Sesquiterpenes Inhibiting the Microglial Activation from Laurus nobilis

May 6, 2014 - The leaves of the plant Laurus nobilis belonging to the family Lauraceae, namely, bay leaves, have been used as a popular spice, and the...
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Sesquiterpenes Inhibiting the Microglial Activation from Laurus nobilis Hongqiang Chen,†,‡ Chunfeng Xie,†,‡ Hao Wang,†,‡ Da-Qing Jin,‡,§ Shen Li,†,‡ Meicheng Wang,† Quanhui Ren,† Jing Xu,*,† Yasushi Ohizumi,∥ and Yuanqiang Guo*,† †

College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research and §School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan S Supporting Information *

ABSTRACT: The inhibitory reagents to inhibit the activation of microglial cells may be potentially useful for the treatment of neurodegenerative diseases. The leaves of the plant Laurus nobilis belonging to the family Lauraceae, namely, bay leaves, have been used as a popular spice, and their extract showed moderate inhibition on microglial activation. A further phytochemical investigation of the leaves led to the isolation of two new (1, 2) and eight known (3−10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, 1H−1H COSY, and NOESY) spectroscopic data analyses and Chem3D modeling. The following biological studies disclosed that these isolated compounds showed inhibitory activities on LPS-induced microglial activation. The results of our phytochemical investigation, including two new sesquiterpenes (1 and 2) and the first report of two compounds (3 and 4) from this species, further revealed the chemical composition of bay leaves as a popular spice, and the biological studies implied that bay leaves, containing bioactive substances with the inhibition of microglial activation, were potentially beneficial to human health. KEYWORDS: Laurus nobilis, bay leaf, sesquiterpenes, microglial activation, neurodegenerative diseases

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INTRODUCTION As human longevity increases, chronic neurodegenerative disorders are having an increasing impact on public health. Among these neurodegenerative disorders, Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common representatives, which have an about 2% incidence of the population over 65 years of age.1 Though the mechanism of neurodegenerative diseases remains unclear, accumulating evidence strongly supports that the pathway leading to neurodegenerative diseases, such as PD and AD, involves the activation of microglial cells.2 Microglial cells, the resident immune cells of the central nervous system (CNS), play a major role in host defense and tissue repair in CNS. The activated microglial cells release neurotoxic and inflammatory mediators, such as nitric oxide (NO), reactive oxygen species (ROS), and proinflammatory cytokines, and overproduction of these inflammatory mediators in the CNS can cause various severe neurodegenerative diseases.3 The activation of microglial cells in the CNS is an early sign that often precedes neuronal death.3,4 Therefore, the inhibition of microglial activation can halt the following neuronal damage in the CNS, and the inhibitory reagents to inhibit the microglial activation may be potentially effective for the treatment of neurodegenerative diseases.5 Laurus nobilis, belonging to the family Lauraceae, is an evergreen tree widely distributed in the Mediterranean area and Europe.6 The leaves of L. nobilis, namely, bay leaves, have been used as not only one of the most popular culinary spices in all western countries but also a folk medicine for asthma, digestive disorders, cardiac diseases, diarrhea, and rheumatic pains.7,8 © 2014 American Chemical Society

Previous phytochemical investigations on L. nobilis revealed the main presence of sesquiterpenes, megastigmane and phenolic components, and flavonoids,7−14 which showed NO inhibitory effects,7,9 cytotoxic activities,8,11−13 and inhibitory effects on alcohol absorption.15 In the course of our survey on biologically active substances in medicinal plants, considerable attention has been given to the occurrence of compounds with inhibitory effects of the microglial activation, since these substances are expected to be potentially useful for the treatment of neurodegenerative diseases. As a continuation of our work on the search for bioactive substances from traditional folk medicines or medicinal food,16−18 we investigated the chemical constituents of the ethyl acetate soluble part of the methanol extract from the leaves of L. nobilis, which showed moderate inhibitory effects on LPS-induced microglial activation. This investigation led to the isolation and identification of two new sesquiterpenes, named laurupenes A and B (1 and 2), and eight known analogues (3−10) (Figure 1). Their structures were elucidated on the basis of spectroscopic data analyses (IR, ESIMS, HR-ESI-MS, and 1D and 2D NMR). This paper herein describes their isolation, structure elucidation, and inhibitory activities on lipopolysaccharide (LPS)-induced microglial activation. Received: Revised: Accepted: Published: 4784

March 31, 2014 May 5, 2014 May 6, 2014 May 6, 2014 dx.doi.org/10.1021/jf501515v | J. Agric. Food Chem. 2014, 62, 4784−4788

Journal of Agricultural and Food Chemistry

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Co. Ltd., China), and a YMC-pack ODS-AM (20 × 250 mm) column (YMC Co. Ltd., Japan). Silica gel was 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 murine microglial BV-2 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). Plant Material. The leaves of L. nobilis were purchased in July 2011 from Bozhou Materia Medica Market, Anhui province, China. A voucher specimen (No. 20110919) 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 L. nobilis (6.0 kg) were powdered and extracted were with MeOH (3 × 48 L) under reflux. The organic solvent was evaporated to obtain a crude extract (375 g). The extract was suspended in H2O (0.7 L) and partitioned with EtOAc (3 × 0.7 L). The EtOAc soluble part (260 g) was subjected to a silica gel column chromatography, using a gradient of acetone in petroleum ether (1%−30%), to give ten fractions (F1−F10) based on TLC analyses. F2 was fractionated by middle pressure liquid chromatography (MPLC) over octadecylsilyl (ODS) eluting with a step gradient from 70% to 90% MeOH in H2O to give four subfractions (F2−1−F2−4). F2−2 was further purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 75% MeOH in H2O) to afford compound 1 (tR = 22 min, 11.4 mg). Compound 2 (tR = 26 min, 4.0 mg) was obtained from the subfraction F2−3 using the same HPLC system (82% MeOH in H2O), and compound 3 (tR = 28 min, 8.1 mg) was obtained from F2−4 (85% MeOH in H2O). F3, F4, F5, F6, and F8 were also subjected to the same MPLC over ODS, eluting with a step gradient from 60% to 90% MeOH in H2O, to give subfractions F3−1−F3−4, F4−1−F4−6, F5−1−F5−5, F6−1−F6−9, and F8−1−F8−8, respectively. The further purification of subfraction F4−2 (73% MeOH in H2O) with the same HPLC system led to afford compound 4 (tR = 24 min, 23.9 mg). Compounds 5 (tR = 21 min, 18.9 mg) and 9

Figure 1. Structures of compounds 1−10 from L. nobilis.

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MATERIALS AND METHODS

General. The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter. The IR spectra were taken on a Bruker Tensor 27 FT-IR spectrometer with KBr disks. The ESIMS spectra were obtained on an LCQ-Advantage mass spectrometer (Finnigan Co. Ltd., U.S.A.). HR-ESI-MS spectra were recorded by an Agilent 6520 Q-TOF LC/MS (Agilent, U.S.A.). 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (400 MHz for 1 H 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

Table 1. 1H and 13C NMR Data of Compounds 1 and 2 (δ ppm in CDCl3 and J in Hz)a 1 position

a

13

2 1

C

1 2

43.7 37.5

3

73.8

4 5 6 7 8

147.8 51.0 83.3 45.6 29.1

9

121.4

10 11 12 13

137.0 139.8 169.8 119.6

14

28.0

15

117.2

OAC

171.0 21.3

13

H

1

C

α α β α

2.63 2.72 1.53 5.52

m m m m

α β α α β

2.71 4.08 2.69 2.57 2.65 5.58

m dd (10.7, 9.4) m m m d (7.6)

76.2 36.2 30.1 146.4 129.6 136.6 52.4 37.1 34.5 153.1 147.4 21.3 109.4

6.22 d (3.3) 5.49 d (3.3) 1.82 s

110.9

5.48 s 5.38 s

113.5

H

β α β α β

3.78 2.08 2.01 2.44 2.21

dd (11.8, 3.3) m m ddd (17.6, 12.8, 4.8) m

β α β α β

6.08 5.54 2.68 1.82 2.11 2.66 1.71

d (15.8) dd (15.8, 10.3) m m m m m

1.72 4.73 4.71 5.30 5.04 4.96 4.87

s s s s s s s

2.06 s

The assignments of NMR data are based on 1H, 13C, DEPT, 1H−1H COSY, HMQC, and HMBC NMR experiments. 4785

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(tR = 23 min, 35.6 mg) were obtained from subfraction F3−2 using the above HPLC system (73% MeOH in H2O). With the same protocols, compounds 6 (tR = 24 min, 32.2 mg) and 10 (tR = 20 min, 13.2 mg) were isolated from subraction F5−1 (65% MeOH in H2O), and compound 7 (tR = 21 min, 64.2 mg) was obtained from F6−1 (62% MeOH in H2O). The further purification of F8−1 with the above HPLC system (60% MeOH in H2O) resulted in the isolation of compound 8 (tR = 15 min, 41.6 mg). Laurupene A (1). Colorless oil; [α]12 D = +27.3 (c 0.55, CH2Cl2); IR (KBr) νmax cm−1 2967, 2930, 1768, 1736, 1666, 1372, 1240, 1137, 1015; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; ESI-MS m/z 289 [M + H]+; HR-ESI-MS m/z 289.1437 [M + H]+, calcd for C17H21O4, 289.1440. Laurupene B (2). Colorless oil; [α]12 D = −17.7 (c 0.26, CH2Cl2); IR (KBr) νmax cm−1 2968, 2929, 1658, 1372, 1276, 1261, 764; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 1; ESI-MS m/z 219 [M + H]+; HR-ESI-MS m/z 219.1742 [M + H]+, calcd for C15H23O, 219.1749. Bioassay for Inhibitory Activities on LPS-Induced Microglial Activation. The activated microglial cells release neurotoxic and inflammatory mediators, such as nitricoxide (NO). The amount of released NO production indicate the level of microglial activation or inhibition degree. Murine microglial BV-2 cells were selected for the experiments. The 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 0.2 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO, U.S.A.) 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-naphthyl)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., U.S.A.) 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.

mentioned spectroscopic features, and the sesquiterpenes isolated from the genus Laurus,8−12 compound 1 might be a sesquiterpene with three rings and an acetoxy group. In order to confirm the above assumption and elucidate the structure, the following HMBC and 1H−1H COSY experiments were performed. In the HMBC spectrum, the long-range correlations of H-1 to C-2, C-3, C-4, C-5, C-6, C-9, C-10, and C-14, and H5 to C-1, C-2, C-3, C-4, C-6, C-7, C-10, and C-15, revealed the fusion of a five-membered ring and a seven-membered ring, which bonded with the α,β-saturated γ-lactone ring via the C-6 and C-7 unit. By the HMBC correlations of H2-15 to C-3, C-4, and C-5, and H2-13 to C-7, C-11, and C-12, the two terminal double bonds were accordingly assigned to C-4 and C-15, and C-11 and C-13, respectively. The oxygenated carbon at δC 73.8 and the residual olefinic carbons at δC 121.4 and 137.0 were ascribable to C-3, C-9, and C-10, respectively, by the interpretation of the HMBC spectrum. Consequently, the substituted acetoxy group was attached at C-3 by the HMBC correlation of H-3 to the carbonyl carbon at δC 171.0. 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 a guaianolide-type sesquiterpene for compound 1 was established.

Figure 2. Selected HMBC and compounds 1 and 2.

1

H−1H COSY correlations of

The relative configuration of 1 was elucidated as follows. For the reported natural guaianolide sesquiterpene, the fivemembered lactone ring, the seven-membered ring, and the other five-membered ring are trans- and cis-fused with each other, respectively, H-1, H-5, and H-7 are in α-positions on the same side, and H-6 is in β-position on the opposite side.19 The NOESY correlations observed for H-5/H-7, H-5/H-1, H-1/H3, H-6/H-2β, H-6/H-8β, and H-9/H3-14 (Figure 3) and the

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RESULTS AND DISCUSSION Compound 1 was isolated as a colorless oil. Its HR-ESI-MS provided the molecular formula C17H20O4, through the presence of a peak at m/z 289.1437 [M + H]+ (calcd for C17H21O4 289.1440). The molecular formula indicated eight unsaturation degrees for 1. The IR spectrum evidenced the presence of an α,β-saturated γ-lactone moiety (1736 cm−1). The 1H NMR spectrum of 1 exhibited two methyl singlets at δH 1.82 (3H, s, H3-14) and 2.06 (3H, s, COCH3), five olefinic protons at δH 5.58 (1H, d, J = 7.6 Hz, H-9), 6.22 and 5.49 (each 1H, d, J = 3.3 Hz, H2-13), and 5.48 and 5.38 (each 1H, s, H2-15), and two oxygenated methine protons at δH 5.52 (1H, m, H-3) and 4.08 (1H, dd, J = 10.7, 9.4 Hz, H-6) (Table 1). The 13C NMR spectrum of 1 showed 17 carbon resonances. From the 1H and 13C NMR spectra, one acetoxy group was obvious. Apart from the two carbons (δC 171.0, 21.3) for the acetoxy group, there were 15 residual resonances in the 13C NMR spectrum, which were classified into one methyl [δC 28.0 (C-14)], four methylenes [δC 37.5 (C-2), 29.1 (C-8), 119.6 (C13), and 117.2 (C-15)], six methines [δC 43.7 (C-1), 73.8 (C3), 51.0 (C-5), 83.3 (C-6), 45.6 (C-7), and 121.4 (C-9)], and four quaternary carbons [δC 147.8 (C-4), 137.0 (C-10), 139.8 (C-11), and 169.8 (C-12)] based on the DEPT and HMQC spectra. According to the unsaturation degrees, the afore-

Figure 3. Key NOESY correlations of compounds 1 and 2.

further Chem3D modeling suggested a conformation for compound 1 as depicted in Figure 3, where the C-3 acetoxy group was in a β-position. The structure of compound 1 was therefore characterized and named laurupene A. Compound 2 possessed a molecular formula C15H22O as determined by the HR-ESI-MS (m/z 219.1742 [M + H]+, calcd for C15H23O, 219.1749), which implied five unsaturation degrees. The 1H NMR spectrum of 2 exhibited one methyl group at δH 1.72 (3H, s, H3-12), eight olefinic protons at δH 4786

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olide (5),21 santamarine (6),8 reynosin (7),8 1β-hydroxy arbusculin A (8),22 zaluzanin D (9),23 and zaluzanin C (10).23 Studies on the inhibition of microglial activation have demonstrated that reagents with inhibitory activities of microglial activation can halt the neuronal damage in the CNS and may be potentially useful for the treatment of neurodegenerative diseases.5 In order to search for and obtain the bioactive substances inhibiting the microglial activation as candidates for the treatment of neurodegenerative diseases, these sesquiterpenes isolated from L. nobilis were evaluated for their inhibitory effects on LPS-induced microglial activation using murine microglial BV-2 cells (Figure 4). The level of

6.08 (1H, d, J = 15.8 Hz, H-5), 5.54 (1H, dd, J = 15.8, 10.3 Hz, H-6), 4.73 and 4.71 (each 1H, s, H2-13), 5.30 and 5.04 (each 1H, s, H2-14), and 4.96 and 4.87 (each 1H, s, H2-15), and one oxygenated methine proton at δH 3.78 (1H, dd, J = 11.8, 3.3 Hz, H-1) (Table 2). The 13C NMR spectrum of 2 showed 15 Table 2. IC50 Values of Compounds 1 and 3−10 Inhibiting the Microglial Activation Indicated by the Amount of NO Productiona compd

IC50 (μM)

compd

IC50 (μM)

1 3 4 5 6

>100 >100 23.3 ± 3.2 39.9 ± 6.0 25.3 ± 1.9

7 8 9 10 SMTb

22.0 30.5 14.0 35.4 8.1

± ± ± ± ±

1.1 0.8 0.5 4.1 1.2

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

a

carbon signals, comprising one methyl (C-12), seven methylenes (C-2, C-3, C-8, C-9, C-13, C-14, and C-15), four methines (C-1, C-5, C-6, and C-7), and three quaternary carbons (C-4, C-10, and C-11) based on the DEPT and HMQC spectra. According to the molecular, unsaturation degrees, and the above spectroscopic features, compound 2 might be a germacrane-type sesquiterpene.8−13 In order to confirm the germacrane-type skeleton and achieve the assignments of proton and carbon signals, a following HMBC experiment was performed. By the interpretation of HMQC and HMBC spectroscopic data, the ten-membered ring and the isopropenyl moiety attached at C-7 were deduced and defined. The additional two terminal double bonds were accordingly assigned to C-4 and C-15, and C-10 and C-14, respectively, by the HMBC correlations of H2-15 to C-3, C-4, and C-5, and H214 to C-1, C-9, and C-10. Based on the above deductions, the germacrane skeleton consisting of one ten-membered ring and an isopropenyl moiety was inferred, where the methylene groups of two terminal double bonds between C-4 and C-15, and C-10 and C-14, replaced the characteristic C-14 and C-15 methyl groups of the common germacrane skeleton. Consequently, the oxygenated carbon at δC 76.2 and the olefinic carbons at δC 129.6 and 136.6 were assigned to C-1, C-5, and C-6, respectively, by the long-range correlations of H-1 to C-9, C-10, C-14, C-2, and C-3, H-5 to C-3, C-4, C-6, C-7, and C-15, and H-6 to C-4, C-5, C-7, C-8, and C-11. The further analyses of 2D NMR spectra led to the assignments of all the protons and carbons. Thus, the planar structure of a germacrane-type sesquiterpene for 2 was established. The relative configuration of 2 was deduced as follows. The coupling constant between H-5 and H-6 (J5,6 = 15.8 Hz) revealed an E-configuration for the C-5−C-6 double bond. The NOESY correlations observed for H-5/H-1, H-5/H-7, H-7/H9β, H-9β/H-1, H-7/H3-12, H-8β/H3-12, and H-6/H-3α (Figure 3) and the Chem3D modeling suggested a conformation for 2 as depicted in Figure 3, where the H-7 and H-1 were both in β-positions. The structure of compound 2 was therefore characterized, which has been named laurupene B. Based on the spectroscopic analyses and the comparison with the literature, the known compounds were identified as entgermacra-4(15),5,10(14)-trien-1α-ol (3),20 15-acetoxycostunolide (4),21 3α-acetoxyeudesma-1,4(15),11(13)-trien-12,6α-

Figure 4. Inhibitory effects of compounds 4−10 on LPS-induced microglial activation. The inhibition levels of microglial activation were indicated by the amount of released NO production in BV-2 cells. BV2 cells were treated with LPS alone or together with each compound at the concentrations indicated. After 20 h incubation, the supernatants were tested by Griess assay and the NO inhibitory rates were calculated. The experiment was performed three times, and the data are expressed as mean ± SD values. The inhibitory rate on NO production was calculated as follows: inhibitory rate (%) = (1 − (LPS/ sample − untreated)/(LPS − untreated)) × 100. ◆ indicates positive control, SMT.

microglial activation or inhibition degree was indicated by the amount of released NO production.24 2-Methyl-2-thiopseudourea sulfate (SMT) was used as a positive control (IC50 8.1 μM). All the evaluated sesquiterpenes exhibited inhibitory effects on LPS-induced microglial activation, and the inhibitory effects indicated by the amount of NO production are shown in Table 3. Compounds 4−10 inhibited LPS-induced microglial activation dose-dependently with IC50 values of 23.3, 39.9, 25.3, 22.0, 30.5, 14.0, and 35.4 μM, respectively. Compounds 1 and 3 showed weak activities (IC50 values >100 μM). Compound 2 was not assayed for the inhibitory effects because of inadequate amount. MTT assay indicated that all the assayed compounds had no significant cytotoxicity to the BV-2 cells at their effective concentration for the inhibition of microglial activation (data not shown). In summary, two new and eight known sesquiterpenes were successfully isolated from the leaves of L. nobilis. Their structures were elucidated by 1D and 2D NMR spectra and Chem3D modeling. Biological studies disclosed that all of the evaluated isolates exhibited inhibitory effects on LPS-induced 4787

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(12) Dall’Acqua, S.; Viola, G.; Giorgetti, M.; Loi, M. C.; Innocenti, G. Two new sesquiterpene lactones from the leaves of Laurus nobilis. Chem. Pharm. Bull. 2006, 54, 1187−1189. (13) Julianti, E.; Jang, K. H.; Lee, S.; Lee, D.; Mar, W.; Oh, K. B.; Shin, J. Sesquiterpenes from the leaves of Laurus nobilis L. Phytochemistry 2012, 80, 70−76. (14) Fiorini, C.; David, B.; Fouraste, I.; Vercauteren, J. Acylated kaempferol glycosides from Laurus nobilis leaves. Phytochemistry 1998, 47, 821−824. (15) Yoshikawa, M.; Shimoda, H.; Uemura, T.; Morikawa, T.; Kawahara, Y.; Matsuda, H. Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity. Bioorg. Med. Chem. 2000, 8, 2071−2077. (16) Xu, J.; Jin, D. Q.; Liu, C.; Xie, C.; Guo, Y.; Fang, L. Isolation, characterization, and NO inhibitory activities of sesquiterpenes from Blumea balsamifera. J. Agric. Food Chem. 2012, 60, 8051−8058. (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, 2075−82. (18) Guo, P.; Li, Y.; Xu, J.; Liu, C.; Ma, Y.; Guo, Y. Bioactive neoclerodane diterpenoids from the whole plants of Ajuga ciliata Bunge. J. Nat. Prod. 2011, 74, 1575−1583. (19) Ando, M.; Ibayashi, K.; Minami, N.; Nakamura, T.; Isogai, K.; Yoshimura, H. Studies on the synthesis of sesquiterpene lactones, 16. The syntheses of 11β,13-dihydrokauniolide, estafiatin, isodehydrocostuslactone, 2-oxodesoxyligustrin, arborescin, 1,10-epiarborescin, 11β,13-dihydroludartin, 8-deoxy-11β,13-dihydrorupicolin B, 8-deoxyrupicolin B, 3,4-epiludartin, ludartin, kauniolide, dehydroleucodin, and leucodin. J. Nat. Prod. 1994, 57, 433−445. (20) Lee, S. O.; Choi, S. Z.; Chio, S. U.; Kim, G. H.; Kim, Y. C.; Lee, K. R. Cytotoxic terpene hydroperoxides from the aerial parts of Aster spathulifolius. Arch. Pharm. Res. 2006, 29, 845−848. (21) Zdero, C.; Bohlmann, F. Sesquiterpene lactones from Dicoma species. Phytochemistry 1990, 29, 183−187. (22) Choi, J. Y.; Choi, E. H.; Jung, H. W.; Oh, J. S.; Lee, W. H.; Lee, J. G.; Son, J. K.; Kim, Y.; Lee, S. H. Melanogenesis inhibitory compounds from Saussureae radix. Arch. Pharm. Res. 2008, 31, 294− 299. (23) Krishna Kumari, G. N.; Masilamani, S.; Ganesh, M. R.; Aravind, S. Microbial transformation of zaluzanin D. Phytochemistry 2003, 62, 1101−1104. (24) 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.

microglial activation and compound 9 exerted the most inhibition against microglial activation indicated by the amount of NO production, which was comparable to the positive control (SMT). The results of our chemical investigation, including two new sesuqiterpenes (1 and 2) and the first report of two compounds (3 and 4) from this species, further revealed the chemical composition of L. nobilis, and the biological screening of these isolates exhibited that the leaves of L. nobilis, used as a popular spice, may be beneficial to human health, while the current biological data suggest that these sesquiterpenes from L. nobilis, especially compounds 4, 6, 7, and 9 with strong inhibitory activities of microglial activation, may probably be considered as candidate agents for neurodegenerative diseases. Further biological studies on these compounds are still underway by our group.

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ASSOCIATED CONTENT

S Supporting Information *

The 1D and 2D NMR and HR-ESI-MS spectra of compounds 1 and 2. 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: [email protected]. *Tel/fax: +86-22-23502595. E-mail: [email protected]. Author Contributions ‡

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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REFERENCES

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dx.doi.org/10.1021/jf501515v | J. Agric. Food Chem. 2014, 62, 4784−4788