CH 146.2, C 39.3, C 117.2, CH 36.9, CH2 37.6, C 39.7, C 32.3, CH2 32.5, CH2 47.1, C 59.6, CH 71.1, CH 42.0, CH2 56.8, CH 30.6, CH 28.0, CH3 16.7, CH3 21.9, CH3 17.0, CH3 16.2, CH3 65.1, CH2 22.7, CH3 23.2, CH3 171.1, C 21.4, CH3 170.9, C 21.4, CH3

154.8, CH 125.4, CH 204.6, C 44.0, C 45.6, CH 32.4, CH2 70.7, CH 48.7, CH 142.3, C 41.7, C 117.6, CH 36.9, CH2 37.7, C 39.7, C 32.1, CH2 36.5, CH2 44.0, C 59.3, CH 71.5, CH 41.1, CH2 57.4, CH 30.4, CH 25.0, CH3 21.9, CH3 22.3, CH3 17.2, CH3 16.8, CH3 15.7, CH3 22.1, CH3 23.0, CH3

36.5, CH2 27.9, CH2 78.8, CH 39.0, C 48.3, CH 33.2, CH2 72.3, CH 48.4, CH 146.3, C 39.4, C 117.1, CH 34.4, CH2 36.9, C 38.9, C 31.4, CH2 36.1, CH2 42.7, C 64.9, CH 215.9, C 42.4, CH2 55.2, CH 30.4, CH 28.2, CH3 15.6, CH3 21.8, CH3 17.0, CH3 16.6, CH3 15.8, CH3 22.3, CH3 22.9, CH3

36.5, CH2 27.9, CH2 78.9, CH 38.8, C 48.5, CH 33.4, CH2 72.6, CH 48.6, CH 146.4, C 39.6, C 117.0, CH 37.8, CH2 35.7, C 38.9, C 30.3, CH2 25.6, CH2 48.3, C 57.6, CH 77.5, CH 41.0, CH2 54.3, CH 30.9, CH 28.2, CH3 15.6, CH3 21.6, CH3 15.7, CH3 15.9, CH3 68.5, CH2 22.5, CH3 23.0, CH3

OAc-28

Figure 1. Structures of arborinane triterpenoids 1−9 isolated from R. philippinensis.

junctions in 1 were established via the NOE correlations from H-3 (δH 4.47) to H-5 (δH 0.96), from H-8 (δH 2.02) to H3-25 (δH 1.06), from H-18 (δH 1.90) to H-21 (δH 1.41) and H3-26 (δH 0.86), and from H2-28 (δH 3.84, 3.76) to H3-27 (δH 0.98) (Figure 3A). The C-19 configuration was established on the basis of the NOE correlation between H2-28 and H-19 (δH 4.44) and the large coupling constant of H-18 (δH 1.90, d, J = 10. 2 Hz), reminiscent of the anti-relationship with H-19

(Figure 3A). This stereochemical assignment was supported by the C-17−C-18 cyclopentanol moiety predominately adopting a C17-endo envelope conformation where H-19 and H-20β (δH 2.09) were eclipsed given the large coupling constant between H-19 and H-20β (J = 9.6 Hz) (Figure 3B). The C-19 absolute configuration of 1 was investigated by employing Mosher’s method.22 Even though the formation of the R-MTPA derivative was observed (Figure S5, Supporting Information), 2561



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the generation of the S-MTPA ester invariably failed. Thus, the absolute configuration of 1 was assumed on the basis of the consistent absolute configuration of arborinane architectures obtained from natural sources (Figures 1 and 3). Consequently, the structure of 1 was established as 3-O-acetylrubiarbonol L. Compound 2 was isolated as a white amorphous powder. The molecular formula was established as C34H54O6 by the sodium adduct HRESIMS ion at m/z 581.3818 (Figure S24, Supporting Information) (calcd [M + Na]+, m/z 581.3818). The 1D NMR data resembled those of rubianol C15 from R. yunnanensis, an arborinane-type triterpenoid possessing an Oacetyl moiety at C-28. However, the 1D NMR spectrum of compound 2 also displayed resonances at δH 2.04 and δC 21.40 and 171.1, implying the presence of an additional acetoxy functionality. Its location at C-3 was based on the deshielded NMR chemical shift of the C-3 signal (δC 80.6) compared to that of rubianol C (δC 78.0)15 and the HMBC cross-peak from H-3 (δH 4.45) to the acetoxy carbonyl carbon (δC 171.1). The orientation of the C-7 hydroxy group was established as βequatorial given the splitting pattern of the H-7 resonance (δH 3.71, td, J = 10.2, 5.4), indicating its anti-relationship with H-8, and the NOE correlation of H-5 (δH 0.97) with H-7 (δH 3.71) (Figure 3A). The assignment of the 2D structure and relative configuration of 2 was carried out the same as for 1 utilizing the 2D hetero- and homonuclear NMR correlations (Figures 2 and

(Table 2), established the molecular formula of 3 as C30H46O3 (calcd [M + Na]+, m/z 477.3345). The 1D NMR data of 3 displayed close similarities to those of rubiarbonone E,14 an arborinane-type triterpenoid from R. yunnanensis possessing a 1,2-enoyl A-ring functionality. Differences in the NMR spectroscopic data included the absence of the C-17 hydroxymethylene motif compared to rubiarbonone E and the presence of a tertiary methyl group (δH 0.81, δC 15.7), implying a difference in the nature of the C-17 substituent. The C-17 resonance at δC 44.0, more shielded compared to that of rubiarbonone E (δC 49.6),14 and the HMBC cross-peaks of the tertiary methyl protons (δH 0.81) with C-16 (δC 36.5), C-17 (δC 44.0), C-18 (δC 59.3), and C-21 (δC 57.4) (Figure 2) confirmed that C-17 in 3 indeed carried a methyl group. The 2D structure was examined by employing the 2D correlations depicted in Figure 2. The configuration of C-17 was established on the basis of the NOE correlations from H-28 (δH 0.81) to H-19 (δH 4.22) (Figure 3A). The configurational details of other stereogenic centers were addressed in a similar fashion by utilizing NOE correlations (Figure 3A). Accordingly, the structure of 3 was established as 28-deoxyrubiarbonone E. The HRESIMS data of compound 4, acquired as a white powder, displayed a sodium adduct ion at m/z 479.3499 (Figure S42, Supporting Information) (calcd [M + Na]+, m/z 479.3501), which, in conjunction with the 13C NMR data (Table 2), led to the assignment of its molecular formula as C30H48O3. The 1D NMR data of 4 shared similarities with those of rubiarbonol B from R. cordifolia var. pratensis23 except for the presence of the 13C NMR resonance for a ketocarbonyl functionality (δC 215.9) and the absence of the C-19 hydroxymethine moiety when compared to rubiarbonol B. This is indicative of the hydroxymethine moiety being oxidized to the corresponding ketocarbonyl functionality, which was confirmed by the HMBC cross-peaks from H-18 (δH 2.18) and H2-20 (δH 1.75, 2.36) to the carbonyl carbon (δC 215.9) (Figure 2). Owing to the absence of the C-19 methine proton, the configurational assignment of C-17 in 4 was addressed using an alternative approach. The well-resolved H-20 resonance at δH 2.36 was assigned to the α-oriented diastereotopic proton based on the H-20α−C-20−C-21−H21 dihedral angle (32°) and the vicinal coupling constant (3JH‑20α,H‑21 = 7.8 Hz) calculated via the Karplus equation (Figure 3B).24 The C-20 diastereotopic proton resonating at δH 1.74 was thus assigned a β orientation, and its NOE correlation with H3-28 implied that H-20β and H3-28 were cofacial (Figure 3B). The configurational analysis of the core structure in 4 was accomplished as shown in Figure 3A. Consequently, the structure of 4 was assigned as 19-dedihydrorubiarbonol B, the first arborinane-type triterpenoid carrying a ketocarbonyl motif at C-19. Compound 5 was obtained as a white powder. The molecular formula was deduced as C30H48O3 from the HRESIMS data exhibiting the sodium adduct ion at m/z 479.3495 (Figure S51, Supporting Information) (calcd [M + Na]+, m/z 479.3501) and the 13C NMR data (Table 2). An inspection of the NMR data (Tables 1 and 2, Figure 2) revealed similarities to rubianol F15 possessing the 19,28-oxide ring. Compared to rubianol F,15 however, the NMR data of 5 lacked resonances of the C-2 oxymethine group but exhibited signals of a methylene moiety instead (δH 1.66, 1.76; δC 27.9). This was confirmed via the COSY correlations starting from H-1 to H-3 (Figure 2).The configuration of the O-heterocyclic moiety was established on the basis of the NOE correlation from H3-27

Figure 2. Key COSY () and HMBC (→) cross-peaks for the elucidation of the 2D structures of 1−9.

3A). The C-19 absolute configuration of 2 was established via utilization of Mosher’s ester protocol.22 Notably, the C-7 carbinol functionality was not susceptible to esterification (Figures S16-1 and S17-1 expanded, Supporting Information), and only the (R)- and (S)-MTPA esters of the C-19 carbinol moiety were obtained, even though clean conversions were not achieved (Figures S16-1 and S17-1). The chemical shift difference of H-18 and H-20, assigned via 1D-TOCSY and COSY analyses (Figures S16-1,-2 and S17-1,-2, Supporting Information), are shown in Figure 4 and are reminiscent of a (19R) absolute configuration. These data collectively support the structure of 2 as 3-O-acetylrubianol C. Compound 3 was acquired as a white amorphous powder. The sodium adduct HRESIMS ion at m/z 477.3339 (Figure S33, Supporting Information), along with the 13C NMR data 2562



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Figure 3. (A) Key NOE correlations (denoted with yellow dotted lines) in the establishment of the relative configuration and (B) configurational and conformational analysis of the cyclopentanol (1, H-20β was assigned on the basis of the NOE correlation between the a proton and H2-28) and cyclopentanone moieties (4).

to H2-28 (Figure 3A) and the 13C NMR chemical shift of the bridgehead C-19 (δC 77.5), similar to that of rubianol F (δC 77.5).15 The full stereochemical assignment was based on the NOE analysis (Figure 3A), and the structure of compound 5 was consequently assigned as 2-deoxyrubianol F. Compounds 6 and 7 were acquired as white powders. Their molecular formulas were established as C 32H52O4 and C32H52O5 on the basis of the HRESIMS sodium adduct ions at m/z 523.3756 and 539.3711 (Figures S61 and S71,

Supporting Information) (calcd [M + Na]+, m/z 523.3763 and 539.3712) and 13C NMR data (Table 4). The 1D NMR spectroscopic data of 6 and 7 were analogous to those of rubiarbonol B and rubiarbonol A,23 respectively. The only difference was the presence of an O-acetyl moiety for compounds 6 and 7 as deduced from the respective NMR resonances (δH/δC 2.05/21.4, δC 171.0 for 6; δH/δC 2.06/21.5, δC 170.9 for 7). The deshielded C-3 NMR chemical shift values and HMBC cross-peaks of H-3 (δH 4.47 and 4.69 for 6 and 7, 2563



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10 and 30 μM and compound 2 at 30 μM appeared to hamper the PDGF-stimulated proliferation. To preclude whether the observed inhibitory activity against PDGF-induced proliferation was attributed to the cytotoxicity of the tested compounds, their cytotoxicity was assessed using the MTT assay. The treatment with active molecules 5, 8, and 9 at two different concentrations (10 and 30 μM) and 2 at one concentration (30 μM) showed their cytotoxicity on VSMCs (Figure 5B), leading to the optimization of their appropriate concentrations capable of inhibiting PDGF-induced proliferation but without cytotoxicity. The lower concentration (5 μM) of compounds 5 (62.6 ± 10.7%, p < 0.01) and 9 (41.1 ± 4.7%, p < 0.05) effectively impeded PDGF-insulted proliferation, and these compounds did not exhibit cytotoxicity (Figure 5C,D). This suggested the importance of determining an optimal concentration level of these arborinane-type triterpenoids for their potential clinical application as remedies for cardiovascular diseases. All the purified compounds were also tested for their antimigration activity against PDGF-stimulated VSMCs utilizing the scratch wound−healing assay.26 The PDGF insult (denoted as C in Figure 5E, upper) exhibited 82.3% wound closure, indicating that the growth factor induced a significant migration of smooth cells. As shown in Figure 5B,D, 5 and 8 at both 10 and 30 μM and 2 at 30 μM appeared to be cytotoxic. Thus, although the treatment with these compounds seemed to inhibit migration, this might be caused by the cytotoxicity of the treated compounds against VSMCs. Compound 7 suppressed the PDGF-induced migration in a concentrationdependent manner (38.5 ± 3.0% at 10 μM and 57.6 ± 3.2% at 30 μM, Figure 5E, upper) without exhibiting cytotoxicity (Figure 5B). The scratch wound−healing assay upon treatment with 7 was photographed (Figure 5E, lower) to further verify the dose-dependent inhibitory activity against the abnormal migration of VSMCs triggered by the addition of PDGF. The treatment of PDGF (25 ng/mL) enhanced the VSMC migration as the wound area was filled with the migrated smooth cells (Figure 5E, lower, expressed as a control), and the dose-dependent inhibition of compound 7 was corroborated on the basis of how much the migration was impeded [Figure 5E, lower; third (C7 10 μM) and fourth (C7 30 μM) photographs]. A growing body of evidence points to the vital role of triterpenoids from diverse plant species in the development of drug leads targeting the alleviation of cardiovascular diseases and related complications.13−16,27−29 In line with such research, the current findings demonstrate that some arborinane triterpenoid-based chemotypes may serve as a drug framework capable of inhibiting PDGF-induced proliferation and migration, often leading to cardiovascular complications and manifestations. Triterpenoids, however, also exerted cytotoxicity at certain concentrations as witnessed not only in the current study but in many other investigations.30 Therefore, it is indispensable to determine optimal concentration levels of triterpenoid-based pharmacophores for the development of nontoxic cardiovascular drugs. Upon the basis of this study, this may be facilitated, presumably in conjunction with biochemical insights regarding the pathophysiology of cardiovascular dysfunction and its causative cytokines and growth factors. Moreover, this study may inspire the investigation of underutilized plant species for the discovery of potent new pharmacophores.

Figure 4. Mosher’s analysis of C-19 in compound 2. ΔδS−R values for the protons of the S- and R-MTPA derivatives in pyridine-d5.

respectively) with the acetoxy carbons (δC 171.0 and 170.9 for 6 and 7, respectively) permitted the acetoxy functionality to be connected to C-3 for both compounds. The stereochemical features of 6 and 7 were assessed using the key NOE correlations shown in Figures 3A and S4 in Supporting Information for 6 and 7, respectively. The structures of compounds 6 and 7 were thus defined as 3-O-acetylrubiarbonol B and 3-O-acetylrubiarbonol A. Compounds 8 and 9 were purified as white powders. Molecular formulas of C32H52O4 and C32H48O4 were suggested for 8 and 9, respectively, given their respective HRESIMS sodium adduct ions at m/z 523.3760 (Figure S81, Supporting Information) (calcd [M + Na]+, m/z 523.3763) and m/z 519.3448 (Figure S91, Supporting Information) (calcd [M + Na]+, m/z 519.3450) and 13C NMR data (Table 4). The NMR spectroscopic data of 8 and 9 are reminiscent of those of rubiarbonol B23 and compound 3, with the exception of the presence of certain resonances (δH/δC 2.00/21.7, δC 171.3 for 8; δH/δC 2.01/21.7, δC 171.2 for 9), indicating the presence of acetoxy moieties in 8 and 9. These O-acetyl functionalities were located at C-19 on the basis of the deshielded 13C NMR resonances of C-19 (δC 74.0 and 73.9 for 8 and 9, respectively), compared to those of arborinane triterpenoids carrying a C-19 hydroxy group (ca. δC 71.0). The HMBC cross-peaks of H-19 (δH 5.07 and 5.10 for 8 and 9, respectively) with the acetoxy carbonyl carbons (δC 171.3 for 8; δC 171.2 for 9) reaffirmed the C-19 locations of the O-acetyl moieties (Figure 2). The αorientations of the acetoxy groups were assigned from the NOE correlations of H-19 (δH 5.07 and 5.10 for 8 and 9, respectively) with CH3-27 (δH 0.88 and 0.94 for 8 and 9, respectively) and CH3-28 (δH 0.82 and 0.85 for 8 and 9, respectively) (Figure S1, Supporting Information). Consequently, the structures of 8 and 9 were established as 19-Oacetylrubiarbonol B and 19-O-acetyl-28-deoxyrubiarbonone, respectively. Compounds 1, 2, and 6−9 are O-acetyl esters of known arborinane-type triterpenoids. Proof that they are not artifacts of the isolation/purification process was provided via an HPLCevaporative light scattering detector (ELSD) profile demonstrating their presence in a crude R. philippinensis extract (Figure S2-1, Supporting Information). Compounds 1−9 were evaluated for their inhibitory effects of PDGF-induced proliferation and migration of VSMCs. According to Figure 5A, the addition of PDGF-BB (composed of two B chains; more potent inducer of VSMC proliferation and migration than PDGF-AA and PDGF-AB)25 increased the cell proliferation and treatment with compounds 5, 8, and 9 at 2564



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Figure 5. Antiproliferative and antimigration activities of compounds 1−9 in VSMCs and an investigation of effective and nontoxic concentrations of the test compounds. (A) Inhibitory potential of compounds 1−9 against PDGF-induced VSMC proliferation; cells were treated with each compound at a concentration of 10 or 30 μM for 24 h, stimulated by 25 ng/mL PDGF-BB (composed of two B chains)25 for another 24 h, and the induced proliferation was assessed using the MTT assay. Paclitaxel (1 μM) was used as a positive control (PC). (B) Evaluation of the cytotoxicity of compounds 1−9; VSMCs were treated with compounds 1−9 (10 or 30 μM) for 48 h, and cell viability was assessed by employing the MTT assay. Digitonin (100 μg/mL) was used as a positive control (PC). (C) Dose-dependent antiproliferative potential of compounds 5, 8, and 9 against PDGF-stimulated VSMCs. Cells were treated with different concentrations of compounds 5, 8, and 9 as denoted in parentheses (μM), and the antiproliferative activity was evaluated using the MTT assay. Positive control (PC): Paclitaxel (1 μM). (D) Cell viability of compounds 5, 8, and 9 at various concentrations as indicated in parentheses (μM). Positive control (PC): digitonin (100 μg/mL). (E) Inhibitory effect of compounds 1−9 on PDGF-induced VSMC migration (upper) and photographs verifying the antimigration potential of compound 7 (C7) (lower). Serum-starved VSMCs were treated with compounds 1−9 for 2 h, and migration was induced with 25 ng/mL PDGF-BB for 48 h. The cells were stained 0.5% crystal violet, and the migration area was determined on the basis of analysis using ImageJ software. The values are expressed as means ± standard error of the mean (SEM) of triplicate experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs control (C, only PDGF-BB stimulation) in A, C, and E and vs vehicle (V, no treatment) in B and D.

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DMX 300 (300 MHz) or Bruker Avance III (600 MHz) spectrometer (Billerica, MA). HRESIMS data were generated on a Waters SYNAPT G2 high-resolution mass spectrometer (Milford, MA). Thin-layer chromatography (TLC) was performed on glass plates precoated with silica gel 60 F254 or RP-18 F254 (20 × 20 cm2, 200 μm, 60 Å, Merck, Kenilworth, NJ). Vacuum-liquid chromatography (VLC) was con-

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Jasco DIP-1000 automatic digital polarimeter (Tokyo, Japan), and IR data were recorded on a Thermo Electron US/ Nicolet380 (Madison, WI). NMR experiments were done on a Bruker 2565



Article

Table 3. 1H NMR Spectroscopic Data (600 MHz) for Compounds 6−9a position 1 2 3 5 6 7 8 11 12 15 16 18 19 20 21 22 23 24 25 26 27 28 29 30 OAc-3 OAc-19 a

6b,d

7c,d

8b,d

9b,d

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

1.75, 1.50, 1.78, 1.67, 4.47, 0.99, 1.91, 1.60, 3.76, 2.07, 5.32, 1.76, 1.61, 1.99, 1.62, 1.98, 1.78, 1.62, 4.21, 1.86, 1.69, 1.31, 1.43, 0.87, 0.89, 1.08, 0.97, 0.91, 0.80,

ov m ov ov dd (12.0, 4.2) m dd (12.3, 5.4) q (12.3) m br d (8.8) brd (6.0) ov ov m d (12.3) dd (16.0, 3.4) dd (16.0, 5.6) d (9.6) td (9.6, 3.6) m m q (9.8) m s s s s s s

0.88, d (6.6) 0.83, d (6.6) 2.05, s

1.65, 1.44, 1.82, 1.70, 4.69, 1.06, 2.16, 1.90, 4.01, 2.47, 5.44, 2.59, 2.49, 2.80, 2.00, 2.01, 1.60, 2.35, 5.06, 2.62, 2.16, 1.59, 2.14, 0.92, 0.95, 1.11, 1.33, 1.42, 4.22, 4.09, 1.10, 0.97, 2.06,

m m m m dd (11.4, 4.2) m dd (13.2, 6.0). q (13.2) sextet (5.2) ov brd (4.2) brd (16.0) dd (16.5, 6.3) brd (14.4) ov ov ov d (9.6) td (10.0, 2.4) q (10.0) td (10.0, 3.1) q (10.0) m s s s s s d (11.4) d (11.4) d (6.6) d (6.6) s

1.71, ov 1.37, m 1.73, m 1.63, qd (11.6, 3.5) 3.20, dd (11.6, 4.2) 0.87, ov 1.90, dd (12.0, 4.6) 1.57, q (12.0) 3.7, td (10.7, 5.4) 2.04, brd (7.0) 5.27, brd (6.0) 1.90, ov 1.45, ov 2.04, ov 1.61, ov 1.61, ov 1.51, m 1.90, d (10.8) 5.07, td (10.2, 2.4) 1.93, dd (10.3, 4.2) 1.61,ddd (14.0, 9.0, 3.2) 1.24, q (9.0) 1.43, m 0.975, s 0.81, s 1.03, s 0.97, s 0.88, s 0.82, s

7.14, d (10.5)

1.67, 2.03, 1.78, 3.91, 2.22, 5.42, 1.94, 1.64, 1.91, 1.52, 1.95, 1.52, 1.92, 5.10, 1.96, 1.64, 1.25, 1.46, 1.14, 1.04, 1.28, 1.00, 0.94, 0.85,

0.87, d (6.4) 0.80, d (6.4)

0.89, d (6.3) 0.82, d (6.3)

2.00, s

2.01, s

5.93, d (10.5)

ov dd (13.0, 6.6) q (13.0) m brd (7.5) brd (6.0) ov dd (8.9, 3.1) d (10.1) ov ov m d (9.9) td (9.9, 3.0) dd (9.4, 4.2) dd (9.4, 3.1) q (9.4) m s s s s s s

ov denotes overlapped resonances. bMeasured in CDCl3. cMeasured in pyridine-d5. dCoupling constants were measured with 1D-TOCSY NMR.

ducted on Merck silica gel (70−230 mesh), and medium-pressure liquid chromatography (MPLC) was carried out by utilizing a Biotage Isolera apparatus equipped with a reversed-phase C18 SNAP Cartridge KPC18-HS (400 g, Uppsala, Sweden). HPLC was performed on a Gilson system (Middleton, WI) using a Phenomenex Kinetex 5μ C18 column (250 × 21.20 mm2, 5 μm, Torrance, CA). Plant Material. The roots of R. philippinensis were collected from Bidoup-Nui Ba National Park, Lamdong Province, Vietnam in August 2014 and identified by Dr. Phuong Thien Thuong (Department of Analytical Chemistry and Herbal Standardization, National Institute of Medicinal Materials, Vietnam) and Dr. MinKyun Na (College of Pharmacy, Chungnam National University, Korea). A voucher specimen was deposited at the herbarium of the Vietnam National Institute of Medicinal Materials, Hanoi, Vietnam (VDL20140801) as well as the Laboratory of Pharmacognosy at the College of Pharmacy, Chungnam National University, Daejeon, Korea (CNU1409). Extraction and Isolation. The dried roots of R. philippinensis (1.5 kg) were extracted with EtOH (3 L × 3) at room temperature for 4 days. The crude extract was concentrated under a vacuum to yield a reddish-brown slurry (150 g). The slurry was suspended in H2O (1.5 L) and sequentially partitioned with CH2Cl2 (2 L × 3) and EtOAc (2 L × 3) to yield the CH2Cl2, EtOAc, and H2O extracts. The CH2Cl2 fraction was subjected to silica gel VLC and eluted with n-hexane/ EtOAc (20:1, 10:1, 5:1, 3:1, and 2:1) and CHCl3/MeOH (8:1) to yield six fractions (D-1−D-6). Fraction D-4 was divided into 10

subfractions (D-4-1−D-4-10) using MPLC with a gradient of MeOH/ H2O (10:90 → 100:0, 7 L). Compound 9 (4 mg) was obtained from fraction D-4-8 (150 mg) by HPLC elution with MeOH/H2O (83:17, 4 mL/min, UV 205 nm, tR 55.0 min), followed by MeCN-H2O (75:25, 4 mL/min, UV 205 nm, t R 68.0 min). Fraction D-5 was chromatographed over MPLC with a gradient of MeOH/H2O (10:90 → 100:0, 7 L) to furnish eight subfractions (D-5-1−D-5-8). Compounds 2 (tR 24.6 min, 23 mg), 3 (tR 26.1 min, 18 mg), and 4 (tR 40.0 min, 20 mg) were purified from fraction D-5-5 (300 mg) by employing HPLC with an isocratic condition (MeOH/H2O = 80:20, 6 mL/min, UV 205 nm). The residual fraction from D-5-5 was further purified using an isocratic elution with MeCN/H2O (72:28, 6 mL/ min, UV 205 nm) to afford 5 (tR 37.0 min, 7 mg). Fraction D-5-6 (100 mg) was subjected to HPLC purification eluting with MeOH/H2O (85:15, 4 mL/min, UV 205 nm) to afford 6 (tR 39.0 min, 23 mg) and 8 (tR 51.5 min, 18 mg). Fraction D-5-7 (53 mg) was purified by utilizing an isocratic elution of MeCN/H2O (83:17, 6 mL/min, UV 205 nm) to yield 1 (tR 41.0 min, 21 mg). Fraction D-6 was divided into 12 subfractions (D-6-1−D-6-11) by employing MPLC with a gradient elution of MeOH/H2O (10:90 → 100:0, 7 L). Compound 7 (tR 39.0, 18 mg) was purified from fraction D-6-10 using HPLC eluting with MeOH/H2O (75:25, 4 mL/min, UV 205 nm). Validation of the natural occurrence of acetates 1 and 2 and 6−9 (Figure S2-1, Supporting Information) was done using an LC-10AD series HPLC system (Shimadzu, Kyoto, Japan) equipped with a YMC C18 column 2566



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(250 × 4.6 mm2, 5 μm) and a Sedex 55 evaporative light scattering detector (Sedere, Alfortville, France). The EtOH extract of the R. philippinensis roots (300 mg) was suspended in H2O (5 mL) and partitioned with the CH2Cl2 extract to yield the crude extract. Compounds 1 and 2 and 6−9 (0.5 mg of each compound) were dissolved in 1 mL of HPLC-grade MeOH and filtered for purity and profiling analyses. Each compound and the CH2Cl2 extract (10 μL) were analyzed by utilizing the HPLC−ELSD system with a gradient program of MeOH/H2O (60:40 → 100:0, 1 mL/min, 1 h) (Figure S21, Supporting Information). 3-O-Acetylrubiarbonol L (1). White amorphous powder; [α]22 D + 26 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 242 (3.87); IR (KBr) νmax 2928, 1723, 1263, 1032, cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 523.3760 [M + Na]+ (calcd for C32H52O4Na, 523.3763). 3-O-Acetylrubianol C (2). White amorphous powder; [α]22 D + 15 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 242 (3.47); IR (KBr) νmax 2938, 1726, 1242, 1032 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 581.3818 [M + Na]+ (calcd for C34H54O6Na, 581.3818). 28-Dehydroxyrubiarbonone E (3). White amorphous powder; [α]22 D + 37 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 237 (4.35); IR (KBr) νmax 3443, 2924, 1663, 1375, 1036 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 477.3339 [M + Na]+ (calcd for C30H46O3Na, 477.3345). 19-Dedihydrorubiarbonol B (4). White amorphous powder; [α]22 D − 28 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 242 (3.83); IR (KBr) −1 1 13 νmax 2961, 1722, 1376, 1033 cm ; H and C NMR, see Tables 1 and 2; HRESIMS m/z 479.3499 [M + Na]+ (calcd for C30H48O3Na, 479.3501). 2-Deoxyrubianol F (5). White amorphous powder; [α]22 D + 33 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 243 (4.19), 205 (4.33); IR (KBr) νmax 3380, 2937, 1373, 1041 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 479.3495 [M + Na]+ (calcd for C30H48O3Na, 479.3501). 3-O-Acetylrubiarbonol B (6). White amorphous powder; [α]22 D + 22 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 243 (4.25); IR (KBr) νmax 3330, 2958, 1734, 1376, 1049 cm−1; 1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 523.3756 [M + Na]+ (calcd for C32H52O4Na, 523.3763). 3-O-Acetylrubiarbonol A (7). White amorphous powder; [α]22 D + 50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 243 (4.39); IR (KBr) νmax 2944, 1710, 1371, 1253, 1029 cm−1; 1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 539.3711 [M + Na]+ (calcd for C32H52O5Na, 539.3712). 19-O-Acetylrubiarbonol B (8). White amorphous powder; [α]22 D − 98 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 242 (4.13), 205 (4.17); IR (KBr) νmax 2927, 1705, 1268, 1033 cm−1; 1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 523.3760 [M + Na]+ (calcd for C32H52O4Na, 523.3763). 19-O-Acetyl-28-deoxyrubiarbonone (9). White amorphous powder; [α]22 D − 72 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 232 (4.13), 205 (4.07); IR (KBr) νmax 2925, 1724, 1248, 1029 cm−1; 1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 519.3448 [M + Na]+ (calcd for 32H48O4Na, 519.3450). Mosher’s Analysis. Compound 2 (1.0 mg) was transferred into two NMR tubes and dried completely using a N2 gas stream. Pyridined5 (0.2 mL) and Mosher’s reagents (R- and S-MTPA) were added to each NMR tube immediately under a N2 gas stream. These NMR tubes were gently shaken to initiate the esterification and were kept in a desiccator. The reactions were monitored every 2 h by measuring the 1 H NMR spectrum. The resonances of H-18−H-20 were assigned on the basis of COSY and 1D-TOCSY experiments (Figures S16 and S17, Supporting Information). S-MTPA derivative: δH 5.68 (1H, brt, J = 8.4 Hz H-19), 2.37 (1H, m, H-18), and 1.95 and 1.42 (2H, m, H-20). RMTPA derivative: δH 5.72 (1H, brt, J = 8.4 Hz H-19), 2.38 (1H, m, H18), and 1.80 and 1.27 (2H, m, H-20). Computational Details. All conformational searches and minimizations were carried out using the Macromodel (version 9.9, Schrodinger LLC) module with mixed torsional/low mode sampling

Table 4. 13C NMR Spectroscopic Data (150 MHz) for Compounds 6−9 6a

8a

9a

position

δC, type

δC, type

δC, type

δC, type

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 OAc-3

36.2, CH2 24.3, CH2 80.7, CH 37.8, C 48.2, CH 32.9, CH2 72.1, CH 49.1, CH 146.1, C 39.3, C 117.3, CH 36.6, CH2 37.77, C 39.6, C 32.1, CH2 37.0, CH2 44.0, C 59.5, CH 71.5, CH 41.0, CH2 57.4, CH 30.5, CH 28.1, CH3 16.7, CH3 21.9, CH3 16.9, CH3 16.8, CH3 15.7, CH3 22.1, CH3 23.0, CH3 171.0, C 21.4, CH3

36.6, CH2 24.9, CH2 80.9, CH 38.3, C 49.1, CH 33.7, CH2 72.2, CH 49.7, C 147.3, C 39.9, C 118.1, CH 38.0, CH2 38.7, C 40.6, C 33.4, CH2 33.8, CH2 49.3, C 60.3, CH 71.0, CH 43.8, CH2 58.4, CH 31.1, CH 28.4, CH3 17.2, CH3 22.2, CH3 17.6, CH3 17.1, CH3 63.3, CH2 23.8, CH3 24.0, CH3 170.9, C 21.5, CH3

36.45, CH2 27.9, CH2 78.8, CH 38.9, C 48.2, CH 33.1, CH2 72.3, CH 49.1, CH 146.4, C 39.4, C 117.1, CH 36.1, CH2 37.6, C 39.5, C 31.9, CH2 36.51, CH2 43.3, C 55.6, CH 74.0, CH 38.1, CH2 57.5, CH 30.3, CH 28.1, CH3 15.48, CH3 21.8, CH3 16.9, CH3 16.7, CH3 15.54, CH3 22.0, CH3 22.9, CH3

154.7, CH 125.4, CH 204.4, C 44.0, C 45.7, CH 32.4, CH2 70.7, CH 48.8, CH 142.4, C 41.7, C 117.6, CH 36.0, CH2 37.6, C 39.7, C 32.0, CH2 36.4, CH2 43.3, C 55.6, CH 73.9, CH 38.1, CH2 57.5, CH 30.3, CH 24.9, CH3 21.9, CH3 22.2, CH3 17.2, CH3 16.8, CH3 15.5, CH3 22.0, CH3 22.9, CH3

171.3, C 21.7, CH3

171.2, C 21.7, CH3

OAc-19 a

7b

Measured in CDCl3. bMeasured in pyridine-d5.

in the MMFF force field. The searches were implemented in the gas phase with a 50 kJ/mol energy window limit and a maximum of 10 000 steps to thoroughly investigate all low-energy conformers. The PolakRibiere conjugate gradient (PRCG) method was adopted for minimization processes with 10 000 maximum iterations and a 0.001 kJ (mol Å)−1 convergence threshold on the rms gradient. Each conformer at the grand minimum was used for configurational and conformational analysis as shown in Figure 3 and Supporting Information. The 3D representations were drawn by utilizing Pymol (1.7.x, Open Source). Cell Culture. Rat aortic VSMCs were isolated by enzymatic dispersion with reference to a previous study.31 Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 8 mM HEPES, and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The purity of the VSMC culture was confirmed by the immunocytochemical localization of α-smooth-muscle actin. Animals and Husbandry. Male rats 4 to 5 weeks old were purchased from Samtako Inc. (Osan, South Korea). All experimental protocols using animals were performed in accordance with the Guide for the National Institutes of Health Guide for the Care and Use of Laboratory Animals, approved by the Chungnam National University Animal Care and Use Committee. Animals were housed three per cage and raised in a controlled facility where the temperature (22 ± 2 °C), 2567


Journal of Natural Products humidity (50 ± 5%), and 12 h dark−light cycle (light cycle starting at 6:00 a.m.) were automatically maintained. Antiproliferation Assay. The inhibitory potential of the test compounds against the PDGF-induced proliferation of VSMCs was measured using an MTT assay.32 VSMCs were seeded into 96-well culture plates at 3.5 × 104 cells/mL and cultured in DMEM containing 10% FBS at 37 °C until the cells reached 70% confluence. The culture medium was replaced with serum-free DMEM, and cultivation continued for 24 h. The serum-starved VSMCs were incubated with the indicated concentrations of arborinane-type triterpenoids or paclitaxel (1 μM) for another 24 h. Cellular proliferation was induced by the addition of 25 ng/mL PDGF-BB for another 24 h. The cultured medium was replaced with serum-free DMEM with a 5 mg/mL MTT solution, and cells were incubated for 4 h. The medium was exchanged with DMSO, and the absorbance at 565 nm was measured using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell Viability Assay. The cell viability was determined by utilizing an MTT assay. VSMCs were seeded into 96-well culture plates at 3.5 × 104 cells/mL and incubated in DMEM with 10% FBS at 37 °C until the cells reached 70% confluence, and the cells were incubated with serum-free medium for 24 h. The serum-starved cells were exposed to arborinane triterpenoids of specified concentrations or 100 μg/mL digitonin for 48 h. The VSMC viability was measured by MTT assay as described in the subsection immediately above. Cell Migration Assay. The PDGF-stimulated migration of VSMCs was measured using the scratch wound−healing assay.26 VSMCs were seeded into 12-well culture plates at 5 × 104 cells/mL and cultured in DMEM with 10% FBS at 37 °C. Until cells reached over 90% confluence in the wells, cells were incubated in serum-free DMEM for 24 h. Each cell was scratched with two lines using a sterilized 200 μL tip, washed with warm serum-free DMEM, and photographed to record the clear zone (wound) area at 0 h. The cells were pretreated with arborinane triterpenoids of designated concentrations or paclitaxel (1 μM) for 2 h, and cell migration was induced by the addition of 25 ng/mL PDGF-BB for 48 h. After incubation for 48 h, VSMCs were fixed with 100% MeOH and stained with 0.5% crystal violet. Images of migrated cells were taken to assess the antimigration activity of the tested triterpenoids. Cell migration areas were analyzed using ImageJ software. Statistical Analysis. Bioassay results in the current study were expressed as means ± SEM. One-way ANOVA was used for Dunnett’s multiple comparison test using GraphPad Prism software (San Diego, CA).

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Basic Science Research Program (NRF-2014R1A2A2A01006793), the Global R&D Center (GRDC) (NRF-2010-00719), and the Priority Research Centers Program (2009-0093815) through the National Research Foundation of Korea (NRF) grant funded by the Korean Government. We thank Dr. Phuong Thien Thuong (National Institute of Medicinal Materials, Vietnam) for providing and authenticating the plant material. We also thank Dr. Bui Huu Tai (College of Pharmacy, CNU) for the helpful discussion regarding structural elucidation. We recognize Mr. Nguyen Quoc Tuan (College of Pharmacy, CNU) and Ms. Le Vu Ngoc Han (Pharmaceutical Analysis, CNU) for sharing expertise regarding samples analyses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00489.

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Article

1D (1H, 13C, DEPT, and 1D-TOCSY) and 2D NMR, IR, and HRESIMS spectra, HPLC profiling, and configurational analysis of compounds 7−9 in this study (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(C.-S.M.) Tel: +82 42 821 5923. Fax: +82 42 821 8925. Email: [email protected]. *(M.N.) Tel: +82 42 821 5925. Fax: +82 42 823 6566. E-mail: [email protected]. Author Contributions

K.T.Q., H.-S.P., and J.O. contributed equally. Notes

The authors declare no competing financial interest. 2568



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Arborinane Triterpenoids from Rubia philippinensis Inhibit

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