New Coumarins and Anti-Inflammatory Constituents from the Fruits of Cnidium monnieri

The fruit of Cnidium monnieri is commercially used as healthcare products for the improvement of impotence and skin diseases. Three new coumarins, 3'-O-methylmurraol (1), rel-(1'S,2'S)-1'-O-methylphlojodicarpin (2), and (1'S,2'S)-1'-O-methylvaginol (3), have been isolated from the fruits of C. monnieri, together with 14 known compounds (4–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4–12, and 14–17 exhibited inhibition (IC50 ≤ 7.31 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 7, 9–11, 15, and 17 inhibited fMLP/CB-induced elastase release with IC50 values ≤7.83 µg/mL. This investigation reveals that bioactive isolates (especially 6, 7, 14, and 17) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.

In our studies on the anti-inflammatory constituents of Formosan plants, many species have been screened for in vitro inhibitory activity on neutrophil pro-inflammatory responses, and C. monnieri has been found to be an active species. The MeOH extract of the fruits of C. monnieri showed potent inhibitory effects on superoxide anion generation and elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB).  (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), have been isolated and identified from the fruits of C. monnieri and their structures are depicted in Figure 2.
This paper describes the structural elucidation of the compounds numbered 1 through 3, and the inhibitory activities of all isolates on superoxide generation and elastase release by neutrophils. Figure 1. The chemical structures of new compounds 1-3 isolated from C. monnieri. 5

Discussion
Seventeen compounds, including three new coumarins 1-3, were isolated from the fruits of C. monnieri. Known compounds 5, 7, and 9 were obtained from this plant for the first time. The structures of these compounds were established on the basis of spectroscopic data. Further discovery of new coumarins from the genus Cnidium may not only provide more structure-activity data of coumarins, but may also contribute to enhancing our understanding of the taxonomy and evolution of the genus Cnidium.
Reactive oxygen species (ROS) (e.g., superoxide anion (O 2 •− ), hydrogen peroxide) and granule proteases (e.g., elastase, cathepsin G) 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), osthol (6), osthenol (7), and imperatorin (14) were the most effective among these compounds, with IC 50 values of 0.005 ± 0.0002, 0.09 ± 0.01, and 0.07 ± 0.02 µg/mL, respectively, against fMLP-induced superoxide anion generation. Osthenol (7) and cnidimol A (17) exhibited the most effective among the isolates, with IC 50 values of 3.28 ± 0.90 and 3.20 ± 0.16 µg/mL, respectively, against fMLP-induced elastase release. Compounds 6, 7, 14, and 17 had been tested for their cytotoxicity on the NIH3T3 cell, where 6, 7, 14, and 17 showed no significant activities with ED 50 values >50 µg/mL. The above isolated compounds might support the traditional use of C. monnieri for the treatment of inflammatory processes. Thus, our study suggests C. monnieri and its isolates (especially 6, 7, 14, and 17) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.

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 (Taoyuan, Taiwan). All donors gave written consent. The Medical Ethics Committee of Chang Gung Memorial Hospital approved this consent procedure.

Plant Material
The fruits of C. monnieri were collected from Yanpu, Pingtung County, Taiwan, in October 2010 and identified by Jih-Jung Chen. A voucher specimen (CM-201010) 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 [23]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method [24], 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 [25,26]. In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ , neutrophils (6 × 10 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 [25,26]. Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), neutrophils (6 × 10 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.

Conclusions
Seventeen compounds, including three new coumarins (1-3), were isolated from the fruits of C. monnieri. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) (e.g., superoxide anion (O 2 •− ), hydrogen peroxide) and granule proteases (e.g., elastase, cathepsin G) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of isolates were evaluated by suppressing fMLP/CB-induced O 2 •− generation and elastase release by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 1, 4-12, and 14-17 can significantly inhibit fMLP-induced O 2 •− generation and/or elastase release. Osthol (6) and cnidimol A (17) were the most effective among the isolated compounds, with IC 50 values of 0.005 ± 0.0002 and 3.20 ± 0.16 µg/mL, respectively, against fMLP-induced O 2 •− generation and elastase release. Our study suggests C. monnieri and its isolates (especially 6, 7, 14, and 17) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.