New Sesquiterpene Dimers from Inula britannica Inhibit NF-κB Activation and NO and TNF-α Production in LPS-Stimulated RAW264.7 Cells

• Abstract
• Introduction
• Materials and Methods
• General
• Plant materials
• Extraction and isolation
• Determination of NF-κB activity
• Determination of NO production
• Determination of TNF-α production
• Results and Discussion
• Acknowledgements
• References

Abstract

A bioassay-guided isolation of an ethyl acetate-soluble extract of the aerial parts of Inula britannica var. chinensis (Rupr.) Regel, using an in vitro NF-κB reporter gene assay, led to the isolation of four new sesquiterpene dimers bearing a norbornene moiety, inulanolides A - D (1 - 4), and three known sesquiterpenes, 1,6α-dihydroxyeriolanolide (5), 1-acetoxy-6α-hydroxyeriolanolide (6), and eupatolide (7). The structures of the new compounds were elucidated by spectroscopic methods. Among these compounds, inulanolides B and D (2 and 4) and eupatolide (7), exhibited potent inhibitory activity on the LPS-induced NF-κB activation with IC₅₀ values of 0.49 μM, 0.48 μM, and 1.54 μM, respectively. Consistent with their inhibitory effect on NF-κB activation, compounds 2, 4, and 7 also strongly inhibited the production of NO and TNF-α in the LPS-stimulated RAW264.7 cells with IC₅₀ values in the range of 2 μM.

Introduction

Nuclear factor-κB (NF-κB) is a key regulator of the cellular inflammatory and immune response [1]. In non-stimulated cells, NF-κB is sequestered in the cytoplasm by binding to the inhibitor proteins, IκBα and IκBβ. Many stimuli including cytokines, phorbol esters, lipopolysaccharide (LPS), UV, and oxidants, lead to the phosphorylation, ubiquitination, and subsequent degradation of the IκB proteins. This induced degradation of IκBs allows NF-κB to freely enter the nucleus and to activate the expression of a large number of target genes, such as iNOS, COX-2, inflammatory cytokines, TNF-α, and the cell adhesion molecules, which are important for the inflammatory and immune response [2], [3]. The constitutive activation of NF-κB has been associated with a number of human diseases, including inflammatory diseases and cancer [4]. Therefore, the identification of the inhibitors of NF-κB activation is expected to provide a direction for the development of novel anti-inflammatory and anticancer agents [4].

As part of our ongoing search for inhibitors of NF-κB activation using an NF-κB reporter gene assay system in LPS-stimulated RAW264.7 cells, a methanol extract of the aerial parts of Inula britannica var. chinensis (Rupr.) Regel was found to potently inhibit NF-κB activation. I. britannica var. chinensis (Rupr.) Regel (Compositae) has been used as a traditional medicine in Eastern Asia to treat bronchitis, digestive disorders, and inflammation [5]. A number of bioactive sesquiterpenes and flavonoids have been isolated from this plant [6], [7], [8]. Recently, Han et al. reported that ergolide, a sesquiterpene lactone isolated from this plant inhibited iNOS and COX-2 expression via inactivation of NF-κB [9]. This paper reports the bioassay-guided isolation and structural elucidation of four new sesquiterpene dimers, inulanolides A - D (1 - 4), and three known monomeric sesquiterpenes (5 - 7) (Fig. [1]), as well as a biological evaluation of these compounds on NF-κB activation and NO and TNF-α production in LPS-stimulated RAW264.7 cells.

Materials and Methods

General

Melting points were determined on an Electrothermal 9100 instrument without correction. Optical rotations were measured on a JASCO DIP-370 polarimeter. UV spectra were obtained on a UV-1601 UV-VIS spectrophotometer (Shimadzu). IR spectra were collected on a JASCO report-100 IR spectrometer in KBr pellets. NMR spectra were recorded on Varian UNITY 300 and 500 NMR spectrometers with TMS as internal standard. The HRFAB-MS was obtained on a JMS-HX110A/HX110A Tandem Mass Spectrometer (JEOL). EI- and ESI-MS were obtained on a Micromass Autospec and a Platform quadrupole Mass Spectrometer, respectively. Preparative HPLC was carried out on a Spectraphysics SP8800 (USA).

Plant materials

The aerial parts of Inula britannica var. chinensis (Rupr.) Regel were collected at Yanggu, Gangwon, Korea in August, 2002 [10]. The sample was identified by Professor K. Bae, College of Pharmacy, Chungnam National University. A voucher specimen (No. 030 908) has been deposited in the Korea Research Institute of Bioscience and Biotechnology.

Extraction and isolation

The dried aerial parts of Inula britannica var. chinensis (Rupr.) Regel (4.5 kg) were extracted with MeOH (50 L) three times at room temperature. The MeOH extract (220 g), exhibiting potent inhibitory effect on the NF-κB activation, was suspended in water and partitioned with hexane (8 L × 2) to afford a hexane-soluble extract (68 g). The H₂O layer was further partitioned with EtOAc to obtain an EtOAc-soluble extract (48 g). The EtOAc extract, exhibiting a potent inhibitory effect on the NF-κB activation, was chromatographed on a silica gel column (6 × 10 cm) eluting with CH₂Cl₂-MeOH (100 : 0, 50 : 1, 20 : 1, 10 : 1, 5 : 1, 1 : 1, MeOH, each 2 L) to afford ten fractions (Fr1 - Fr10). The active fractions Fr2 - Fr5 (11 g) were combined and subjected to silica gel column chromatography eluting with hexane-CH₂Cl₂ (20 : 1, 5 : 1) to afford nine fractions (Fr41 - Fr49). The Fr48 was subjected to MPLC (silica gel) eluting with CH₂Cl₂-acetone (20 : 1), producing ten subfractions Fr481 - Fr4810. The Fr482 and Fr483 were combined (0.832 g) and subjected to preparative HPLC (ODS-H80, 150 × 20 mm, YMC, Japan, CH₃CN-H₂O, 40 : 60, flow rate: 5 mL/min), resulting in the purification of compounds 1 (103.0 mg, t_R = 28.62 min), 2 (11.2 mg, t_R = 39.34 min), 6 (200.1 mg, t_R = 7.13 min), and 7 (50.0 mg, t_R = 16.76 min).

The fractions Fr486 - Fr4810 were combined and eluted on Sephadex LH 20 (CHCl₃-MeOH, 1 : 1), resulting in seven subfractions Fr4860 - 1 - Fr4860 - 7. The Fr4860 - 3 and Fr4860 - 4 were combined (0.341 g) and subjected to preparative HPLC (CH₃CN-H₂O, 40 : 60, flow rate: 5 mL/min), leading to the isolation of compounds 3 (86.2 mg, t_R = 26.98 min) and 4 (8.2 mg, t_R = 37.87 min). The Fr4860 - 5 and Fr4860 - 6 were combined (2.08 g) and subjected to MPLC (silica gel) eluting with CH₂Cl₂-acetone (20 : 1), producing seven fractions Fr486056 - 1 - Fr486056 - 7. Compound 5 (170.5 mg) was crystallized from Fr486056 - 6.

Inulanolide A (1): White needles; m. p. 126.1 - 127.0 °C; [α]_D ²⁵: + 56.0° (c 0.10, MeOH); UV (MeOH): λ_max (log ε) = 201.3 (4.63) nm; IR (KBr): ν_max = 3510, 2946, 1765, 1735, 1370, 1028 cm^-1; ¹H- and ¹³C-NMR: see Tables [1] and [2]; EIMS: m/z = 596 (8), 536 (8), 309 (5), 288 (13), 246 (58), 228 (100); HR-FAB-MS: m/z = 597.3067 [M + H]⁺ (calcd. for C₃₄H₄₅O₉ : 597.3064).

Inulanolide B (2): White amorphous powder; m. p. 184.1 - 185.0 °C; [α]_D ²⁵: -200.0° (c 0.01, MeOH); UV (MeOH): λ_max (log ε) = 202.5 (4.80) nm; IR (KBr): ν_max = 3450, 2930, 1760, 1739, 1228, 1032 cm^-1; ¹H- and ¹³C-NMR: see Tables [1] and [2]; EI-MS: m/z = 536 (8), 288 (14), 246 (100), 231 (27), 228 (80); HR-FAB-MS: m/z = 537.2847 [M + H]⁺ (calcd. for C₃₂H₄₁O₇ : 537.2852).

Inulanolide C (3): White amorphous powder; m. p. 161.5 - 162.4 °C; [α]_D ²⁵: -125.0° (c 0.01, MeOH); UV (MeOH): λ_max (log ε) = 203.0 (4.86), 231.0 (4.69) nm; IR (KBr): ν_max = 3450, 2946, 1760, 1729, 1240, 1024 cm^-1; ¹H- and ¹³C-NMR: see Tables [1] and [2]; EI-MS: m/z = 536 (7), 476 (11), 288 (5), 246 (48), 231 (8), 228 (100); HR-FAB-MS: m/z = 537.2852 [M + H]⁺ (calcd. for C₃₂H₄₁O₇ : 537.2852).

Inulanolide D (4): White amorphous powder; m. p. 156.6 - 157.6 °C; [α]_D ²⁵: -110.0° (c 0.01, MeOH); UV (MeOH): λ_max (log ε) = 202.7 (4.93), 233.3 (4.22) nm; IR (KBr): ν_max = 3450, 2956, 1766, 1735, 1240, 1030 cm^-1; ¹H- and ¹³C-NMR: see Tables [1] and [2]; EI-MS: m/z = 536 (7), 288 (32), 246 (72), 231 (8), 228 (100); HR-FAB-MS: m/z = 537.2854 [M + H]⁺ (calcd. for C₃₂H₄₁O₇ : 537.2852).

1,6a-Dihydroxyeriolanolide (5): Colorless cubic crystals; m. p. 175.1 - 177.0 °C; [α]_D ²⁵: + 91.3° (c 0.09, CHCl₃); UV (MeOH): λ_max (log ε) = 213.0 (3.81) nm; ¹H- and ¹³C-NMR (CDCl₃) spectral data in agreement with previous reports [6], [11]; ESI-MS: m/z = 289.4 [M + Na]⁺.

1-Acetoxy-6a-hydroxyeriolanolide (6): Colorless cubes; m. p. 124.0 - 126.1 °C; [α]_D ²⁵: + 101.6° (c 0.26, CHCl₃); UV (MeOH): λ_max (log ε) = 212.1 (3.92) nm; ¹H- and ¹³C-NMR (CDCl₃) spectral data in agreement with previous reports [6], [11]; ESI-MS: m/z = 331.5 [M + Na]⁺.

Eupatolide (7): Colorless amorphous powder; m. p. 182.1 - 188.1 °C; [α]_D ²⁵: + 420.0° (c 0.01, MeOH); UV (MeOH): λ_max (log ε) = 209.2 (4.30) nm; ¹H- and ¹³C-NMR (DMSO-d ₆) spectral data in agreement with previous reports [12], [13]; ESI-MS: m/z = 271.4 [M + Na]⁺.

Determination of NF-κB activity

NF-κB activity was determined as previously described [14], [15]. RAW264.7 cells stably transfected with a plasmid containing 8 copies of κB elements linked to SEAP (secreted alkaline phosphatase) gene were used.

Determination of NO production

RAW264.7 cells were seeded in 96 well plates at 1 × 10⁵ cells/well. After 3 h, the cells were treated with various concentrations of compounds and stimulated for 24 h with or without 1 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO, USA). As a parameter of NO synthesis, the nitrite concentration was measured in the supernatant of RAW264.7 cells by the Griess reaction as previously described [14], [16].

Determination of TNF-α production

RAW264.7 cells were seeded in the 96-well plates at a density of 1 × 10⁴ cells/well, pretreated with different concentrations of compounds for 1 h, then the cells were stimulated with LPS (1 μg/mL) for 18 h. TNF-α production in the supernatant of RAW264.7 cells was quantitated using an OptEIA^TM assay kit according to the manufacturer’s instructions (Pharmingen, San Diego, CA, USA) as previously described [17].

Results and Discussion

Inulanolide A (1) was obtained as white needles and shown to possess a molecular formula of C₃₄H₄₄O₉ (HR-FAB-MS [M + H]⁺, m/z = 597.3067). The ¹³C, DEPT, and HMQC NMR spectra of compound 1 exhibited 34 carbon signals, which revealed the presence of six methyls, eight methylenes, nine methines, eleven quaternary carbons including four carbonyl groups. The ¹H- and ¹³C-NMR spectra of compound 1 (Tables [1] and [2]) displayed the characteristic signals for a guaianolide unit at δ_H = 4.33 (1H, ddd, J = 3.2, 8.4, 11.0 Hz, H-8′), 5.62 (1H, d, J = 3.6 Hz, H-13′), 6.12 (1H, d, J = 3.2 Hz, H-13′), δ_C = 119.57 (C-13′), 172.26 (C-12′) [α-methylene lactone functionality], δ_H = 1.06 (3H, d, J = 7.2 Hz, H-14′), 1.64 (3H, d, J = 1.6 Hz, H-15′), 2.13 (3H, s, H-2′′′), and 4.54 (1H, brs, H-2′) [18]. The HMBC spectrum of compound 1 showed the cross peaks, H-2′/C-1′, C-3′, C-4′, C-1′′′; H-8′/C-12′; H-13′/C-7′, C-11′, C-12′; H-14′/C-1′, C-9′, C-10′; H-15′/C-3′, C-4′, C-5′, which confirmed the presence of this unit (Fig. [2]).

The signals for the 1-acetoxy-6α-hydroxyeriolanolide moiety (6), which was one of the major compounds obtained from this study, were observed at δ_H = 1.13 (3H, d, J = 7.2 Hz, H-15), 1.74 (3H, s, H-14), 1.92 (3H, s, H-2′), 3.94 (1H, m, H-1), 4.00 (1H, m, H-1), 4.24 (1H, d, J = 2.0 Hz, H-6), and 5.08 (1H, m, H-8) [6], [11]. This structure was confirmed by the COSY cross peaks and HMBC correlations (H-1/C-1′′; H-6/C-4, C-5, C-8, C-10; H-7/C-6, C-11, C-13; H-14/C-5, C-9, C-10; H-15/C-3, C-4, C-5) (Fig. [2]).

The connection of the two above units was established using 1D and 2D NMR experiments. Therefore, the absence of C-11 and C-13 exocyclic methylene signals of the 1-acetoxy-6α-hydroxyeriolanolide moiety (6), and the chemical shifts of the quaternary carbons C-11 and C-1′ at δ_C = 56.59 and 64.08, suggested that the two units of compound 1 are connected through these moieties. In addition, the HMBC cross peaks (Fig. [2]), H-7/C-3′; H-2′/C-11, C-13; H-3′/C-7, C-13; H-13/C-1′, C-3′, C-5′, suggested that compound 1 is a Diels-Alder-type condensation product of the two units and has a bridged ring system, a norbornene moiety [19], [20]. The retro-Diels-Alder fragmentation pattern, A (m/z = 288) and 6′ (m/z = 309), was detected in the EI mass spectrum (see Supporting Information).

The relative configuration of compound 1 was determined by a NOESY NMR experiment (Fig. [3]), and the comparison of the chemical shifts and coupling constants with the literature values [21], [22], [23]. Thus, the chemical shifts of H-7′ (δ_H = 2.87), and H-8′ (δ_H = 4.30), the coupling constant between H-7′ and H-8′ (11.0 Hz), and the allylic couplings observed between H-7′ and H-13′ (3.2 and 3.6 Hz), were indicative of a trans-fused lactone ring of the guaianolide unit [21]. The NOESY spectrum of compound 1 displayed the cross peaks, H-2′/H-14′ and H-8′/H-14′, but no NOE correlation between H-2′ to H-10′ indicated a β-orientation of the H-14′ methyl group (Fig. [3]) [19]. This configuration was also supported by the MM2 energy minimized structure of 1 (the calculated distances between key protons were consistent with the observed NOE correlations) (Fig. [3]). Therefore, the structure of this new sesquiterpene dimer, inulanolide A (1), was assigned as depicted in Fig. [1].

Inulanolide B (2) was obtained as a white amorphous powder and shown to possess a molecular formula of C₃₂H₄₀O₇ (HR-FAB-MS [M + H]⁺, m/z = 537.2847). The ¹H- and ¹³C-NMR data of compound 2 (Tables [1] and 2) are comparable to those of inulanolide A (1) except for the 1-acetoxy-6α-hydroxyeriolanolide moiety (6) of the latter compound, indicating that compound 2 is also a sesquiterpene dimer and is based on a guaianolide skeleton [18]. An analysis of the remaining ¹H- and ¹³C-NMR signals of compound 2 suggested that these signals are attributed to eupatolide (7), which was also identified in this study [13]. The presence of an eupatolide moiety (7) was also confirmed by the salient COSY cross peaks (H-5/H-6, H-6/H-7, H-7/H-8) and HMBC correlations (H-6/C-4, C-5, C-8; H-8/C-6, C-7, C-10, C-11; CH₃ - 14/C-1, C-9, C-10; CH₃ - 15/C-3, C-4, C-5) (see Supporting Information).

The presence of the norbornene moiety and the relative configuration of compound 2 were also determined in a manner comparable to those for inulanolide A (1). The similar retro-Diels-Alder fragmentation pattern to that of compound 1, was observed in the EI mass spectrum of compound 2 (see Supporting Information). Accordingly, the structure of this new compound, inulanolide B (2), was assigned as depicted. An attempt to determine the absolute configurations of inulanolides A and B (1 and 2) using the Mosher ester procedure failed as a result of the inability to derivatize compounds 1 and 2 to the corresponding (S)- and (R)-MTPA esters [24], [25].

The inulanolides C (3) and D (4) had the same molecular formula of C₃₂H₄₀O₇ (HR-FAB-MS [M + H]⁺, 3: m/z = 537.2852, 4: m/z = 537.2854). The ¹H- and ¹³C-NMR data of compounds 3 and 4 (Tables [1] and [2]) indicated that they are also sesquiterpene dimers and both have the guaianolide skeleton. The ¹H-NMR spectrum of compound 3 displayed proton signals for an exomethylene at δ_H = 4.99 (1H, s, H-14) and 5.21 (1H, s, H-14), and an olefinic proton signal at δ_H = 5.41 (1H, brs, H-6). In the aliphatic region, an oxygenated methine signal at δ_H = 4.84 (1H, m, H-8) and the signals for a branched pentol moiety suggested the presence of a modified 1,6α-dihydroxyeriolanolide (5) [6], [11], which was confirmed using 2D NMR techniques. In the ¹H-NMR spectrum of compound 4, the characteristic aromatic proton signals of an ABX system at δ_H = 7.01 (1H, d, J = 8.0 Hz, H-9), 7.09 (1H, dd, J = 1.5, 8.0 Hz, H-8), and 7.24 (1H, d, J = 1.5 Hz, H-6), and the signals of a branched pentol moiety were observed. An analysis of the 1D and 2D NMR data suggested the presence of a free carboxyl and a trisubstituted phenyl groups, which were closely related to the 1,6α-dihydroxyeriolanolide (5) portion of compound 3.

The presence of the norbornene moiety and the relative configuration of compounds 3 and 4 were determined using NMR and EI mass techniques (see Supporting Information). Interestingly, the ¹H-NMR spectrum of 4 displayed a relative upfield shift of the H-2′′ signal at δ_H = 1.16 and the downfield shift of the H-3′ signal at δ_H = 3.69 due to the shielding and deshielding effects of the phenyl ring of compound 4, respectively, supporting the proposed structure of compound 4. Therefore, the structures of compounds 3 and 4, inulanolides C and D, were elucidated.

Compounds 1 - 7 from I. britannica were examined for their effects on the LPS-induced NF-κB activation using an NF-κB reporter gene assay system [14], [15]. As shown in Table [3], all the sesquiterpene dimers, inulanolides A - D (1 - 4), and the monomer, eupatolide (7), inhibited NF-κB activation with IC₅₀ values ranging from 0.48 μM to 6.96 μM. Because NF-κB is a major transcription factor that is involved in the induction of iNOS and the expression of the pro-inflammatory cytokines [2], [3], the effects of compounds 1 - 7 on the NO and TNF-α production in LPS-stimulated RAW264.7 cells were examined. As shown in Table [3], all the compounds inhibited NO and TNF-α production with IC₅₀ values ranging from 1.52 μM to 41.52 μM, and 3.21 μM to 79.26 μM, respectively, except for compound 5. Consistent with the inhibitory activity on NF-κB activation, inulanolides B and C (2 and 3), and eupatolide (7) had a significant inhibitory effect on NO and TNF-α production. The cell viability measured by the MTT assay after Griess assay showed that all compounds had no significant cytotoxicity to the RAW264.7 cells at their effective concentrations for the inhibition of NO production (Table [3]).

Many natural NF-κB inhibitors contain a lactone ring conjugated with an exomethylene group, which can react with biological nucleophile, especially the sulfhydryl group of cysteine residues by a Michael-type reaction [26]. Compounds 1 - 7 also contain a lactone ring conjugated with an exomethylene group. Among them dimeric compounds 1 - 4 share a common structural entity, guaianolide, and compound 7 belongs to the germacranolide group. However, compounds 5 and 6, which are belong to the secoeudesmanolides, are largely inactive in the NF-κB reporter gene assay. According to a recent report on the quantitative structure-activity relationship of sesquiterpene lactones, the eudesmanolide group generally showed weak activity in the NF-κB DNA binding assay [26]. Compounds 5 and 6 were inactive up to 100 μM against NF-kB activation induced by LPS, however, they showed a comparable activity in the LPS-induced NO production with aminoguanidine. One possible speculation on this result would be that these compounds may affect the post-transcriptional level including an influence on the enzymatic activity of iNOS protein [27]. In a previous study [17], compounds with the guaianolide moiety obtained from Artemisia sylvatica also exhibited potent inhibitory activity on NF-κB activation. Although the number of dimeric sesquiterpenoids examined in this study might not be sufficient to clarify the structure-activity relationship, the presence of a guaianolide moiety appears to be important for the enhanced activity. Our results might provide a partial scientific explanation for the use of this plant in Asia for treating inflammatory diseases [5].

Acknowledgements

This study was supported in part by a grant from the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program and a research grant from the Plant Diversity Research Center of 21st Frontier Research Program funded by the Korean Ministry of Science and Technology. We are grateful to the Korea Basic Science Institute for the provision of certain spectroscopic instruments used in this investigation.

References

• 1 Baldwin A S. The NF-kappaB and I kappa B proteins: new discoveries and insights.  Annu Rev Immunol. 1996;  14 649-83
  CrossrefPubMedSearch in Google Scholar
• 2 Baeuerle P A, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappaB transcription factor.  Cell. 1988;  53 211-7
  CrossrefPubMedSearch in Google Scholar
• 3 Baeuerle P A, Henkel T. Function and activation of NF-kappaB in the immune system.  Annu Rev Immunol. 1994;  12 141-79
  CrossrefPubMedSearch in Google Scholar
• 4 Makarov S S. NF-κB as a therapeutic target in chronic inflammation: recent advances.  Mol Med Today. 2000;  6 441-8
  CrossrefPubMedSearch in Google Scholar
• 5 Bensky D, Gamble A, Kaptchuk T J, Bensky L L. Chinese herbal medicine: Materia Medica. Seattle; Eastland Press 1993: pp 193-4
  Search in Google Scholar
• 6 Zhou B N, Bai N S, Lin L Z, Cordell G A. Sesquiterpene lactones from Inula britannica .  Phytochemistry. 1993;  34 249-52
  CrossrefPubMedSearch in Google Scholar
• 7 Park E J, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica .  Planta Med. 1998;  64 752-4
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Park E J, Kim Y, Kim J. Acetylated flavonol glycosides from the flower of Inula britannica .  J Nat Prod. 2000;  63 34-6
  CrossrefPubMedSearch in Google Scholar
• 9 Han J W, Lee B G, Kim Y K, Yoon J W, Jin H K, Hong S. et al . Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW264.7 macrophages through the inactivation of NF-κB.  Br J Pharmacol. 2001;  133 503-12
  CrossrefPubMedSearch in Google Scholar
• 10 Lee C B. Illustrated Flora of Korea. Seoul; Hyangmoonsa 1993: p 729
  Search in Google Scholar
• 11 Jeske F, Luneck S, Jakupovic J. Secoeudesmanolides from Inula britannica .  Phytochemistry. 1993;  34 1647-9
  CrossrefPubMedSearch in Google Scholar
• 12 Dolejs L, Herout V. On terpenes. CXLV. Constitution of eupatoriopicrin, a germacranolide from Eupatorium cannabinum L.  Coll Czech Chem Commun. 1962;  27 2654-61
  PubMedSearch in Google Scholar
• 13 Uchiyama T, Miyase T, Ueno A, Usmanghani K. Terpenic glycosides from Pluchea indica .  Phytochemistry. 1989;  28 3369-72
  CrossrefPubMedSearch in Google Scholar
• 14 Koo T H, Lee J -H, Park Y J, Hong Y S, Kim H S, Kim K W. et al . A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-κB by targeting IκB phosphorylation.  Planta Med. 2001;  67 103-7
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Lee J -H, Koo T H, Hwang B Y, Lee J J. Kaurane diterpene, kamebakaurin, inhibits NF-kappa B by directly targeting the DNA-binding activity of p50 and blocks the expression of antiapoptotic NF-kappa B target genes.  J Biol Chem. 2002;  277 18 411-20
  PubMedSearch in Google Scholar
• 16 Schmidt H HHW, Kelm M. Determination of nitrite and nitrate by the Griess reaction. In: Feelisch M, Stramler J, editors Methods in nitric oxide research. New York; John Wiley & Sons Ltd 1996: pp 491-7
  Search in Google Scholar
• 17 Jin H Z, Lee J H, Lee D, Hong Y S, Kim Y H, Lee J J. Inhibitors of the LPS-induced NF-κB activation from Artemisia sylvatica .  Phytochemistry. 2004;  65 2247-53
  CrossrefPubMedSearch in Google Scholar
• 18 Al-Easa H S, Rizk A M, Ahmed A A. Guaianolides from Picris radicata .  Phytochemistry. 1996;  43 423-4
  CrossrefPubMedSearch in Google Scholar
• 19 Kamperdick C, Phuong N M, Adam G, Sung T V. Guaiane dimers from Xylopia vielana .  Phytochemistry. 2003;  64 811-6
  CrossrefPubMedSearch in Google Scholar
• 20 Kamperdick C, Phuong N M, Sung T V, Adam G. Guaiane dimers from Xylopia vielana .  Phytochemistry. 2001;  56 335-40
  CrossrefPubMedSearch in Google Scholar
• 21 Bohlmann F, Zdero C, King R M, Robinson H. Pseudoguaianolides and other sesquiterpene lactones from Gaillardia species.  Phytochemistry. 1984;  23 1979-88
  CrossrefPubMedSearch in Google Scholar
• 22 Gao F, Wang H, Mabry T J, Watson W H, Kashyap R P. Sesquiterpene lactones and a C₂₀ aliphatic lactone from Texas bitterweed, Hymenoxys odorata .  Phytochemistry. 1990;  29 551-60
  CrossrefPubMedSearch in Google Scholar
• 23 Ahmed A A, Ahmed A M. Jasonol, a rare tricyclic eudesmane sesquiterpene and six other new sesquiterpenoids from Jasonia candicans .  Tetrahedron. 1998;  54 8144-52
  PubMedSearch in Google Scholar
• 24 Dale J A, Mosher H S. Nuclear magnetic resonance enantiomer reagents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxy-α-trifluoromethylphenylacetate (MTPA) esters.  J Am Chem Soc. 1973;  95 512-9
  CrossrefPubMedSearch in Google Scholar
• 25 Su B N, Park E J, Mbwambo Z H, Santarsiero B D, Mesecar A D, Fong H HS. et al . New chemical constituents of Euphorbia quinquecostata and absolute configuration assignment by a convenient Mosher ester procedure carried out in NMR tubes.  J Nat Prod. 2002;  65 1278-82
  CrossrefPubMedSearch in Google Scholar
• 26 Siedle B, Garcia-Pineres A J, Murillo R, Schulte-Mönting J, Castro V, Rüngeler P. et al . Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-kB.  J Med Chem. 2004;  47 6042-54
  CrossrefPubMedSearch in Google Scholar
• 27 Zidorn C, Dirsch V M, Rüngeler P, Sosa S, Della Loggia R, Merfort I. et al . Anti-inflammatory activities of hypocretenolides from Leotodon hispidus .  Planta Med. 1999;  65 704-8
  Thieme ConnectPubMedSearch in Google Scholar

Dr. Jung Joon Lee

Anticancer Research Laboratory

Korea Research Institute of Bioscience and Biotechnology

P.O. Box 115

Yuseong

Daejeon 305-600

Korea

Fax: +82-42-860-4585

Email: jjlee@kribb.re.kr

References

• 1 Baldwin A S. The NF-kappaB and I kappa B proteins: new discoveries and insights.  Annu Rev Immunol. 1996;  14 649-83
  CrossrefPubMedSearch in Google Scholar
• 2 Baeuerle P A, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappaB transcription factor.  Cell. 1988;  53 211-7
  CrossrefPubMedSearch in Google Scholar
• 3 Baeuerle P A, Henkel T. Function and activation of NF-kappaB in the immune system.  Annu Rev Immunol. 1994;  12 141-79
  CrossrefPubMedSearch in Google Scholar
• 4 Makarov S S. NF-κB as a therapeutic target in chronic inflammation: recent advances.  Mol Med Today. 2000;  6 441-8
  CrossrefPubMedSearch in Google Scholar
• 5 Bensky D, Gamble A, Kaptchuk T J, Bensky L L. Chinese herbal medicine: Materia Medica. Seattle; Eastland Press 1993: pp 193-4
  Search in Google Scholar
• 6 Zhou B N, Bai N S, Lin L Z, Cordell G A. Sesquiterpene lactones from Inula britannica .  Phytochemistry. 1993;  34 249-52
  CrossrefPubMedSearch in Google Scholar
• 7 Park E J, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica .  Planta Med. 1998;  64 752-4
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Park E J, Kim Y, Kim J. Acetylated flavonol glycosides from the flower of Inula britannica .  J Nat Prod. 2000;  63 34-6
  CrossrefPubMedSearch in Google Scholar
• 9 Han J W, Lee B G, Kim Y K, Yoon J W, Jin H K, Hong S. et al . Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in RAW264.7 macrophages through the inactivation of NF-κB.  Br J Pharmacol. 2001;  133 503-12
  CrossrefPubMedSearch in Google Scholar
• 10 Lee C B. Illustrated Flora of Korea. Seoul; Hyangmoonsa 1993: p 729
  Search in Google Scholar
• 11 Jeske F, Luneck S, Jakupovic J. Secoeudesmanolides from Inula britannica .  Phytochemistry. 1993;  34 1647-9
  CrossrefPubMedSearch in Google Scholar
• 12 Dolejs L, Herout V. On terpenes. CXLV. Constitution of eupatoriopicrin, a germacranolide from Eupatorium cannabinum L.  Coll Czech Chem Commun. 1962;  27 2654-61
  PubMedSearch in Google Scholar
• 13 Uchiyama T, Miyase T, Ueno A, Usmanghani K. Terpenic glycosides from Pluchea indica .  Phytochemistry. 1989;  28 3369-72
  CrossrefPubMedSearch in Google Scholar
• 14 Koo T H, Lee J -H, Park Y J, Hong Y S, Kim H S, Kim K W. et al . A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-κB by targeting IκB phosphorylation.  Planta Med. 2001;  67 103-7
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Lee J -H, Koo T H, Hwang B Y, Lee J J. Kaurane diterpene, kamebakaurin, inhibits NF-kappa B by directly targeting the DNA-binding activity of p50 and blocks the expression of antiapoptotic NF-kappa B target genes.  J Biol Chem. 2002;  277 18 411-20
  PubMedSearch in Google Scholar
• 16 Schmidt H HHW, Kelm M. Determination of nitrite and nitrate by the Griess reaction. In: Feelisch M, Stramler J, editors Methods in nitric oxide research. New York; John Wiley & Sons Ltd 1996: pp 491-7
  Search in Google Scholar
• 17 Jin H Z, Lee J H, Lee D, Hong Y S, Kim Y H, Lee J J. Inhibitors of the LPS-induced NF-κB activation from Artemisia sylvatica .  Phytochemistry. 2004;  65 2247-53
  CrossrefPubMedSearch in Google Scholar
• 18 Al-Easa H S, Rizk A M, Ahmed A A. Guaianolides from Picris radicata .  Phytochemistry. 1996;  43 423-4
  CrossrefPubMedSearch in Google Scholar
• 19 Kamperdick C, Phuong N M, Adam G, Sung T V. Guaiane dimers from Xylopia vielana .  Phytochemistry. 2003;  64 811-6
  CrossrefPubMedSearch in Google Scholar
• 20 Kamperdick C, Phuong N M, Sung T V, Adam G. Guaiane dimers from Xylopia vielana .  Phytochemistry. 2001;  56 335-40
  CrossrefPubMedSearch in Google Scholar
• 21 Bohlmann F, Zdero C, King R M, Robinson H. Pseudoguaianolides and other sesquiterpene lactones from Gaillardia species.  Phytochemistry. 1984;  23 1979-88
  CrossrefPubMedSearch in Google Scholar
• 22 Gao F, Wang H, Mabry T J, Watson W H, Kashyap R P. Sesquiterpene lactones and a C₂₀ aliphatic lactone from Texas bitterweed, Hymenoxys odorata .  Phytochemistry. 1990;  29 551-60
  CrossrefPubMedSearch in Google Scholar
• 23 Ahmed A A, Ahmed A M. Jasonol, a rare tricyclic eudesmane sesquiterpene and six other new sesquiterpenoids from Jasonia candicans .  Tetrahedron. 1998;  54 8144-52
  PubMedSearch in Google Scholar
• 24 Dale J A, Mosher H S. Nuclear magnetic resonance enantiomer reagents. Configurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and α-methoxy-α-trifluoromethylphenylacetate (MTPA) esters.  J Am Chem Soc. 1973;  95 512-9
  CrossrefPubMedSearch in Google Scholar
• 25 Su B N, Park E J, Mbwambo Z H, Santarsiero B D, Mesecar A D, Fong H HS. et al . New chemical constituents of Euphorbia quinquecostata and absolute configuration assignment by a convenient Mosher ester procedure carried out in NMR tubes.  J Nat Prod. 2002;  65 1278-82
  CrossrefPubMedSearch in Google Scholar
• 26 Siedle B, Garcia-Pineres A J, Murillo R, Schulte-Mönting J, Castro V, Rüngeler P. et al . Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-kB.  J Med Chem. 2004;  47 6042-54
  CrossrefPubMedSearch in Google Scholar
• 27 Zidorn C, Dirsch V M, Rüngeler P, Sosa S, Della Loggia R, Merfort I. et al . Anti-inflammatory activities of hypocretenolides from Leotodon hispidus .  Planta Med. 1999;  65 704-8
  Thieme ConnectPubMedSearch in Google Scholar

Dr. Jung Joon Lee

Anticancer Research Laboratory

Korea Research Institute of Bioscience and Biotechnology

P.O. Box 115

Yuseong

Daejeon 305-600

Korea

Fax: +82-42-860-4585

Email: jjlee@kribb.re.kr

• Supplementary Material