Anti-Inflammatory Constituents from Bidens frondosa

A new polyacetylene glucoside (3E,5E,11E)-tridecatriene-7,9-diyne-1,2,13-triol-2-O-β-d-glucopyranoside (1), a new phenylpropanoid glucoside 2′-butoxyethylconiferin (2), and a new flavonoid glycoside 8,3′,4′-trihydroxyflavone-7-O-(6′′-O-p-coumaroyl)-β-d-glucopyranoside (3), have been isolated from Bidens frondosa together with fifty-three known compounds 4–56. The structures of these compounds were established by spectroscopic methods. mainly ESIMS, 1D- and 2D-NMR spectroscopic data. and comparison with literature data. Compounds 1–34, 36, 39, 43, 47, 51, and 52 were tested for inhibition of nuclear factor kappa B (NF-κB) in 293-NF-κB-luciferase report cell line induced by lipopolysaccharide (LPS), and compounds 1, 2, 3, 9, 15, 21, 24 and 51 were tested for the production of TNF-α, IL-1β, IL-6, IL-10 in RAW 264.7 macrophages induced by LPS. In conclusion, the isolated compounds 1, 2, 3, 9, 15, 21, 24 and 51 exhibited significant activity in anti-inflammatory activity assays.


Introduction
Bidens frondosa (L.) is an annual herbaceous plant growing widely on nutrient-rich mudsoils or muddy sandsoils at the shores of rivers and lakes, which is one of species of genus Bidens from the family Asteraceae. It is native to North America, and now distributed throughout China. It has attracted a great deal of attention for its wide range of biological activities, such as antibacterial [1], antioxidant [2], antimalarial [3] and antidiarrheal [4]. Previous studies reported that B. frondosa contained acetylene [3], polyenic D-glucosides [5,6], chalcones and aurones [2,3,7,8], flavones and terpenes [9]. However, a literature survey reveals that there are no reports on the anti-inflammatory activity of B. frondosa, and the research on chemical constituents of B. frondosa is also limited. Thus we decided to isolate chemical constituents of B. frondosa and to investigate its anti-inflammatory activity.

Anti-Inflammatory Activity
Compounds 1-34, 36, 39, 43, 47, 51, and 52 were evaluated for their anti-inflammatory activities in a luciferase assay (Table S1). Compared with 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 have found the luciferase activity of compounds (1, 2, 3, 9, 15, 21, 24 and 51) group were significantly decreased by comparing with the LPS group, which showed that they had significant inhibitory effect on NF-κB activity. In addition, the luciferase activity decreased with the increase of sample concentration, which indicated that the inhibition of NF-κB activity was dose-dependent. In conclusion, compounds 1, 2, 3, 9, 15, 21, 24 and 51 showed significant inhibitory effect on NF-κB in 293-NF-κB-luciferase report cell line induced by LPS (Figure 4). The effects of compounds 1, 2, 3, 9, 15, 21, 24 and 51 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. We have found the content of inflammatory cytokines TNF-α, IL-6, IL-1, IL-10 of the LPS group were significantly increased by comparing with the experimental results of the Cell group, which indicated that the monocyte RAW264.7 induced by LPS was constructed successfully. Compared with the LPS group, compounds 1, 2, 3, 9, 15, 21, 24 and 51 exhibited significant inhibitory activity on the production of above inflammation factors tested in vitro at concentrations of 1, 10 and 100 μg/mL, and the inhibition activity was dose-dependent ( Figures 5-8).

Plant Material
The plant of B. Frondosa was collected from Jiujiang, Jiangxi Province, during July 2013, and identified by Ceming Tan (Jiujiang Forest Herbarium, Jiangxi, China). A voucher specimen (No. 20130729) was deposited at the Department of Pharmacognosy of the Second Military Medical University.

Extraction and Isolation
The air-dried aerial parts of B. frondosa (10 kg) were extracted three times with 80% EtOH (100 L) under reflux. After removal of the solvent by evaporation under vacuum, the residue was suspended in water (10 L) and then successively partitioned with petroleum ether, EtOAc and n-BuOH (3 × 15 L), respectively.

Luciferase Assay
The NF-κB 293 cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum (FBS). The cells were pretreated with these forty compounds at concentrations of 1, 10 and 100 μg/mL for 4 h and then stimulated with 10 μg/mL lipopolysaccharide (LPS) for 24 h. The cells were rinsed twice with phosphate-buffered saline (PBS, pH 7.4) and lysed with passive lysis buffer (Promega, Madison, WI, USA). Then inhibitory effect on NF-κB was analyzed using the luciferase assay system (Promega) according to the manufacturer′s instructions [60].

Measurement of TNF-α, IL-1β, IL-6, and IL-10
The cells were cultured in serum-free medium for 8 h and then incubated in medium containing 1, 10 and 100 μg/mL of compounds 1, 2, 3, 9, 15, 21, 24 and 51 for 2 h. The cells were then treated with 10 μg/mL of LPS for 24 h. Ibuprofen (1, 10 and 100 μg/mL) was used as a positive control. The supernatants of cell culture were harvested and centrifuged at 3000× g at 4 °C for 2 min for the analysis of TNF-α, IL-1β, IL-6, and IL-10. Enzyme-linked immunosorbent assays for detecting the cytokines in the supernatants were carried out according to the instructions provided by the manufacturer. Finally, the standard provided with the kits was used to quantify each cytokine in the supernatants [61].    Compounds 1, 2, 3, 9, 15, 21, 24 and 51 showed significant activities in anti-inflammatory assays. And they exhibited good anti-inflammatory effects in a dose-dependent manner. The observed potential anti-inflammatory activity warrants further investigations.