Characterization and NO Inhibitory Activities of Chemical Constituents

Sep 4, 2014 - †State Key Laboratory of Medicinal Chemical Biology and College of ... College of Science, Northwest A&F University, Yangling 712100, ...
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Characterization and NO Inhibitory Activities of Chemical Constituents from an Edible Plant Petasites tatewakianus Meicheng Wang,†,‡ Qiang Zhang,§ Hao Wang,† Quanhui Ren,† Yihang Sun,† Chunfeng Xie,† Jing Xu,*,†,‡ Da-Qing Jin,⊥ Yasushi Ohizumi,∥ and Yuanqiang Guo*,†,‡ †

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, ‡Tianjin Key Laboratory of Molecular Drug Research, and ⊥School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China § College of Science, Northwest A&F University, Yangling 712100, People’s Republic of China ∥ Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan S Supporting Information *

ABSTRACT: Petasites tatewakianus is an edible plant belonging to the family Compositae. In our continuous search for NO inhibitors, which may be useful for the development of anti-inflammatory agents, the chemical constituents of the leaves of the edible plant P. tatewakianus were investigated. This phytochemical investigation led to the isolation of 3 new (1−3) and 10 known (4−13) sesquiterpenes and 2 other types of known compounds (14 and 15). Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analyses, and the absolute configurations of compounds 1 and 3 were confirmed by comparing their experimental CD spectra with those calculated by the time-dependent density functional theory (TDDFT) method. The following biological studies disclosed that these isolated compounds showed inhibitory activities on LPS-induced NO production in murine microglial BV-2 cells. The results of our phytochemical investigation, including two new bakkenolide sesquiterpenes (1 and 2), one new sesquiterpene with an unusual carbon skeleton (3), and the first report of compounds 5−7 and 10−15 from this species, further revealed the chemical composition of P. tatewakianus as an edible plant, and the biological studies implied that P. tatewakianus, containing bioactive substances with the inhibitory activities of NO production, was potentially beneficial to human health. KEYWORDS: edible plant, Petasites tatewakianus, sesquiterpenes, NO inhibitory activities, TDDFT CD calculations

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INTRODUCTION Petasites tatewakianus Kitam, belonging to the family Compositae, is a perennial herbaceous plant mainly distributed in northeast China, Japan, Korea, Far East region of Russia, and Sakhalin Island.1 This plant has been consumed as a wild vegetable in northeast China and Japan and cultivated as a popular leafy vegetable in recent years.2,3 Previous phytochemical investigations on P. tatewakianus led to the isolation and identification of several sesquiterpenes, which showed neuroprotective and weak cytotoxic activities.2−4 However, to the best of our knowledge, though several sesquiterpenes were isolated and their bioactivities were investigated, phytochemical and pharmacological studies on the edible plant P. tatewakianus are limited, and there have been no reports on the NO inhibitory effects of the chemical constituents or the extract from this plant. In our search for pharmacologically active substances in medicinal plants,5,6 much attention has been given to the occurrence of compounds with inhibitory effects of NO production, since NO plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent.7 As a continuation of our search for inhibitors of NO production from plants, the chemical constituents of the leaves of the edible plant P. tatewakianus were investigated. This phytochemical investigation led to the isolation of 3 new sesquiterpenes (1−3), named tatewakipenes A−C (1−3), and 12 known compound © 2014 American Chemical Society

(4−15) (Figure 1). Their structures were elucidated on the basis of the spectroscopic data analyses (IR, ESIMS, HR-ESIMS, and 1D and 2D NMR) and the time-dependent density functional theory (TDDFT) CD calculations. This paper herein describes the isolation, structure elucidation, and NO inhibitory activities of chemical constituents from the edible plant P. tatewakianus.

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MATERIALS AND METHODS

General. The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, U.S.). The IR spectra were taken on a Bruker Tensor 27 FT-IR spectrometer with KBr discs (Bruker, Germany). ECD spectra were obtained on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., U.K.). The ESIMS spectra were acquired on an LCQ-Advantage mass spectrometer (Finnigan Co. Ltd., U.S.). HRESIMS spectra were recorded by IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA). 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (400 MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. HPLC separations were performed on a CXTH system, equipped with a UV3000 detector at 210 nm (Beijing Chuangxintongheng Instruments Co. Ltd., China) and a YMC-pack ODS-AM (250 × 20 mm) column (YMC Co. Ltd., Japan). Silica gel was Received: Revised: Accepted: Published: 9362

July 19, 2014 August 30, 2014 September 4, 2014 September 4, 2014 dx.doi.org/10.1021/jf5034224 | J. Agric. Food Chem. 2014, 62, 9362−9367

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murine microglial BV-2 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). Plant Material. The leaves of P. tatewakianus were collected in July 2013 from Changbai Mountain area, Jilin province, China. A voucher specimen (No. 20130716) was identified by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China) and deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China. Extraction and Isolation. The air-dried leaves of P. tatewakianus (7.9 kg) were powdered and extracted with MeOH (3 × 48 L) under reflux. The organic solvent was evaporated to obtain a crude extract (910 g). The crude extract was suspended in H2O (0.9 L) and partitioned with EtOAc (3 × 0.9 L). The EtOAc soluble part (277 g) was subjected to a silica gel column chromatography (⌀ 9 × 70 cm), using a gradient of acetone in petroleum ether (1%−30%), to give seven fractions (F1−F7) based on TLC analyses. F1 was fractionated by middle pressure liquid chromatography (MPLC) over octadecylsilyl (ODS) eluting with a step gradient from 75% to 95% MeOH in H2O to give three subfractions (F1−1−F1−3). F1−3 was further purified by preparative HPLC (YMCpack ODS-AM, 250 × 20 mm, 84% MeOH in H2O) to produce compounds 1 (tR = 19 min, 11.3 mg), 2 (tR = 26 min, 12.4 mg), 5 (tR = 17 min, 15.6 mg), 10 (tR = 31 min, 11.6 mg), 11 (tR = 36 min, 12.1 mg), 12 (tR = 24 min, 13.2 mg), and 13 (tR = 29 min, 43.7 mg). By the above HPLC system, the purification of F1−1 (75% MeOH in H2O) afforded compounds 4 (tR = 34 min, 25.5 mg) and 7 (tR = 31 min, 24.4 mg). Fraction F3 was subjected to the same MPLC (65−90% MeOH in H2O)

Figure 1. Structures of compounds 1−15 from P. tatewakianus. used for column chromatography (200−300 mesh, Qingdao Marine Chemical Group Co. Ltd., China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Chemical Reagent Company, China. Biological reagents were from Sigma Company. The

Table 1. NMR Data of Compounds 1−4 (δ ppm in CDCl3 and J in Hz)a 1 13

C

70.4

β 5.06 dt (11.8, 4.8)

69.4

β 5.10 dt (11.6, 4.8)

25.6

2

26.4

26.8

29.5

4 5 6

35.3 43.3 45.8

α 1.81 m β 1.70 m α 1.68 m β 1.37 m α 1.58 m

20.1

3

α 1.84 m β 1.70 m α 1.67 m β 1.36 m α 1.59 m

α 1.70 m β 1.75 m α 1.45 m β 1.60 m α 2.20 m β 1.33 m α 1.55 m

7 8 9

54.8 177.8 80.8

10 11 12

51.9 147.6 70.6

13

108.4

14 15 OR-1

15.5 19.6 176.3 34.0 18.7 19.1

OR-9

1 2 3 4 5 6 1 2 3 4 5

169.8 21.2

H

2.23 d (14.3) 1.95 d (14.3)

α 5.80 d (11.2) β 2.67 dd (11.2, 4.8) 4.71 d (13.0) 4.61 d (13.0) 5.22 s 5.18 s 0.90 d (6.7) 1.10 s 2.33 set (7.1) 1.11 d (7.1) 1.09 d (7.1)

2.06 s

1

C

29.5 35.3 43.2 45.7

13

4

1

C

13

3 1

position

a

2 1

H

29.0 35.0 40.0 27.2

2.24 d (14.3) 1.96 d (14.3)

54.8 177.6 80.8

125.7 175.6 65.5

α 5.77 d (11.2) β 2.74 dd (11.2, 4.8)

51.6 147.7 70.6

4.70 d (13.2) 4.66 d (13.2) 5.21 s 5.18 s 0.91 d (6.6) 1.12 s

108.2 15.5 19.6 165.7 114.1 162.1 33.6 11.7 18.8 169.9 21.1

41.0 159.0 72.5

H

2.75 d (13.6) 2.09 d (13.6)

4.44 dd (10.8, 3.7) 4.03 dd (10.8, 8.9) β 1.86 m

13.8

4.67 d (17.2) 4.62 d (17.2) 2.04 s

14.9 24.6

0.90 d (7.2) 0.94 s

5.44 s 2.12 q (7.4) 1.03 t (7.4) 2.14 s 2.03 s

168.3 128.0 137.8 15.8 20.7

13

C

70.3 26.7 29.4 35.1 43.2 45.6 54.9 177.4 80.7 51.4 147.6 70.5 108.3 15.5 19.5 167.2 128.1 136.7 15.5 20.3 169.9 20.9

6.06 qq (7.2, 1.5) 2.00 dq (7.2, 1.5) 1.90 quint (1.5)

The assignments of NMR data are based on 1H, 13C, DEPT, 1H−1H COSY, HMQC, and HMBC NMR experiments. 9363

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to obtain four subfractions F3−1−F3−4, and the following purification of F3−2 by the same HPLC system (94% MeOH in H2O) yielded compound 3 (tR = 29 min, 4.5 mg), 8 (tR = 17 min, 14.7 mg), 9 (tR = 19 min, 17.1 mg), and 15 (tR = 26 min, 5.4 mg). Compounds 6 (tR = 26 min, 14.7 mg) and 14 (tR = 22 min, 15.4 mg) were isolated from F5−3 (87% MeOH in H2O), which was obtained from F5 by the above MPLC. Tatewakipene A (1). Colorless oil; [α]27 D : −75.7 (c 0.3, CH2Cl2); CD (CH3CN): 201 (Δε −29.30) nm; IR (KBr) νmax 2964, 2933, 1780, 1733, 1635, 1265, 1154, and 1045 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1; ESIMS m/z 401 [M + Na]+; HR-ESIMS m/z 401.1935 [M + Na]+, calcd. for C21H30NaO6, 401.1940. Tatewakipene B (2). Colorless oil; [α]27 D : −129.6 (c 0.2, CH2Cl2); IR (KBr) νmax 2964, 2926, 1780, 1747, 1696, 1650, 1233, 1145, and 1046 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1; ESIMS m/z 427 [M + Na]+; HR-ESIMS m/z 427.2095 [M + Na]+, calcd. for C23H32NaO6, 427.2097. Tatewakipene C (3). Colorless oil; [α]27 D : +10.9 (c 0.1, CH2Cl2); CD (CH3CN): 205 (Δε −0.73), 227 (Δε +2.84) nm; IR (KBr) νmax 2925, 2857, 1750, 1637, and 1276 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1; ESIMS m/z 357 [M + Na]+; HR-ESIMS m/z 357.2032 [M + Na]+, calcd. for C20H30NaO4, 357.2042. Computation. For those compounds needed to determine the absolute configurations, their calculated ECD spectra were performed using TDDFT method as follows. A preliminary conformational search was carried out in Conflex 6.7 using MMFF94 force field.8 Conformers within 6 kcal/mol were saved and further optimized on B3LYP/631+G(d,p) level in the Gaussian 09 package.9 The stable conformers with populations greater than 1% were submitted to ECD calculation by the TDDFT [B3LYP/6-31+G(d,p)] method with CPCM model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half bandwidth of 0.3 eV. The final ECD curve were generated according to the Boltzmann distribution of each conformer. Bioassay for NO Production. Murine microglial BV-2 cells were cultured at 37 °C in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/mL penicillin/streptomycin under a water-saturated atmosphere of 95% air and 5% CO2. The cells were seeded in 96-well culture plates (5 × 104 cells/well) and allowed to adhere for 24 h at 37 °C. The cells were incubated for 20 h with or without 0.15 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO) in the absence or presence of the test compounds. 2-Methyl-2-thiopseudourea, sulfate (SMT) was used as a positive control. As a parameter of NO synthesis, the nitrite concentration was measured by the Griess reaction using the supernatant of the BV-2 cells. Briefly, 50 μL of the cell culture supernatant was reacted with 50 μL of Griess reagent [1:1 mixture of 0.1% N-(1-naphtyl)ethylenediamine in H2O and 1% sulfanilamide in 5% phosphoric acid] in a 96-well plate, and the absorbance was read with a microplate reader (Thermo Fisher Scientific Inc. America) at 550 nm. The experiment was performed three times, and the IC50 values for the inhibition of NO production were determined on the basis of linear or nonlinear regression analysis of the concentration−response data curves.

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= 11.2 Hz, H-9)]. The 13C NMR spectrum of 1 showed 21 carbon resonances. From the 1H and 13C NMR spectra, one acetyl group and one isobutyryl group were evident from the above methyl signals and corresponding carbon resonances (δC 169.8 and 21.2 and 176.3, 34.0, 18.7, and 19.1). Apart from the above six signals for two acyl groups, there are additional 15 resonances exhibited for the parent skeleton in the 13C NMR spectrum, which comprised two methyls [δC 15.5 (C-14) and 19.6 (C-15)], five methylenes [δC 26.4 (C-2), 29.5 (C-3), 45.8 (C-6), 70.6 (C-12), and 108.4 (C-13)], four methines [δC 70.4 (C-1), 35.3 (C-4), 80.8 (C-9), and 51.9 (C-10)], and four quaternary carbons [δC 43.3 (C-5), 54.8 (C-7), 177.8 (C-8), and 147.6 (C-11)] based on the DEPT and HMQC spectra. The above spectroscopic features and the 15 skeletal carbons displayed in the 13C NMR spectrum suggested that compound 1 should be a sesquiterpene with two acyloxy groups (one acetoxy and one isobutyryloxy group).10−16 On comparing the chemical shifts from C-1 to C-15 of compound 1 with those of bakkenolide sesquiterpenes reported in the literature,14−16 the presence of the same parent skeleton of bakkenolide-type was obvious. The following experiments and interpretation of HMQC and HMBC spectra corroborated the above deductions, which verified that compound 1 was a bakkenolide-type sesquiterpene with two acyloxy groups. The positions of the acyloxy groups were determined via interpretation of the HMBC data. The HMBC correlation of the proton signal at δH 5.06 (H1) with the carbonyl signal at δC 176.3 (CO of the isobutyryloxy group), indicated the presence of the isobutyryloxy group at C-1. Similarly, the long-range coupling of the proton signal at δH 5.80 (H-9) with the carbonyl carbon signal at δC 169.8 demonstrated that the acetoxy group was located at C-9. By further analyzing the HMQC, HMBC, and 1H−1H COSY spectra (Figure 2), all the proton and carbon signals were assigned unambiguously, which resulted in the establishment of the planar structure for 1.

Figure 2. Selected HMBC and 1H−1H COSY correlations of compounds 1 and 3.

The relative configuration of compound 1 was established based on the NOESY spectrum and Chem3D modeling (Figure 3). NOESY correlations observed for H-10/H3-15, H3-15/H3-

RESULTS AND DISCUSSION

Compound 1 was obtained as a colorless oil. Its HR-ESIMS provided the molecular formula, C21H30O6, through the presence of a peak at m/z 401.1935 [M + Na]+ (calcd. for C21H30NaO6, 401.1940). The molecular formula indicated seven unsaturation degrees for 1. The 1H NMR spectrum of 1 exhibited five methyl signals [δH 0.90 (3H, d, J = 6.7 Hz, H3-14), 1.10 (3H, s, H3-15), 2.06 (3H, s, COCH3-9), and 1.11 and 1.09 (each 3H, d, J = 7.1 Hz, COCH(CH3)2-1)] (Table 1), two olefinic protons of a terminal double bond [δH 5.22 and 5.18 (each 1H, s, H2-13)], a set of oxygenated methylene protons [δH 4.61 and 4.71 (each 1H, d, J = 13.0 Hz, H2-12)], and two oxygenated methine protons [δH 5.06 (1H, dt, J = 11.8, 4.8 Hz, H-1) and 5.80 (1H, d, J

Figure 3. Key NOESY correlations of compounds 1 and 3. 9364

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carbon signals, the following HMQC and HMBC experiments were performed. By interpretation of the 2D NMR spectra, the same bakkenolide-type sesquiterpene skeleton as that in 1 was defined, and the acyloxy groups, especially the methylsenecioyloxy group, were validated. The HMBC correlations of the proton signals at δH 5.10 (H-1) and 5.77 (H-9) with the carbonyl carbons at δC 165.7 and 169.9, respectively, demonstrated that the methylsenecioyloxy group was attached at C-1, and the acetoxy group was at C-9. Further analyses of the HMQC, HMBC, and 1H−1H COSY spectroscopic data led to the assignments of all the proton and carbon signals. Thus, the planar structure with a bakkenolide-type sesquiterpene skeleton for 2 was disclosed. The same relative configuration was inferred for compounds 2 and 1 on the basis of comparison of their NOESY spectra. Compounds 2 and 1 had the same parent skeleton and there were no additional chiral carbons in compound 2, so, from the biosynthetic point of view, the absolute configuration of compound 2 was also defined as 1S, 4S, 5R, 7R, 9R, 10R, which was also supported by the identical laeotropic optical rotation of two compounds. Therefore, the structure of compound 2 was characterized and named tatewakipene B. Compound 3 possessed a molecular formula C20H30O4 as determined by the HR-ESIMS (m/z 357.2032 [M + Na]+ calcd. for C20H30NaO4, 357.2042), which implied six unsaturation degrees. The 1H NMR spectrum of compound 3 exhibited five methyl signals [δH 2.04 (3H, s, H3-13), 0.90 (3H, d, J = 7.2 Hz, H3-14), 0.94 (3H, s, H3-15), 2.00 (3H, dq, J = 7.2, 1.5 Hz, COCH(CH3)CH(CH3)-9), and 1.90 (3H, quint, J = 1.5 Hz, COCH(CH3)CH(CH3)-9], one olefinic proton [δH 6.06 (1H, qq, J = 7.2, 1.5 Hz, COCH(CH3)CH(CH3)-9)], and two sets of oxygenated methylene protons [δH 4.44 (1H, dd, J = 10.8, 3.7 Hz, H-9a) and 4.03 (1H, dd, J = 10.8, 8.9 Hz, H-9b), and 4.62 and 4.67 (each 1H, d, J = 17.2, H2-12)] (Table 1). The 13C NMR spectrum of 3 showed 20 carbon resonances. From the 1H and 13 C NMR spectra, one angeloyl group in compound 3 was deduced from the above characteristic protons and the corresponding carbons (δC 168.3, 128.0, 137.8, 15.8, and 20.7). Apart from the five carbon signals for the angeloyl group, there were 15 residual resonances displayed in the 13C NMR spectrum, which were classified into three methyls [δC 13.8 (C-13), 14.9 (C-14), and 24.6 (C-15)], six methylenes [δC 25.6 (C-1), 20.1 (C-2), 29.0 (C-3), 27.2 (C-6), 65.5 (C-9), and 72.5 (C-12)], two methines [δC 35.0 (C-4) and 41.0 (C-10)], and four quaternary carbons [δC 40.0 (C-5), 125.7 (C-7), 175.6 (C-8), and 159.0 (C11)] based on the DEPT and HMQC spectra. According to the unsaturation degrees, the aforementioned spectroscopic features, and the sesquiterpenes isolated from the genus Petaistes,11−17 compound 3 might be a bicyclic sesquiterpene with an angeloyloxy group. In order to confirm the above assumption and elucidate the structure, the following HMBC and 1H−1H COSY experiments were performed. By interpretation of the HMBC spectrum, one ring, the α,β-saturated γ-lactone ring, was validated based on the long-range correlations of H2-12 to C-7, C-8, C-11, and C-13, and H3-13 to C-7, C-11, and C-12. The other ring was also deduced and defined from the HMBC correlations of H-10 to C-1, C-2, C-4, and C-5, H3-14 to C-3, C4, and C-5, and H3-15 to C-4, C-5, and C-10. The above two rings, one α,β-saturated γ-lactone ring and one six-membered ring, were connected through the methylene unit of C-6, which was demonstrated by the long-range couplings of the methylene protons at δH 2.75 and 2.09 (each 1H, d, J = 13.6 Hz, H2-6) to the carbons of two rings at δC 35.0 (C-4), 40.0 (C-5), 41.0 (C-10), 125.7 (C-7), 175.6 (C-8), and 159.0 (C-11). Accordingly, the

14, H-1/H-10, H-9/H-4, H-4/H-2α, and H-9/H-13a, but not for H-9/H3-15, H-9/H-1, and H-4/H3-15, and the Chem3D modeling suggested a conformation for compound 1 as depicted in Figure 3, where the six-membered ring A and the fivemembered ring B were cis-fused and existed in a chair and envelope conformation, and the lactone ring C was perpendicular to the five-membered ring B. The conformations of the three rings and the NOESY correlations required that the C-15 methyl group was β-axially oriented, the C-14 methyl group and the proton H-10 were β-equatorially oriented, the C-1 isobutyryloxy group was α-equatorially oriented, and the C-9 acetoxy group was in a β-position. Thus, the relative configuration of 1 was assigned as shown in Figure 3. The absolute configuration of 1 was tentatively established by comparison of its experimental CD spectrum with those calculated by the TDDFT method. The optimized geometries were obtained by systematic conformational search with the MMFF94 force field and further optimized on B3LYP/6-31+G(d,p) level by Gaussian 09 package.8,9 Then the ECD spectra were calculated at the CAM-B3LYP/SVP level with CPCM model in acetonitrile. The calculated ECD spectrum of 1a (Figure 4) matches the experimental result very well. Thus,

Figure 4. Calculated ECD spectra of 1a (1S, 4S, 5R, 7R, 9R, 10R)- and 1b (1R, 4R, 5S, 7S, 9S, 10S)-isomers and the experimental ECD spectrum of 1 in acetonitrile.

the absolute configuration of 1 was determined as 1S, 4S, 5R, 7R, 9R, 10R. The structure of 1 was therefore characterized and named tatewakipene A. Compound 2, a colorless oil, possessed a molecular formula of C23H32O6 as determined from the HR-ESIMS (427.2095 [M + Na]+, calcd. for C23H32NaO6, 427.2097). From the 1H NMR spectrum of 2, four methyl groups, six olefinic protons, and two oxygenated methine protons were displayed (Table 1). The 13C NMR spectrum of 2 showed 23 carbon resonances. From the 1H and 13C NMR spectra, one acetoxy was apparent from the methyl singlet and the corresponding carbon signals. In addition, one methylsenecioyloxy group was also deduced and defined from the observation of the following carbon signals (δC 165.7, 114.1, 162.1, 33.6, 11.7, and 18.8) and the corresponding proton signals (Table 1) based on the reported sesquiteprnes with acyloxy groups from the genus Patesites in the literature.14−17 Apart from the above 7 resonances for the substituent groups, the remaining 15 carbon resonances displayed in the 13C NMR spectrum constitute a bakkenolide-type sesquiterpene skeleton, which was the same as that of compound 1.17 In order to confirm the above deductions and achieve the assignments of the proton and 9365

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three methyl carbons at δC 13.8, 14.9, and 24.6 and the remaining oxygenated methylene carbon at δC 65.5 were assigned to C-13, C-14, C-15, and C-9, respectively, based on the HMBC correlations of the methyl and methylene protons to the corresponding carbons of the two rings. The skeleton of bicyclic sesquiterpene for 3 was therefore elucidated, which possessed an unusual skeleton of 7,9-seco-bakkenolide. After defining the 7,9seco-bakkenolide skeleton, the HMBC correlations of the methylene protons H2-9 to the carbonyl carbon at δC 168.3 indicated that the angeloyloxy group was attached at C-9. By further analyzing the HMQC, HMBC, and 1H−1H COSY spectra (Figure 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of 7,9-secobakkenolide sesquiterpene for compound 3 was established. The relative configuration of compound 3 was established based on the NOESY spectrum. NOESY correlations observed for H-10/H3-15, H3-15/H3-14, H-1β/H-10, H-9a/H-4, H-4/H2α, H-3β/H3-15, H-3β/H3-14, and H-1β/H-3β (Figure 3), but not for H-9a(b)/H3-15, and the Chem3D modeling revealed that the six-membered ring had the same conformation as those in compounds 1 and 2, where the H-10 proton was β-equatorially oriented, the C-14 methyl group was in a β-position with an equatorial orientation, and the C-15 methyl group was in a βposition with an axial orientation. Thus, the relative configuration of 3 was assigned as depicted in Figure 3. After the relative configuration was determined, the absolute configuration of 3, a new sesquiterpene with an unusual carbon skeleton, was also considered to elucidate. As in the case of compound 1, the calculated CD spectra were performed using time-dependent density functional theory (TDDFT) method, and the experimental CD spectrum was recorded (Figure 5). By comparison of

one (12),22 petasipaline B (13),23 5-hydroxy-3,7,4′-trimethoxyflavone (14),24 and pregn-4-ene-3,20-dione (15).25 Studies on the role of NO in the process of inflammation have demonstrated that reagents with NO inhibitory activities may be useful for the development of anti-inflammatory agents.7 In order to search and obtain the bioactive substances inhibiting NO production as candidates of anti-inflammatory agents, these compounds isolated from P. tatewakianus were evaluated for their inhibitory effects on LPS-induced NO production in murine microglial BV-2 cells by the Griess reaction.26 2-Methyl-2thiopseudourea sulfate (SMT) was used as a positive control (IC50 3.6 μM). All the evaluated isolates exhibited inhibitory effects on LPS-induced NO production and the inhibitory effects are shown in Table 2. Compounds 10, 12, and 14 inhibited LPSTable 2. IC50 Values of Compounds 1, 2, and 4−15 Inhibiting NO Production in BV-2 Cells compound

IC50 (μM)

compound

IC50 (μM)

1 2 4 5 6 7 8 9

>100 >100 65.8 ± 2.5 >100 >100 >100 84.1 ± 2.5 55.1 ± 4.1

10 11 12 13 14 15 SMTa

38.7 ± 3.1 64.4 ± 3.1 47.0 ± 2.0 62.4 ± 3.2 45.8 ± 3.6 52.0 ± 1.5 3.6 ± 0.2

a

SMT (2-methyl-2-thiopseudourea, sulfate) was used as a positive control. Data are presented based on three experiments. Compound 3 was not assayed for the inhibitory effects because of inadequate amount.

induced NO production dose-dependently with IC50 values of 38.7, 47.0, and 45.8 μM, respectively. Compounds 4, 8, 9, 11, 13, and 15 showed moderate inhibitory effects and compounds 1, 2, and 5−7 showed weak activities (IC50 values >100 μM). Compound 3 was not investigated for the NO inhibitory effects because of inadequate amount. Among these bioactive compounds, eight compounds (1, 2, and 4−9) had the same parent skeleton, and the analysis of the preliminary structure activity relationship (SAR) inhibiting NO production revealed that the presence of the C-1 cis-methylthioacryloyloxy group seems to increase NO inhibitory effects markedly. MTT assay indicated that all the assayed compounds had no significant cytotoxicity to the BV-2 cells at their effective concentrations for the inhibition of NO production (data not shown). In summary, 3 new sesquiterpenes (1−3), including one with an unusual carbon skeleton (3), and 12 known compounds (4− 15) were successfully isolated from the leaves of P. tatewakianus. Their structures were elucidated on the basis of the spectroscopic data analyses and the TDDFT CD calculations. Biological studies disclosed that all of the evaluated isolates exhibited inhibitory effects on LPS-induced NO production and compound 10 exerted the most inhibition against LPS-induced NO production in BV-2 cells. The results of our chemical investigation, including two new bakkenolide sesquiterpenes (1 and 2), one new sesquiterpene with an unusual carbon skeleton (3), and the first report of compounds 5−7 and 10−15 from this species, further revealed the chemical composition of P. tatewakianus as an edible plant, and the biological studies implied that P. tatewakianus, containing bioactive substances with the inhibitory activities of NO production, may be potentially beneficial to human health.

Figure 5. Calculated ECD spectra of 3a (4S, 5R, 10S)- and 3b (4R, 5S, 10R)-isomers and the experimental ECD spectrum of 3 in acetonitrile.

its experimental and calculated CD spectra, the absolute configuration of 3 was established as 4S, 5R, 10S. Thus, compound 3 was characterized as a new sesquiterpnene with an unusual carbon skeleton and named tatewakipene C. Based on the spectroscopic analyses and the comparison with the literature, the known compounds were identified as bakkenolide B (4),18 japonipene A (5),17 bakkenolide III (6),14 valerilactone A (7),18 petatewalide A (8),3 bakkenolide D (9),19 14-acetoxy-7β-senecioyloxy-notonipetranone (10),20 tussilagone (11),21 [1S-[1α(R*), 3aβ, 7α, 7aα] ]-octahydro-1(1-hydroxyethyl)-4-methylene-7-(1-methylethyl)-2H-inden-29366

dx.doi.org/10.1021/jf5034224 | J. Agric. Food Chem. 2014, 62, 9362−9367

Journal of Agricultural and Food Chemistry

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Article

(17) Wang, S.; Jin, D. Q.; Xie, C.; Wang, H.; Wang, M.; Xu, J.; Guo, Y. Isolation, characterization, and neuroprotective activities of sesquiterpenes from Petasites japonicus. Food Chem. 2013, 141, 2075−2082. (18) Xu, J.; Yang, B.; Guo, Y.; Jin, D. Q.; Guo, P.; Liu, C.; Hou, W.; Zhang, T.; Gui, L.; Sun, Z. Neuroprotective bakkenolides from the roots of Valeriana jatamansi. Fitoterapia 2011, 82, 849−853. (19) Abe, N.; Onoda, R.; Shirahata, K.; Kato, T.; Woods, M. C.; Kitahara, Y.; Ro, K.; Kurihara, T. Structures of bakkenolides- B, - C, and D as determined by the use of a nuclear Overhauser effect. Tetrahedron Lett. 1968, 16, 1993−1997. (20) Kikuchi, M.; Suzuki, N. Studies on the constituents of Tussilago farfara L. II. Structures of new sesquiterpenoids isolated from the flower buds. Chem. Pharm. Bull. 1992, 40, 2753−2755. (21) Park, H. R.; Yoo, M. Y.; Seo, J. H.; Kim, N. Y.; Kang, J. Y.; Cui, L.; Lee, C. H.; Lee, H. S. Sesquiterpenoids isolated from the flower buds of Tussilago farfara L. inhibit diacylglycerol acyltransferase. J. Agric. Food Chem. 2008, 56, 10493−10497. (22) Pedro, J. N.; J. Roberto, V.; Mirna, R. G.; Luisa, U. R.; Juan, D. H. Minor oplopanes from Senecio mexicanus. Phytochemistry 1989, 28, 2397−2401. (23) Koji, H. Oplopane sesquiterpenes from Petasites palmatus. Phytochemistry 1989, 28, 3373−3376. (24) Tong, X.; Cheng, Y. Chemical constituents from Acorus tatarinowii. Tianran Chanwu Yanjiu Yu Kaifa 2011, 23, 404−409. (25) Qi, S.; Su, G.; Zhang, S. Pregnane steroids in Gorgonian Subergorgia suberosa from the South China Sea. Tianran Chanwu Yanjiu Yu Kaifa 2008, 20, 1−4. (26) Schmidt, H. H. H. W.; Kelm, M. Determination of nitrite and nitrate by the Griess reaction. In Methods in Nitric Oxide Research; Feelisch, M., Stamler, J. S., Eds.; John Wiley and Sons: West Sussex, U.K., 1996; pp 491−497.

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR and HR-ESIMS spectra of compounds 1−3. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel./fax: +86-22-23502595. E-mail address: xujing611@ nankai.edu.cn *Tel./fax: +86-22-23502595. E-mail address: victgyq@nankai. edu.cn. Funding

This work was supported by the National Natural Science Foundation of China (No. 21372125). Notes

The authors declare no competing financial interest.

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