Bioactive Diterpenoids and Flavonoids from the Aerial Parts of

Jun 23, 2014 - known compounds, were isolated from the aerial parts of Scoparia dulcis. The 7S absolute configuration of the new diterpenoids 1−4 an...
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Bioactive Diterpenoids and Flavonoids from the Aerial Parts of Scoparia dulcis Qing Liu,†,‡ Qi-Ming Yang,† Hai-Jun Hu,§ Li Yang,† Ying-Bo Yang,† Gui-Xin Chou,†,‡ and Zheng-Tao Wang*,†,‡ †

The MOE Key Laboratory of Standardization of Chinese Medicines, and SATCM Key Laboratory of New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, People’s Republic of China ‡ Shanghai R&D Center for Standardization of Chinese Medicines, Shanghai 201203, People’s Republic of China § Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Six new diterpenoids, 4-epi-7α-O-acetylscoparic acid A (1), 7α-hydroxyscopadiol (2), 7α-O-acetyl-8,17β-epoxyscoparic acid A (3), neo-dulcinol (4), dulcinodal-13-one (5), and 4-epi-7αhydroxydulcinodal-13-one (6), and a new flavonoid, dillenetin 3-O(6″-O-p-coumaroyl)-β-D-glucopyranoside (10), along with 12 known compounds, were isolated from the aerial parts of Scoparia dulcis. The 7S absolute configuration of the new diterpenoids 1−4 and 6 was deduced by comparing their NOESY spectra with that of a known compound, (7S)-4-epi-7-hydroxyscoparic acid A (7), which was determined by the modified Mosher’s method. The flavonoids scutellarein (11), hispidulin (12), apigenin (15), and luteolin (16) and the terpenoids 4-epi-scopadulcic acid B (9) and betulinic acid (19) showed more potent α-glucosidase inhibitory effects (with IC50 values in the range 13.7−132.5 μM) than the positive control, acarbose. In addition, compounds 1, 11, 12, 15, 16, and acerosin (17) exhibited peroxisome proliferator-activated receptor gamma (PPAR-γ) agonistic activity, with EC50 values ranging from 0.9 to 24.9 μM.

T

ulcers.7 Phytochemical investigations on S. dulcis have resulted in the isolation of several alkaloids, diterpenoids, flavonoids, steroids, and triterpenoids.7 The antihyperglycemic effects of S. dulcis were reported,8,9 and two diterpenoids, scoparic acids A and D,10,11 and a flavonoid, 8-hydroxytricetin-7-glucuronide,12 were found to exhibit antidiabetic potential in vitro. As a part of a continuing search for biologically active natural diterpenoids from traditional Chinese medicines,13−18 the present paper describes a systematic phytochemical investigation on the aerial parts of S. dulcis, with the isolated compounds evaluated for their α-glucosidase inhibitory and PPAR-γ agonistic activities.

ype 2 diabetes is characterized by hyperglycemia resulted from insulin resistance. The thiazolidinediones (TZDs), well-known insulin resistance ameliorating agents, have been used for treatment of type 2 diabetes since the 1980s. In the past several years, a compelling case has been made that peroxisome proliferator-activated receptor gamma (PPAR-γ) is the major functioning receptor for the common actions of TZDs in diabetes.1 PPAR-γ is a member of the nuclear hormone receptor superfamily that can be activated by many kinds of ligands, such as flavonoids, terpenoids, and unsaturated fatty acids,2−4 as well as TZDs themselves. Its activation results in increased insulin sensitivity partly by reversing lipotoxicityinduced insulin resistance.5 In addition, some α-glucosidase inhibitors, insulin secretagogues, and peptide analogues are also utilized as antidiabetic drugs in clinical practice. Therefore, screening of new α-glucosidase inhibitors and PPAR-γ agonists from herbal medicine seems a reasonable strategy for the discovery of antidiabetic agents or their lead compounds. Scoparia dulcis L. (Scrophulariaceae) is an edible perennial herb with serrated leaves and white flowers and is distributed widely in tropical and subtropical regions of Asia, Africa, and the Americas.6 In traditional and folk medicines, the fresh or dried forms of this plant are used for treatment of bronchitis, cancer, diabetes, diarrhea, fever, hemorrhoids, hepatosis, hypertension, insect bites, stomachache, tuberculosis, and © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 70% aqueous acetone extract of the dried aerial parts of S. dulcis was partitioned with petroleum ether and EtOAc, successively. The petroleum ether and EtOAc extracts then were subjected to column chromatography over silica gel, MCI gel CHP20P, Sephadex LH-20, YMC C18 gel, and semipreparative HPLC, to afford six new diterpenoids (1−6), a new flavonoid (10) (Figure 1), and 12 known compounds. The known compounds were identified as the diterpenoids (7S)-4Received: February 16, 2014 Published: June 23, 2014 1594

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and 21.3 (C-2″)] group in the molecule. In addition, the 13C and DEPT NMR spectra displayed 20 other carbon signals, represented by three methyls, seven methylenes, five methines, and five quaternary carbons. The above spectroscopic characteristics resembled those of scoparic acid A (8).10 HMBC correlations of H-18 (δH 1.35) with C-19 (δC 182.1), H-6 (δH 5.30) with C-7′ (δC 165.6), H-17a (δH 5.26) and H17b (δH 5.07) with C-7 (δC 75.2), C-8 (δC 140.4), and C-9 (δC 56.7), and H-14 (δH 5.43) with C-12 (δC 38.2), C-15 (δC 59.4), and C-16 (δC 16.7) confirmed that 1 is an analogue of scoparic acid A (8). The main difference observed was that 1 contains an additional acetyl group in its structure. The HMBC correlation of H-7 (δH 5.29) with C-1″ (δC 169.3) indicated the acetyl group to be at the C-7 position. In the NOESY spectrum (Figure 2), H-20 (δH 1.49) correlated with H-9 (δH 1.97), but not with H-5 (δH 2.88) or H-18 (δH 1.35), while H-5 (δH 2.88) correlated with H-6 (δH 5.30) and H-18 (δH 1.35), but not with H-7 (δH 5.29), and H-7 (δH 5.29) showed a weak correlation with H-9 (δH 1.97). This suggested that H-9 and the carboxyl group at the C-4 position are both β-oriented (for scoparic acid A, the carboxyl group at C-4 is α-oriented), while the acetoxy group at the C-7 position is α-oriented. Thus, the structure of 1 was elucidated as 4-epi-7α-O-acetylscoparic acid A. Compound 2 was obtained as a white, amorphous powder and assigned a molecular formula of C27H38O5 by HRESIMS at m/z 465.2623 ([M + Na]+, calcd for 465.2617) and the 13C NMR spectroscopic data. The 1H and 13C NMR data (Tables 1 and 2) were very similar to those of scopadiol,27 except for the loss of one methylene signal and the appearance of one additional oxygen-bearing methine signal at δC 75.7 (C-7) and δH 4.16 (1H, d, J = 2.4 Hz, H-7). HMBC correlations (Figure 1) of H-7 (δH 4.16) with C-5 (δC 35.9), C-6 (δC 74.7), C-9 (δC 56.6), and C-17 (δC 118.3) demonstrated that an additional hydroxy group is attached to the C-7 position in 2. In the NOESY spectrum (Figure 2), H-7 (δH 4.16) correlated with H17a (δH 5.03) and H-9 (δH 1.88), but not with H-5 (δH 2.47). In turn, H-20 (δH 1.43) correlated with H-9 (δH 1.88) and H19 (δH 0.89), indicating that the hydroxy group at the C-7 position and the hydroxymethyl group at the C-4 position are both α-oriented. Accordingly, the structure of 2 was established as 7α-hydroxyscopadiol. Compound 3 was isolated as a white, amorphous powder, with the molecular formula C29H38O8 deduced by HRESIMS at m/z 553.2187 ([M + K]+, calcd for 553.2204) and the 13C NMR spectroscopic data. Similar to 1, the 1H and 13C NMR spectroscopic characteristics (Tables 1 and 2) indicated that 3 is also a labdane-type diterpene with a benzoyl group and an acetyl group present. However, distinct from 1, the 1H and 13C NMR spectra revealed only the presence of a single CC double bond, but exhibited additional signals arising from an isolated methylene group at δH 2.71 (1H, d, J = 4.8 Hz, H-17a), 2.53 (1H, d, J = 4.8 Hz, H-17b), and δC 49.5 (C-17), respectively. These data suggested that 3 contains an epoxy group. HMBC correlations (Figure 1) of H-17a (δH 2.71) and H-17b (δH 2.53) with C-7 (δC 74.5), C-8 (56.7), and C-9 (52.9) supported this inference. In addition, HMBC correlations of H-18 (δH 1.38) with C-4 (δC 46.8), C-5 (δC 38.6), and C-19 (δC 182.6), H-6 (δH 5.31) with C-7′ (δC 165.9), H-7 (δH 4.47) with C-1″ (169.1), and H-14 (δH 5.42) with C-12 (δC 39.8), C-15 (δC 59.2), and C-16 (16.6) were observed. Thus, the planar structure of 3 could be elucidated as shown in Figure 1. The relative configurations of the carboxyl

Figure 1. Key HMBC correlations of compounds 1−6 and 10.

epi-7-hydroxyscoparic acid A (7), scoparic acid A (8),10 and 4epi-scopadulcic acid B (9),19 the flavonoids scutellarein (11),20 hispidulin (12),21 homoplantaginin (13),22 apigenin-8-C-α-Larabinopyranoside (14),23 apigenin (15), luteolin (16), acerosin (17),24 and pectolinarin (18),25 and the triterpenoid betulinic acid (19)26 by comparison of their spectroscopic data with those reported in the literature or with pure authentic samples isolated by our laboratory previously. Compound 1 was obtained as a white, amorphous powder. Its molecular formula of C29H38O7 was deduced by HRESIMS at m/z 537.2305 ([M + K]+, calcd for 537.2255) and 13C NMR spectroscopic data, indicating 11 degrees of unsaturation. The IR spectrum showed absorptions at 3434 (OH), 1745 (CO), 1721 (CO), and 1451 (benzene ring) cm−1. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) in combination with the HMBC spectrum (Figure 1), in which correlations of H-2′, 6′ (δH 8.03) with C-7′ (δC 165.6) and of H-2″ (δH 2.09) with C-1″ (δC 169.3) were observed, suggested the presence of both a benzoyl [δC 130.0 (C-1′), 129.8 (C-2′, 6′), 128.5 (C-3′, 5′), 133.2 (C-4′), and 165.6 (C-7′)] and an acetyl [δC 169.3 (C-1″) 1595

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Table 1. 1H NMR Spectroscopic Data of Compounds 1−7 in CDCl3 (600 MHz, δ in ppm, J values in Hz) position 1a 1b 2a 2b 3a 3b 5 6 7a 7b 8 9 11a 11b 12a 12b 14a 14b 15a 15b 16a 16b 17a 17b 18a 18b 19 20 2′, 6′ 3′, 5′ 4′ 2″

1 1.82 1.29 1.77 1.67 1.86 1.77 2.88 5.30 5.29

m brd (13.3) m m m m d (2.0) t (2.4) d (2.5)

2

3

1.76 1.19 1.75 1.58 1.73 1.21 2.47 5.51 4.16

m brd (13.9) m m m brd (13.6) d (2.2) t (2.4) d (2.4)

1.74 1.34 1.83 1.64 1.82

1.96 m 1.83 m 5.43 t (6.9)

1.88 1.91 1.77 2.11 1.85 5.43

brs dd (10.6, 4.1) m brt (11.1) m t (6.9)

1.05 1.93 1.80 2.02

4.19 d (6.9)

4.13 d (6.9)

4.19 d (6.7)

1.71 s

1.68 s

1.70 s

5.26 d (1.8) 5.07 d (1.8) 1.35 s

5.03 4.90 3.66 3.10 0.89 1.43 8.01 7.45 7.57

2.71 d (4.8) 2.53 d (4.8) 1.38 s

1.97 m 1.81 m

1.49 8.03 7.46 7.58 2.09

s d (7.4) t (7.4) t (7.4) s

d (2.1) d (2.1) d (11.4) d (11.4) s s d (7.4) t (7.4) t (7.4)

m brs m m m

2.85 d (2.0) 5.31 brs 4.47 brs

t (6.4) m m m

5.42 t (6.7)

1.73 8.10 7.46 7.57 2.14

s d (7.5) t (7.5) t (7.5) s

4 1.80 1.56 1.70 1.56 1.49 1.34 1.82 5.54 3.69

5

m m m m brd (12.2) td (13.0, 2.6) d (1.9) t (2.0) t (2.9)

6

1.68 m

m m m m m m d (2.0) t (2.4) t (3.1)

2.46 m

1.88 1.63 2.16 1.10 1.62 5.81 1.93 1.74 2.52

1.76 d (12.2) 1.49 brd (12.2)

1.88 d (12.3) 1.55 d (12.3)

1.77 d (12.1) 1.50 d (12.1)

3.01 2.16 1.84 1.62 2.64 2.21 1.11

2.28 2.16 1.81 1.71 2.22 1.77 1.12

3.04 2.16 1.87 1.65 2.77 2.24 1.12

dd (15.9, 12.8) dd (15.9, 5.9) m m m dd (13.3, 3.5) s

d (12.3) m brd (12.3) m d (2.5) q (2.8) dt (15.3, 3.3) m m

1.84 1.66 1.81 1.75 1.49 1.45 2.53 5.00 3.68

dd (16.1, 6.2) dd (16.1, 12.0) m m brt (12.5) m s

7 1.93 1.28 1.78 1.67 1.96 1.66 3.02 5.31 4.20

m brd (12.2) m m m m brs brs brs

1.95 2.10 1.74 2.29 1.85 5.51

m m m brt (10.8) m t (6.3)

2.45 m

dd (16.0, 12.8) dd (16.0, 5.9) m m m td (13.2, 3.8) s

4.19 d (6.3) 1.77 s

1.06 s

1.14 s

9.37 s

5.06 brs 4.93 brs 1.36 s

1.04 1.50 8.05 7.49 7.61

9.88 1.34 7.99 7.49 7.61

1.28 1.54 8.01 7.48 7.61

1.47 8.00 7.47 7.60

s s d (7.5) t (7.5) t (7.5)

s s dd (7.4, 1.2) t (7.4) t (7.4)

s s dd (7.5, 1.2) t (7.5) tt (7.5, 1.2)

s d (7.5) t (7.5) t (7.5)

H-6 (δH 5.54), suggesting that H-8 and H-19 are β-oriented, while H-5 and H-6 are α-oriented. The small coupling constant (J = 3.0 Hz) between H-7 (δH 3.69) and H-8 (δH 2.46) demonstrated that H-7 and H-8 are cis-oriented, with H-7 being β-oriented. Accordingly, the structure of 4 was determined as shown in Figure 1, and this compound has been named neodulcinol. Compound 5 was obtained as a white, amorphous powder. Its molecular formula was determined to be C27H34O4 by HRESIMS at m/z 461.2083 ([M + K]+, calcd for 461.2094) and confirmed by the 13C NMR spectroscopic data. In the same way as scopadulane-type diterpenoids, its 1H NMR spectrum exhibited signals due to a monosubstituted benzene ring [δH 7.99 (2H, dd, J = 7.4, 1.2 Hz, H-2′,6′), 7.61 (1H, t, J = 7.4 Hz, H-4′), and 7.49 (2H, d, J = 7.4 Hz, H-3′,5′)] and three methyl singlets [δH 1.34 (3H, s, H-20), 1.14 (3H, s, H-18), and 1.12 (3H, s, H-17)]. In addition, the 1H and 13C NMR spectra displayed aldehyde group signals [δH 9.88 (1H, s) and δC 205.9]. These spectroscopic characteristics (Tables 1 and 2) resembled those of dulcinodal.28 However, the 13C NMR spectrum of 5 showed a carbonyl signal (δC 213.0) instead of an oxygen-bearing carbon at δC 74.9 (C-13), indicating that the C-13 oxygen-bearing methine in dulcinodal is oxidized to a ketone in 5. This structure was confirmed by the HMBC correlations (Figure 1) of H-14a (δH 2.28), H-14b (δH 2.16), and H-17 (δH 1.12) with C-13 (δC 213.0). In the same manner as dulcinodal, the aldehyde group was proved to be β-oriented

group at the C-4 position, the C-8, C-17 epoxy group, and the acetoxy group at the C-7 position were determined to be α-, β-, and α-oriented, respectively, by the NOESY correlations of H20 (δH 1.73) with H-18 (δH 1.38) and H-9 (δH 1.05), H-9 (δH 1.05) with H-17b (δH 2.53), and H-17a (δH 2.71) with H-7 (δH 4.47) (Figure 2). Therefore, the structure of 3 was established as 7α-O-acetyl-8,17β-epoxyscoparic acid A. Compound 4 was obtained as a white, amorphous powder and assigned a molecular formula of C27H36O4 by HRESIMS at m/z 463.2264 ([M + K]+, calcd for 463.2251) and the 13C NMR spectroscopic data. The 1H NMR spectrum exhibited signals for four methyl singlets at δH 1.50 (3H, s, H-20), 1.11 (3H, s, H-17), 1.06 (3H, s, H-18), and 1.04 (3H, s, H-19) and a monosubstituted benzene ring at δH 8.05 (2H, d, J = 7.5 Hz, H2′,6′), 7.61 (1H, t, J = 7.5 Hz, H-4′), and 7.49 (2H, d, J = 7.5 Hz, H-3′,5′). The 13C and DEPT NMR spectra displayed in total 27 carbon signals, including two carbonyl carbons at δC 214.9 (C-13) and 166.5 (C-7′) and two oxygen-bearing methines at δC 74.7 (C-6) and 71.3 (C-7). These spectroscopic characteristics were almost the same as those of iso-dulcinol.19 The HMBC correlations (Figure 1) of H-6 (δH 5.54) with C-7 (δC 71.3) and C-7′ (δC 166.5), in combination with the 1H−1H COSY correlations from H-5 (δH 1.82) through H-6 (δH 5.54) to H-7 (δH 3.69), demonstrated that the hydroxy group is attached to the C-7 position. In the NOESY spectrum (Figure 2), H-20 (δH 1.50) correlated with H-8 (δH 2.46) and H-19 (δH 1.04), while H-18 (δH 1.06) correlated with H-5 (δH 1.82) and 1596

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Table 2. 13C NMR Spectroscopic Data of Compounds 1−7 in CDCl3 (150 MHz, δ in ppm) position

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′, 6′ 3′, 5′ 4′ 7′ 1″ 2″

38.3 18.2 40.2 46.8 38.9 73.9 75.2 140.4 56.7 38.2 26.0 38.2 140.0 123.1 59.4 16.7 121.9 19.7 182.1 25.7 130.0 129.8 128.5 133.2 165.6 169.3 21.3

38.7 18.5 38.0 38.6 35.9 74.7 75.7 145.2 56.6 38.4 26.6 38.7 140.6 123.1 59.4 16.6 118.3 70.8 20.9 26.0 130.3 129.7 128.5 133.1 166.4

38.3 17.6 40.1 46.8 38.6 73.0 74.5 56.7 52.9 38.7 27.3 39.8 139.2 123.8 59.2 16.6 49.5 19.6 182.6 25.6 130.3 130.0 128.5 133.2 165.9 169.1 21.1

35.1 18.8 44.3 33.8 44.8 74.7 71.3 40.0 52.6 39.1 46.6 52.3 214.9 38.4 36.7 25.0 19.9 33.6 25.1 21.3 130.2 129.7 128.6 133.3 166.5

34.0 18.7 35.8 49.5 53.3 69.6 34.6 35.4 52.2 39.1 45.6 52.2 213.0 42.4 36.6 23.2 19.6 24.4 205.9 20.8 129.9 129.6 128.7 133.4 165.8

34.4 17.3 35.3 49.2 37.2 76.5 70.9 40.0 52.6 38.2 46.6 52.3 214.5 38.4 36.6 25.1 19.8 205.5 18.0 22.0 129.8 129.7 128.7 133.4 166.1

37.8 18.4 38.8 47.5 39.4 78.3 75.4 144.4 57.4 38.4 26.9 38.8 141.3 123.0 59.5 16.4 118.1 19.3 184.0 25.3 129.7 129.9 128.6 133.5 167.3

COSY correlations from H-5 (δH 2.53) through H-6 (δH 5.00) to H-7 (δH 3.68), suggested that 6 has a hydroxy group at the C-7 position. In the NOESY spectrum (Figure 2), H-20 (δH 1.54) correlated with H-8 (δH 2.46) and H-19 (δH 1.28), but not with the aldehyde group (δH 9.37), which suggested that H8 (δH 2.45) and H-19 (δH 1.28) are β-oriented and the aldehyde group is α-oriented. Moreover, the small coupling constant (J = 3.1 Hz) between H-7 (δH 3.69) and H-8 (δH 2.45) demonstrated that H-7 and H-8 are cis-oriented. Accordingly, the structure of 6 was elucidated as 4-epi-7αhydroxydulcinodal-13-one. Compound 10 was obtained as a yellow, amorphous powder. Its molecular formula C32H30O14 was deduced by HRESIMS at m/z 637.1556 ([M − H]−, calcd for 637.1557) and the 13C NMR spectroscopic data. The IR spectrum indicated the presence of hydroxy (3393 cm−1), carbonyl (1691 cm−1), and phenyl (1655 and 1605 cm−1) groups. The 1H and 13C NMR data (Table 3) exhibited signals for three aromatic rings [an Table 3. 1H and 13C NMR Spectroscopic Data of Compound 10 in CD3OD (600 and 150 MHz for 1H and 13C NMR, respectively, δ in ppm, J values in Hz) positon 2 3 4 5 6 7 8 9

by the NOESY correlation (Figure 2) of the aldehyde proton (δH 9.88) with H-20 (δH 1.34). On the basis of the evidence obtained, the structure of 5 was determined as dulcinodal-13one. Compound 6 was isolated as a white, amorphous powder and designated with an elemental formula of C27H34O5 by HRESIMS at m/z 477.2058 ([M + K]+, calcd for 477.2043), confirmed by the 13C NMR spectroscopic data. The 1H and 13C NMR spectra (Tables 1 and 2) were very similar to those of 5. The only difference was that 6 exhibited two oxygen-bearing methine signals at δC 76.5 (C-6) and 70.9 (C-7), with 5 having only one oxygen-bearing methine resonance at δC 69.6 (C-6). The HMBC correlations (Figure 1) of H-6 (δH 5.00) with C-7 (δC 70.9) and C-7′ (δC 166.1), in combination with the 1H−1H

δH

6.15 brs 6.30 brs

δC

positon

158.4 135.5 179.3 162.9 100.0 166.1 94.9 158.3

OMe-3′ OMe-4′ 1″ 2″ 3″ 4″ 5″ 6″a 6″b

10 1′ 2′ 3′ 4′ 5′ 6′

7.83 d (1.6)

6.90 d (8.6) 7.65 dd (8.6, 1.6)

105.6 123.7 114.0 149.5 152.9 111.8 123.7

1‴ 2‴, 6‴ 3‴, 5‴ 4‴ 7‴ 8‴ 9‴

δH 3.88 s 3.76 s 5.35 d (7.4) 3.51 m 3.52 m 3.39 t (9.3) 3.53 m 4.31 dd (11.9, 2.3) 4.25 dd (11.9, 5.9) 7.31 d (8.5) 6.80 d (8.5) 7.39 d (15.9) 6.08 d (15.9)

δC 56.6 56.2 104.0 77.9 75.9 71.6 75.7 64.0

127.0 131.2 116.8 161.2 146.5 114.7 168.7

ABX spin system at δH 7.83 (1H, d, J = 1.6 Hz, H-2′), 7.65 (1H, dd, J = 8.6, 1.6 Hz, H-6′), and 6.90 (1H, d, J = 8.6 Hz, H-

Figure 2. Key NOESY correlations of compounds 1−7. 1597

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5′); an AB spin system at δH 7.31 (2H, d, J = 8.5 Hz, H-2‴ and 6‴) and 6.80 (2H, d, J = 8.5 Hz, H-3‴ and 5‴); and an AX spin system at δH 6.30 (1H, brs, H-8) and 6.15 (1H, brs, H-6)], a trans-CC bond [δH 7.39 (1H, d, J = 15.9 Hz, H-7‴) and 6.08 (1H, d, J = 15.9 Hz, H-8‴)], and a β-glucopyranosyl moiety [anomeric proton at δH 5.35 (1H, d, J = 7.4 Hz)], which was confirmed as D-glucose by acid hydrolysis. The HMBC correlations of H-6″ (δH 4.31 and δH 4.25) and H-7‴ (δH 7.39) with C-9‴ (δC 168.7) and H-2‴ (δH 7.31) with C-7‴ (δC 146.5) (Figure 1) indicated that 10 possesses a 6″-O-pcoumaroyl-β-D-glucopyranosyl moiety in its structure. Other spectroscopic characteristics in combination with the HMBC correlations of OMe-3′ (δH 3.88) with C-3′ (δC 149.5) and OMe-4′ (δH 3.76) with C-4′ (δC 152.9) suggested the presence of a 3′,4′-dimethoxyquercetin (dillenetin) unit.29 In addition, the HMBC correlation of H-1″ (δH 5.35) with C-3 (δC 135.5) was also observed. Accordingly, the structure of 10 was established as dillenetin 3-O-(6″-O-p-coumaroyl)-β-D-glucopyranoside. The 1H and 13C NMR data of the known compound (7S)-4epi-7-hydroxyscoparic acid A (7) (Tables 1 and 2) were completely assigned on the basis of the 2D NMR data inclusive of HSQC, HMBC, and 1H−1H COSY experiments, since there is no literature report on its NMR data to date. Its relative configuration was determined by a NOESY experiment (Figure 2), and the absolute configuration of C-7 was determined as 7S by the modified Mosher’s method (Figure 3). On comparison with the configuration of 7, the absolute configuration of C-7 in 1−4 and 6 was also determined to be S.

activities of these compounds except for 15 and 16 are being reported for the first time. Table 4. PPAR-γ Agonistic Effects of Compounds 1, 3, and 7−19 compound 1 3 7 8 9 10 11 12 13 14 15 16 17 18 19 rosiglitazoned

EC50 (μM)a 0.9 >100 >100 >100 >100 >100 16.4 7.3 >100 >100 24.9 2.3 4.3 >100 >100 0.03

± 0.3

± 1.1 ± 1.6

± 6.1 ± 0.4 ± 0.4

± 0.005

Emaxb 24.7 −c − − − − 3.4 11.3 − − 3.4 7.6 2.0 − − 11.8

± 4.2

± 1.0 ± 2.1

± 0.8 ± 2.0 ± 0.4

± 0.2

a

EC50 is the concentration required to produce half the maximum-fold activation, and values are represented as means ± SD based on four independent experiments. bEmax is the maximum-fold activation. cNot determined. dPositive control.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Krüss-P800-T polarimeter. UV spectra were recorded on a JASCO J-180 spectrometer. IR spectra were obtained with a Nicolet-380 spectrometer. 1D and 2D NMR spectra were run on a Bruker AVANCE-III instrument operating at 600 MHz for 1H and 150 MHz for 13C, respectively, with tetramethylsilane as internal standard. HRESIMS were obtained on a Waters UPLC Premior QTOF spectrometer, and UPLC-MS analysis was performed on an Acquity Waters Ultra-Performance liquid chromatographic system equipped with a Waters UPLC column (Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm) and a Micromass ZQ 2000 ESI mass spectrometer. GC analysis was run on a Thermo Trace GC Ultra spectrometer equipped with a TR-5MS GC column (60 m × 0.25 mm, i.d., 2.5 μm) and a H2 flame ionization detector. Column chromatography was performed with silica gel (200−300 mesh, Qingdao Makall Group Co., Ltd., Qingdao, People’s Republic of China), MCI gel CHP 20P (70−150 μm, Mitsubishi Chemical Co., Tokyo, Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and YMC gel ODS-A-HG (50 μm, YMC Co., Ltd., Kyoto, Japan). Semipreparative HPLC was conducted on an LC-3000 semipreparative gradient HPLC system (Beijing Tong Heng Innovation Technology Co., Ltd., Beijing, People’s Republic of China), equipped with a UV−vis detector and a YMC-Pack PRO C18 column (250 × 10 mm, i.d., 5 μm). Thin-layer chromatography (TLC) was carried out on HSGF254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, People’s Republic of China). Plant Material. The aerial parts of S. dulcis were collected and authenticated by Mr. Yi-Lin Zhu (Guangxi University of Chinese Medicine) in October 2012, in Nanning, Guangxi Zhuang Autonomous Region, People’s Republic of China. A voucher specimen (No. YGC-Nanning1210) was deposited in the herbarium of the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine. Extraction and Isolation. The dried aerial parts of S. dulcis (3.7 kg) were extracted three times (each 5 days) with aqueous acetone (15 L, water−acetone, 3:7) at room temperature. After removal of the organic solvent under reduced pressure, the residue (about 4 L) was

Figure 3. ΔδH values (δS − δR) of the MTPA esters of 7.

All isolated compounds except 2 and 4−6 were evaluated for their inhibitory effects against α-glucosidase and agonistic activities for PPAR-γ. 4-epi-Scopadulcic acid B (9) was found to exhibit an inhibitory effect against α-glucosidase, with an IC50 value of 14.6 ± 1.5 μM, more potent than the positive control, acarbose, for which the IC50 value was 3760 ± 157.2 μM. The known α-glucosidase inhibitors scutellarein (11), hispidulin (12), apigenin (15), luteolin (16), and betulinic acid (19) were also found to be active in this work.30−33 At variance from the present study, in some literature, 15 has shown a weak inhibitory effect against α-glucosidase, while 19 was inactive.31,34 This may be due to the origin of the α-glucosidase used. For example, luteolin (16) exhibits unequal inhibitory effects against yeast α-glucosidase and rat small intestinal αglucosidase.31 The flavonoids 11, 12, 15, 16, and acerosin (17) and the new diterpenoid 4-epi-7α-O-acetylscoparic acid A (1) showed potent agonistic activities for PPAR-γ, with EC50 values of 16.4 ± 1.1, 7.3 ± 1.6, 24.9 ± 6.1, 2.3 ± 0.4, 4.3 ± 0.4, and 0.9 ± 0.3 μM, respectively (Table 4). The PPAR-γ agonistic 1598

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(KBr) νmax 3393, 2936, 1691, 1655, 1605, 1514, 1452, 1361, 1273, 1255, 1206, 1171, 1085, 1021, 833 cm−1; 1H and 13C NMR data see Table 3; negative-ion HRESIMS m/z 637.1556 [M − H]− (calcd for C32H29O14, 637.1557). Acid Hydrolysis of Compound 10. Acid hydrolysis of compound 10 was carried out using a method described previously.35 By comparison with the retention times of authentic samples (tR, Dglucose = 49.55 min, and tR, L-glucose = 49.64 min), the configuration of the β-glucopyranosyl moiety in compound 10 was determined to be D (tR = 49.55 min). Determination of the Absolute Configuration of Compound 7. The absolute configuration of compound 7 was determined by the modified Mosher’s method.36,37 Compound 7 (about 1.5 mg) was dissolved in pyridine-d5 (0.5 mL) and transferred into a dried NMR tube, and then 20 μL of (R)-(−)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (R-MTPC; Sigma-Aldrich Co., St. Louis, MO, USA) was added. After reacting for 6 h at room temperature, the SOMTPA ester derivative of 7 (7s) was obtained. The same procedure was applied to 7 with S-MTPC (Sigma-Aldrich Co.) to yield the ROMTPA ester derivative, 7r. The 1H NMR data of 7s and 7r (see Supporting Information) were assigned on the basis of the HSQC and 1 H−1H COSY spectra. α-Glucosidase Inhibitory Activity Assay. The assay was carried out according to a reported method.38 α-Glucosidase (from Saccharomyces cerevisiae) was purchased from Sigma-Aldrich Co. and dissolved in potassium phosphate buffer (pH 6.8) with a concentration of 0.25 units/mL. Test samples and the positive control, acarbose, were also dissolved in potassium phosphate buffer (pH 6.8). PPAR-γ Agonistic Activity Assay. The Hek-293 cell line (ATCC, Manassas, VA, USA) was cultured in DMEM containing 10% FBS at 37 °C in a humidified 5% CO2 incubator and seeded into 96-well plates at a concentration of 1 × 104 cells/well. The expression plasmid pCMX-Gal-mPPARγ-LBD and the Gal4 reporter vector MH100 × 4TK-Luc were cotransfected with a transfection reagent, FuGENE 6 (Promega, Madison, WI, USA). Renilla was also transfected as the internal control. After transfection, cells were incubated with the test compounds at different concentrations for 24 h; then the cells were collected, and the luciferase activity was determined by a dualluciferase reporter assay system (Promega).

partitioned with petroleum ether (5 L × 4) and EtOAc (5 L × 4), successively, to give dried petroleum ether (29 g) and EtOAc (108 g) extracts, respectively. The petroleum ether extract (29 g) was subjected to column chromatography (CC) over silica gel (8.4 × 43 cm), eluted with a gradient mixture of petroleum ether−EtOAc (50:1 to 1:1), to afford five fractions (Frs. 1−5). Fr. 4 (2.8 g) was chromatographed with silica gel, eluted with CHCl3, to produce 19 (78 mg). Fr. 5 (2.1 g) was subjected to CC over Sephadex LH-20 (4.0 × 44 cm), eluted with CH2Cl2−MeOH (1:1), and then silica gel, eluted with petroleum ether−EtOAc (10:1 to 5:1), to afford two subfractions (Fr. 5-1 and Fr. 5-2). Fr. 5-1 was purified by semipreparative HPLC, eluted with MeOH−H2O (85:15, monitored at 238 nm), to yield 5 (4 mg), 6 (3 mg), and 9 (9 mg). Fr. 5-2 was subjected to semipreparative HPLC, eluted with MeOH−H2O (93:7, monitored at 238 nm), to give 4 (2 mg). The EtOAc extract (108 g) was subjected to CC over MCI gel CHP 20P (8.4 × 40 cm), eluted with a gradient mixture of MeOH−H2O (0:1 to 1:0), giving four fractions (Frs. A−D). Fr. C (19.6 g) was chromatographed sequentially with silica gel (5.4 × 31 cm), eluted with CH2Cl2−MeOH (30:1 to 3:1), Sephadex LH-20, eluted with MeOH, and YMC C18, eluted with MeOH−H2O (3:7 to 9:1), to afford 11 (26 mg), 13 (17 mg), 14 (9 mg), 16 (42 mg), and 18 (7 mg). Fr. D (10.5 g) was subjected to CC over MCI gel CHP 20P (5.4 × 35 cm), eluted with a gradient mixture of MeOH−H2O (6:4 to 1:0), to afford five subfractions (Fr. D1−Fr. D5). Further CC over silica gel, eluted with CH2Cl2−MeOH (40:1 to 10:1), and YMC C18, eluted with MeOH−H2O (6:4 to 1:0), gave 2 (1 mg), 3 (15 mg), 10 (14 mg), 12 (25 mg), 15 (452 mg), and 17 (8 mg) from Fr. D2−Fr. D4. CC with Sephadex LH-20, eluted with MeOH−H2O (8:2), and YMC C18, eluted with MeOH−H2O (7:3 to 1:0), yielded 1 (5 mg), 7 (612 mg), and 8 (16 mg) from Fr. D5. 4-epi-7α-O-Acetylscoparic acid A (1): white, amorphous powder; [α]20 D −4.3 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (4.07), 230 (4.11) nm; IR (KBr) νmax 3434, 2983, 2946, 2871, 1745, 1721, 1451, 1390, 1371, 1314, 1273, 1228, 1176, 1104, 1070, 1025, 982, 713 cm−1; 1 H and 13C NMR data see Tables 1 and 2; positive-ion HRESIMS m/z 537.2305 [M + K]+ (calcd for C29H38O7K, 537.2255). 7α-Hydroxyscopadiol (2): white, amorphous powder; [α]20 D +2.8 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 203 (4.13), 230 (4.09) nm; IR (KBr) νmax 3392, 2927, 2869, 1715, 1472, 1450, 1388, 1365, 1275, 1109, 1069, 1050, 1025, 712 cm−1; 1H and 13C NMR data see Tables 1 and 2; positive-ion HRESIMS m/z 465.2623 [M + Na]+ (calcd for C27H38O5Na, 465.2617). 7α-O-Acetyl-8,17β-epoxyscoparic acid A (3): white, amorphous powder; [α]20 D +6.9 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 203 (4.00), 230 (4.03) nm; IR (KBr) νmax 3434, 2987, 2945, 2872, 1748, 1720, 1451, 1392, 1371, 1275, 1226, 1177, 1108, 1027, 1012, 984, 714 cm−1; 1H and 13C NMR data see Tables 1 and 2; positive-ion HRESIMS m/z 553.2187 [M + K]+ (calcd for C29H38O8K, 553.2204). neo-Dulcinol (4): white, amorphous powder; [α]20 D +187.5 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 202 (4.11), 230 (4.23) nm; IR (KBr) νmax 3434, 2929, 2867, 1708, 1451, 1272, 1110, 1026, 739, 714 cm−1; 1H and 13C NMR data see Tables 1 and 2; positive-ion HRESIMS m/z 463.2264 [M + K]+ (calcd for C27H36O4K, 463.2251). Dulcinodal-13-one (5): white, amorphous powder; [α]20 D −16.7 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 203 (4.09), 230 (4.12) nm; IR (KBr) νmax 3361, 2924, 2852, 1712, 1659, 1632, 1467, 1274, 1106, 1025, 711 cm−1; 1H and 13C NMR data see Tables 1 and 2; positiveion HRESIMS m/z 461.2083 [M + K]+ (calcd for C27H34O4K, 461.2094). 4-epi-7α-Hydroxydulcinodal-13-one (6): white, amorphous powder; [α]20 D +7.5 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 202 (3.91), 230 (3.96) nm; IR (KBr) νmax 3361, 2924, 2852, 1712, 1659, 1632, 1467, 1274, 1106, 1025, 711 cm−1; 1H and 13C NMR data see Tables 1 and 2; positive-ion HRESIMS m/z 477.2058 [M + K]+ (calcd for C27H34O5K, 477.2043). Dillenetin 3-O-(6″-O-p-coumaroyl)-β-D-glucopyranoside (10): yellow, amorphous powder; [α]D20 −88.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.70), 269 (4.39), 315 (4.48) nm; IR

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ASSOCIATED CONTENT

S Supporting Information *

NMR and HRESIMS spectra of the new compounds, 1H NMR data of 7s and 7r, and key acquisition parameters of HMBC spectra for compounds 2 and 4−6 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-51322507. Fax: +86-21 51322505. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University (IRT1071).

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REFERENCES

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