New Labdane-Type Diterpenoids and Anti-Inflammatory Constituents from Hedychium coronarium

Four new labdane-type diterpenoids: hedychicoronarin (1), peroxycoronarin D (2), 7β-hydroxycalcaratarin A (3), and (E)-7β-hydroxy-6-oxo-labda-8(17),12-diene-15,16-dial (4), have been isolated from the rhizomes of Hedychium coronarium, together with 13 known compounds (5–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 3, 5, 6, and 10 exhibited inhibition (IC50 values ≤4.52 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3–6, 10, and 11 inhibited fMLP/CB-induced elastase release with IC50 values ≤6.17 μg/mL.


Introduction
Hedychium coronarium Koenig (Zingiberaceae) is a perennial herb distributed in India, Southeast Asian countries, southern China, Japan, and Taiwan [1]. H. coronarium, popularly called "White Butterfly Flower" or "Butterfly Ginger", is used as a folk medicine for treatment of headache, contusion, inflammation, insomnia, stomach disorders, and sharp pain due to rheumatism in China [2,3].
Human neutrophils are known to play crucial roles in host defence against microorganisms and in pathogenesis of different diseases, such as rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), asthma, and ischemia-reperfusion injury [21][22][23][24]. In response to diverse stimuli, activated neutrophils secrete a series of cytotoxins, such as the superoxide anion radical (O 2 •− ), a precursor to other reactive oxygen species (ROS), granule proteases, bioactive lipids, and neutrophil elastase, a major contributor to destruction of tissue in chronic inflammatory disease [21,25,26]. Suppression of the extensive or inappropriate activation of neutrophils by drugs has been proposed as a method to ameliorate inflammatory diseases. The effects on neutrophil pro-inflammatory responses of compounds isolated from the rhizomes of H. coronarium 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. LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive control for superoxide anion generation and elastase release. Diphenyleneiodonium, an NADPH oxidase inhibitor, was used as a positive control for superoxide anion generation. From the results of our biological tests, the following conclusions can be drawn:  2, and 6-9), coronarin A (6) (with a hydroxy group at C-7 and a furan-3-yl group at C-12) exhibited more effective inhibition than its analogues, 1, 2, and 7-9 (without any substituent at C-7 and with a 2-oxo-5-substituted-tetrahydrofuran-3-yl group at C-12) against fMLP-induced O 2 •− generation and elastase release. (g) Calcaratarin A (5) displayed the most effective among the isolates, with IC 50 values of 2.25 ± 0.42 and 2.36 ± 0.41 μg/mL, respectively, against fMLP-induced O 2 •− generation and elastase release. Our study suggests H. coronarium and its isolates (especially 3, 5, 6, and 10) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases. Thus, the detailed mechanism of action of these compounds appears worthy of further investigation.

Plant Material
The rhizomes of H. coronarium were collected from Taitung District Agricultural Research and Extension Station, Taitung County, Taiwan, in June 2009 and identified by J. F. Chen. A voucher specimen (Chen 3011) 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. LY294002 (purity >99%, Sigma, St. Louis, MO, USA) was used as a positive control.

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 [27]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method [28], 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 [29,30]. 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 [30]. 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.