New Sesquiterpenenoids from Ainsliaea yunnanensis

Investigation of the ethanol extract of the whole plant of Ainsliaea yunnanensis led to the isolation of four new dimeric sesquiterpene lactones, ainsliadimer F–I (1–4), together with seven known dimeric sesquiterpene lactones (5–11) and ten sesquiterpenes (12–21). Their structures were elucidated by spectroscopic methods. The relative stereochemistry of ainsliadimer F was further confirmed by single crystal X-ray diffraction analysis. Compounds 1–21 were tested for the inhibition of nuclear factor kappa B (NF-κB) in the 293-NF-κB-luciferase reporter cell line induced by lipopolysaccharide (LPS), and Compounds 5, 18, 20 and 21 were further tested for the production of TNF-α, IL-1β, IL-6 and IL-10 in RAW 264.7 macrophages induced by LPS. Compounds 5, 18, 20 and 21 exhibited significant activity in anti-inflammatory activity assays.


Introduction
The plants of Asteraceae are well known to contain biologically-active sesquiterpenoids [1][2][3][4].Within this family, the genus Ainsliaea comprises 70 species, 48 of which are indigenous to China.Phytochemical studies have indicated that sesquiterpenoids are characteristic constituents of Ainsliaea species [5][6][7][8], and guaiane-type sesquiterpene, as one of the most important chemical types of sesquiterpenes, has attracted much interest due to the structural diversity and varied biological activities, such as anti-inflammatory [9] and antitumor [10,11].A. yunnanensis, a plant of Ainsliaea, is mainly distributed in Yunnan province of China, which has been used in Chinese folk medicine to treat rheumatism, lumbago and gonitis [12].Some chemical constituents of this plant have been reported previously [12,13].
Their structures were show in Figure 1.These compounds were tested for anti-inflammatory activities, and Compounds 5, 18, 20 and 21 exhibited significant activities in anti-inflammatory activity assays.These compounds were tested for anti-inflammatory activities, and Compounds 5, 18, 20 and 21 exhibited significant activities in anti-inflammatory activity assays.

Structure Elucidation of New Compounds
Compound 1 was obtained as colorless prisms, and its molecular formula was established to be C30H34O7 by its HRESI (m/z 529.2206 [M + Na] + ) and 13 C-NMR spectra.The 13

Structure Elucidation of New Compounds
Compound 1 was obtained as colorless prisms, and its molecular formula was established to be C 30 H 34 O 7 by its HRESI (m/z 529.2206 [M + Na] + ) and 13 C-NMR spectra.The 13   ).The structure and relative configuration of 1 were further confirmed by the X-ray diffraction analysis (Figure 2).Compound 1 was named as ainsliadimer F. and 150.2 were ascribed to three exocyclic double bonds; the signals at δC 83.9, 82.6 were ascribed to two oxygen-substituted methines; the signal at δC 68.4 was linked to one oxygen atom; and the signal at δC 59.2 was ascribed to one methoxyl group.Analysis of NMR data (Table 1) revealed a dimeric sesquiterpene skeleton consisting of guaianolide moieties, and it was quite close to the known compound ainsliadimer D (10) [17], except for a methoxy group in Compound 2, replacing the hydroxyl group in ainsliadimer D. This was confirmed by the HMBC correlations of C-1′′(δC 59.2) with H-15 (δH 3.94, 3.74).The structure of 2 was established on the basis of 1D-and 2D-NMR spectra ( 1 H, 13 C, DEPT, COSY, HSQC and HMBC).Accordingly, the structure of Compound 2 was named as ainsliadimer G. Compound 2 was a natural chemical constituent and not an isolation artifact, which was tested by HPLC (Figure S30) in a crude extract.and 150.2 were ascribed to three exocyclic double bonds; the signals at δ C 83.9, 82.6 were ascribed to two oxygen-substituted methines; the signal at δ C 68.4 was linked to one oxygen atom; and the signal at δ C 59.2 was ascribed to one methoxyl group.Analysis of NMR data (Table 1) revealed a dimeric sesquiterpene skeleton consisting of guaianolide moieties, and it was quite close to the known compound ainsliadimer D (10) [17], except for a methoxy group in Compound 2, replacing the hydroxyl group in ainsliadimer D. This was confirmed by the HMBC correlations of C-1 ˝1(δ C 59.2) with H-15 (δ H 3.94, 3.74).The structure of 2 was established on the basis of 1D-and 2D-NMR spectra ( 1 H, 13 C, DEPT, COSY, HSQC and HMBC).Accordingly, the structure of Compound 2 was named as ainsliadimer G. Compound 2 was a natural chemical constituent and not an isolation artifact, which was tested by HPLC (Figure S30) in a crude extract.Compound 3 was obtained as a colorless powder and showed the [M + Na] + ion peak at m/z 571.2310 in its HRESI, corresponding to a molecular formula of C 32 H 36 O 8 .The 13 C-NMR spectrum showed 32 carbon resonances, classified into eleven quaternary carbons, eight methines, including two oxygen-substituted carbons, twelve methylenes, including two oxygen-bearing carbons, and one methyl.Analysis of NMR data (Table 1) showed it to be quite close to Compound 2, except for an ethoxy group in Compound 3 replacing a methoxy group in Compound 2. This was confirmed by HMBC correlations of H-15 (δ H 3.96, 3.76) with C-1" (δ C 66.8).Accordingly, the structure of Compound 3 was named as ainsliadimer H. Compound 3 was a natural chemical constituent, which was also tested by HPLC (Figure S31) in a crude extract.
Compound 4 was obtained as colorless prisms, and its molecular formula was established to be C 30 H 32 O 7 by its HRESI (m/z 527.2043 [M + Na] + ) and 13 C-NMR spectra.The 13 C-NMR spectrum exhibited 30 carbon resonances, classified into eleven quaternary carbons, eight methines, including two oxygen-substituted carbons, ten methylenes and one methyl.The 1 H-and 13 C-NMR spectra of 4 were quite close to those of ainsliadimer D (10) [17], except for one hydroxymethyl at the position C-4 in ainsliadimer D was replaced by one methyl in Compound 4. This was confirmed by HMBC correlations of Me-15 (δ H 1.30) with C-3 (δ C 208.4), C-4 (δ C 47.0), C-5 (δ C 55.1).The structure of 4 was established on the basis of 1D-and 2D-NMR spectra and was named ainsliadimer I.
The guaiane-type sesquiterpenes have wide-spread occurrence in the genus Ainsliaea species.Among them, Compounds 1-11 were dimeric guaiane-type sesquiterpene lactones; they were used as important chemotaxonomic markers in this genus [15][16][17].They are generally biosynthesized from two monomeric sesquiterpene lactones through a six-membered cyclohexene of the 3,4-dihydro-2H-pyran ring via hetero Diels-Alder cycloaddition reactions, except ainsliadimer A (7), which represents the single dimeric sesquiterpene lactone with a five-membered cyclopentane ring [16].On the other hand, Compound 11 has been reported to be present in the genera Gochnatia; we now report its presence for the first time in the Ainsliaea family, although the other dimeric guaiane-type sesquiterpene lactones were only isolated from Ainsliaea species.This is an interesting finding, as Gochnatia could be an alternative source of these compounds.
To conclude, the isolation of different sesquiterpene lactones is consistent with the chemical profile of the other Ainsliaea species.Of these, dimeric guaiane-type sesquiterpene lactones structurally characterized by a six-membered ring are systematically important and might serve as the common characteristic constituents of both Ainsliaea and Asteraceae families.

Evaluation of Biological Activity
Compounds 1-21 were assessed for inhibitory activity in a luciferase assay.Compared to the cell group, the luciferase activity of the LPS group was significantly enhanced, which indicated that the inflammatory cell model induced by LPS was constructed successfully.Then, we found that the luciferase activity of Compounds 5, 18, 20 and 21 was significantly decreased by comparing to the LPS group, which showed that they had significant inhibitory effect on NF-κB activity.The effects of Compounds 5, 18, 20 and 21 on the inflammatory response were investigated further.The anti-inflammatory effects were evaluated by investigating the inhibitory activity of the compounds on the production of TNF-α, IL-1β, IL-6 and IL-10 in RAW 264.7 macrophages induced by LPS.For all assays, ibuprofen was used as a positive control.Compared to the LPS group, these compounds were tested their inhibitory ratio of TNF-α, IL-1β, IL-6 and IL-10 release in LPS-stimulated RAW 264.7 macrophages in vitro at three different concentration (1 µg/mL, 10 µg/mL, 100 µg/mL).The concentration of TNF-α in the RAW 264.7 cells pretreated with Compounds 5, 18, 20 and 21 was reduced to 67.9%, 66.5%, 65.7% and 63.2% at 10 µg/mL and 33%, 15.5%, 16.9% and 37.7% at 100 µg/mL, respectively, while the inhibitory rates of the positive control ibuprofen were 73.8% and 23.0%.In the same way, the concentrations of IL-1β, IL-6 and IL-10 in the RAW 264.7 cells pretreated with Compounds 5, 18, 20 and 21 have a similar effect at the same concentration.The inhibition activity was dose-dependent (Figures 3-7).The results suggested that the basal skeleton of guaiane-type sesquiterpene lactone (18, 20, 21) might be crucial for the inhibitory activity on NF-κB production in RAW 264.7 cells.The functional groups such as hydroxyl groups linked at C-8 were found to decrease the inhibitory activity.The presence of the double bonds from the C-4 and C-11 contribution to the NF-κB decreased the activity according the results of zaluzanin C (14) and dihydroestafiatol (21), while the glucosyl group at C-3 slightly rose the inhibitory activity on NF-κB production.The significant inhibitory activity of gochnatiolide A (5) might be owed to the hydroxyl group linked at C-10 and the decrease of the methyl groups compared to other dimeric guaiane-type sesquiterpene lactones.The possible structure-activity relationships of these compounds should be further studied in the future, and it will be a significant topic.

Plant Material
The plant materials of A. yunnanensis were collected from Chuxiong, Yunnan province, during June 2013, and authenticated by Professor Ceming Tan (Jiujiang Forest Herbarium, Jiujiang, China).A voucher specimen (No. 20130629) was deposited at the Department of Pharmacognosy of the Second Military Medical University.

Extraction and Isolation
The air-dried A. yunnanensis (15 kg) was extracted three times with 80% ethanol (150 L) under reflux.After removal of the solvent by evaporation under vacuum, the residue was suspended in water (15 L) and then successively partitioned with petroleum, EtOAc and n-BuOH (3 ˆ15 L), respectively.

Figure 2 .
Figure 2. Single-crystal X-ray structure of Compound 1. Compound 2 was isolated as colorless prisms.The molecular formula of Compound 2 was determined as C31H34O8 from the quasi-molecular ion peak [M + Na] + at m/z 557.2148 in its positive HRESI.The 13 C-NMR spectrum exhibited 31 carbon resonances, classified into eleven quaternary carbons, eight methines, including two oxygen-substituted carbons, eleven methylenes and one methoxyl group.The signals at δC 206.3, 222.5 were ascribed to two ketone groups.The signals at δC 170.1, 169.4 were ascribed to two ester carbonyls.The signals at δC 114.1, 119.2, 121.5, 138.6, 139.5and 150.2 were ascribed to three exocyclic double bonds; the signals at δC 83.9, 82.6 were ascribed to two oxygen-substituted methines; the signal at δC 68.4 was linked to one oxygen atom; and the signal at δC 59.2 was ascribed to one methoxyl group.Analysis of NMR data (Table1) revealed a dimeric

Molecules 2016 ,
21, 1031 6 of 11LPS group, which showed that they had significant inhibitory effect on NF-κB activity.The effects of Compounds 5, 18, 20 and 21 on the inflammatory response were investigated further.The anti-inflammatory effects were evaluated by investigating the inhibitory activity of the compounds on the production of TNF-α, IL-1β, IL-6 and IL-10 in RAW 264.7 macrophages induced by LPS.For all assays, ibuprofen was used as a positive control.Compared to the LPS group, these compounds were tested their inhibitory ratio of TNF-α, IL-1β, IL-6 and IL-10 release in LPS-stimulated RAW 264.7 macrophages in vitro at three different concentration (1 μg/mL, 10 μg/mL, 100 μg/mL).The concentration of TNF-α in the RAW 264.7 cells pretreated with Compounds 5, 18, 20 and 21 was reduced to 67.9%, 66.5%, 65.7% and 63.2% at 10 μg/mL and 33%, 15.5%, 16.9% and 37.7% at 100 μg/mL, respectively, while the inhibitory rates of the positive control ibuprofen were 73.8% and 23.0%.In the same way, the concentrations of IL-1β, IL-6 and IL-10 in the RAW 264.7 cells pretreated with Compounds 5, 18, 20 and 21 have a similar effect at the same concentration.The inhibition activity was dose-dependent (Figures3-7).