Euphormins A and B, New Pyranocoumarin Derivatives from Euphorbia formosana Hayata, and Their Anti-Inflammatory Activity

Euphormin-A (1) and euphormin-B (2), two new pyranocoumarin derivatives, and forty known compounds (3–42) were isolated from Euphorbia formosana Hayata (Euphorbiaceae). The chemical structures of all compounds were established based on spectroscopic analyses. Several isolates were evaluated for their anti-inflammatory activity. Compounds 1, 2, 10, 18, 25, and 33 significantly inhibited against superoxide anion generation and elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB). Furthermore, compounds 25 and 33 displayed the most potent effects with IC50 values of 0.68 ± 0.18 and 1.39 ± 0.12 µM, respectively, against superoxide anion generation when compared with the positive control (2.01 ± 0.06 µM).


Euphormin-B (Compound 2)
Compound 2 was isolated as a brown crystal with an elemental composition of C 15 Table 1). The NMR data for 2 were similar to those of compound 1 except for the disappearance of a methoxy signal, suggesting that 2 is an analog of 1 ( Figure 2). The position of the methoxy group at C-1 was elucidated using the HMBC correlations of δ 3.74 (OCH 3 , s) to δ 168.2 (C-1) and of δ 7.03 (H-7) to δ 168.2 (C-1) and 117. In summary, a bioassay-guided separation of E. formosana roots resulted in the isolation of two new pyranocoumarins 1 and 2, showing potential anti-inflammatory activity. In addition, forty known compounds 3-42 were isolated and elucidated. The present study certainly enriches the chemical diversity and provides more chemotaxonomic evidence for E. formosana.

Anti-Inflammatory Activity
Overexpression of neutrophils has already been regarded to display significant correlations with various human diseases, such as rheumatoid arthritis, ischemia, reperfusion injury, chronic obstructive pulmonary disease, and asthma [52][53][54][55][56]. In response to diverse stimuli, activated neutrophils secreted a series of cytotoxins, such as superoxide anion and elastase [57]. This study evaluated several constituents inhibiting superoxide anion generation and elastase release in human neutrophils responding to fMLP/CB ( Table 2). The results showed that the new pyranocoumarin derivatives 1 and 2 were promising anti-inflammatory compounds against superoxide anion generation with IC 50 values of 4.51 ± 0.45 and 3.68 ± 0.05 µM, respectively, indicating that pyranocoumarins were the potential active anti-inflammatory components in the water fraction of this plant. More importantly, two polyphenolic compounds 25 and 33 exhibited more potent antiinflammatory activity against superoxide anion generation with IC 50 values of 0.68 ± 0.18 and 1.39 ± 0.12 µM, respectively. The results of compounds 25 and 33 suggested comparable anti-inflammatory activities with the positive control (2.01 ± 0.06 µM). Besides, compound 18 exhibited moderate inhibitory activity against elastase release. Based on our present study, the compounds isolated from E. formosana were promising candidates for further pharmaceutical developments as new anti-inflammatory entities.

General Experimental Procedures
Melting points were measured on a Fisher Scientific melting point apparatus and were uncorrected. UV spectra were recorded on a Hitachi UV-3010 spectrophotometer in MeOH solution. IR spectra were recorded on a Jasco FT-IR-410 spectrophotometer as KBr discs. The 1 H-and 13 C-NMR spectra were recorded on a Bruker Avance-400 spectrometer. Chemical shifts values are given with tetramethylsilane as an internal reference.

Plant Material
E. formosana roots were collected by Dr. Yi Jen Hsieh at Tzu Chi University, Hualien, Taiwan. A voucher specimen (No. EFR-1) was deposited at the Department of Laboratory Medicine and Biotechnology, School of Medicine, Tzu Chi University, Taiwan.

Preparation of Human Neutrophils
Blood was taken from healthy human donors (20-32 years old) by venipuncture using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Neutrophils were isolated by a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Purified neutrophils that contained > 98% viable cells, as determined by the Trypan blue exclusion method, were resuspended in calcium (Ca 2+ )-free Hank's balanced salt solution (HBSS) buffer at pH 7.4 and were maintained at 4 • C until use.

Measurement of Superoxide Anion Generation
The assay for the generation of superoxide anion was based on the SOD-inhibited reduction of ferricytochrome c [57,58]. In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ , neutrophils (6 × 10 5 cells/mL) were equilibrated at 37 • C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO) for 5 min. Cells were activated with 100 nM fMLP during preincubation with 1 µg/mL cytochalasin B (fMLP/CB) for 3 min. Changes in the 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). Calculations were based on the differences in 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
Azurophilic granule degranulation was determined by elastase release, as described previously [57,58]. 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-pnitroanilide (100 µM), neutrophils (6 × 10 5 /mL) were equilibrated at 37 • C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO, as control) for 5 min. Cells were activated by 100 nM fMLP and 0.5 µg/mL CB, and changes in absorbance at 405 nm were continuously monitored to assay elastase release. The results were expressed as the percentage elastase release in the fMLP/CB-activated, drug-free control system.

Statistical Analysis
Results were expressed as mean ± S.E.M. Computation of 50% inhibitory concentration (IC 50 ) was computer-assisted (PHARM/PCS v.4.2). Statistical comparisons were made between groups using the Student's t test. Values of p less than 0.05 were considered to be statistically significant

Conclusions
Two new compounds (1 and 2) and forty known compounds  were isolated from the roots of E. formosana. The chemical structures of these isolates were elucidated based on their spectroscopic data. The anti-inflammatory activity of the isolated compounds was evaluated. The results showed that compounds 1, 2, 10, 25, and 33 inhibited fMLP-induced superoxide generation. In addition, new compounds 1 and 2 showed promising antiinflammatory activity against superoxide anion generation, with IC 50 values of 4.51 ± 0.45 and 3.68 ± 0.05 µM, respectively. Among the isolates, compounds 25 and 33 were the most potent with IC 50 values of 0.68 ± 0.18 and 1.39 ± 0.12 µM, respectively, against superoxide anion generation. Furthermore, compound 18 exhibited good anti-inflammatory activity against elastase release, with IC 50 values of 8.07 ± 1.40 µM. Based on the above results, E. formosana should be a helpful herbal medicine for patients with the inflammationrelated disease.