New Flavones, a 2-(2-Phenylethyl)-4H-chromen-4-one Derivative, and Anti-Inflammatory Constituents from the Stem Barks of Aquilaria sinensis

In the current study, two new flavones, 4′-O-geranyltricin (1) and 3′-O-geranylpolloin (2), and a new 2-(2-phenylethyl)-4H-chromen-4-one derivative, 7-hydroxyl-6-methoxy-2-(2-phenylethyl)chromone (3), have been isolated from the stem barks of A. sinensis, together with 21 known compounds 4–24. The structures of new compounds 1–3 were determined through spectroscopic and MS analyses. Compounds 2, 3, 5, 6, and 8–10 exhibited inhibition (IC50 ≤ 12.51 μM) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3, 6, 8, 10, and 19 inhibited fMLP/CB-induced elastase release with IC50 values ≤ 15.25 μM. This investigation reveals bioactive isolates (especially 2, 3, 5, 6, 8, 9, 10, and 19) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.


Results
Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of stem barks of A. sinensis on a silica gel column and preparative thin-layer chromatography (TLC) afforded three new compounds 1-3 and twenty-one known compounds 4-24.

Results
Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of stem barks of A. sinensis on a silica gel column and preparative thin-layer chromatography (TLC) afforded three new compounds 1-3 and twenty-one known compounds 4-24.
Human neutrophils are known to play an important role in the host defense against microorganisms and in the pathogenesis of various diseases such as rheumatoid arthritis, ischemia-reperfusion injury, asthma, and chronic obstructive pulmonary disease [46,47]. In response to different stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anion (O 2 ‚´) , a precursor of other reactive oxygen species (ROS), granule proteases, bioactive lipids [46,48,49]. Suppression of the extensive or inappropriate activation of neutrophils by drugs has been proposed as a way to ameliorate inflammatory diseases. Reactive oxygen species (ROS) [e.g., superoxide anion (O 2 ‚´) and hydrogen peroxide] and granule proteases (e.g., elastase, cathepsin G, and proteinase-3) produced by human neutrophils are involved in the pathogenesis of a variety of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of compounds isolated from the stem barks of A. sinensis were evaluated by suppressing fMet-Leu-Phe/ cytochalasin B (fMLP/CB)-induced superoxide anion (O 2 ‚´) generation and elastase release by human neutrophils. The inhibitory activity data on neutrophil pro-inflammatory responses are summarized in Table 1.   1, 2, and 4-8), only apigenin 7,4 1 -dimethyl ether (7) (with a 4 1 -methoxyphenyl moiety) at 10 µg/mL alone elicited superoxide anion generation and elastase release by human neutrophils in the absence of fMLP/CB; (f) sakuranetin (9) and velutin (6) were the most effective among the isolated compounds, with IC 50 values of 1.74˘0.17 and 1.78˘0.35 µM, against fMLP-induced superoxide anion generation; (g) 7-hydroxy-6-methoxy-2-(2-phenylethyl)chromone (3), velutin (6), and 3 1 -hydroxygenkwanin (8) were the most effective among the isolated compounds, with IC 50 value of 3.91˘0.87, 4.26˘0.12, and 4.56˘0.63 µM, against fMLP-induced elastase release.
Granule proteases (e.g., elastase, cathepsin G) and reactive oxygen species (ROS) e.g., superoxide anion (O 2 ‚´) , hydrogen peroxide] and produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Inhibition of the inappropriate activation of neutrophils by drugs has been proposed as a way to ameliorate inflammatory diseases. Based on the results of our biological tests (Table 1) Our study suggests A. sinensis and its isolates (especially 3, 5, 6, and 8-10) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. More experiments should be performed to deduce the action modes of these compounds.

Ethics Statement
Blood was taken from healthy human donors (20-30 years old) by venipuncture, using a protocol (No. 102-1595A3) approved by the Institutional Review Board at Chang Gung Memorial Hospital. All donors gave written consent. The Medical Ethics Committee of Chang Gung Memorial Hospital approved this consent procedure.

Plant Material
The stem barks of A. sinensis was collected from Pingtung County, Taiwan, in August 2013 and identified by Prof. J.J. Chen. A voucher specimen (AS 201308) was deposited in the Department of Pharmacy, Tajen University, Pingtung, Taiwan.

Biological Assay
The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation and elastase release in fMLP/CB-activated human neutrophils in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS.

Preparation of Human Neutrophils
Human neutrophils from venous blood of healthy, adult volunteers (20-28 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes [50]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method [51], were re-suspended in a calcium (Ca 2+ )-free HBSS buffer at pH 7.4 and were maintained at 4˝C prior to use.

Measurement of Superoxide Anion Generation
The assay for measurement of superoxide anion generation was based on the SOD-inhibitable reduction of ferricytochrome c [52,53]. In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ , neutrophils (6ˆ10 5 /mL) were equilibrated at 37˝C for 2 min and incubated with different concentrations (10-0.01 µg/mL) of compounds or DMSO (as control) for 5 min. Cells were incubated with cytochalasin B (1 µg/mL) for 3 min prior to the activation with 100 nM formyl-L-methionyl-L-leucyl-L-phenylalanine for 10 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).

Measurement of Elastase Release
Degranulation of azurophilic granules was determined by measuring elastase release as described previously [53,54]. Experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM), neutrophils (6ˆ10 5 /mL) were equilibrated at 37˝C for 2 min and incubated with compounds for 5 min. Cells were stimulated with fMLP (100 nM)/CB (0.5 µg/mL), and changes in absorbance at 405 nm were monitored continuously in order to assay elastase release. The results were expressed as the percent of elastase release in the fMLP/CB-activated, drug-free control system.

Statistical Analysis
Results are expressed as the mean˘SEM, and comparisons were made using Student's t-test. A probability of 0.05 or less was considered significant. The software SigmaPlot was used for the statistical analysis.