Limonoids from the Seeds of Swietenia macrophylla and Their Anti-Inflammatory Activities

A new limonoid, swietemacrophin (1), was isolated from the seeds of Swietenia macrophylla, together with five known compounds 2–6. The structure of 1 was determined through extensive 1D/2D-NMR and mass-spectrometric analyses. Swietemacrophin (1), humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), 3-O-tigloylswietenolide (4), and swietemahonin E (5) exhibited inhibition (IC50 values ≤ 45.44 μM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Compounds 1, 4, 5, and swietenine (6) showed potent inhibition with IC50 values ≤ 36.32 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation.

Reactive oxygen species (ROS) [e.g., superoxide anion (O2 •− ), hydrogen peroxide] and granule proteases (e.g., elastase, cathepsin G) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Nitric oxide (NO) is a mediator in the inflammatory response involved in host defense [9]. Suppression of the extensive or inappropriate activation of neutrophils and/or macrophages by drugs has been proposed as a way to ameliorate inflammatory diseases. The effects on pro-inflammatory responses of isolates were evaluated by suppressing fMLP-induced O2 •− generation by human neutrophils and by inhibiting LPS-induced NO release by murine macrophages.
In a screening program searching for anti-inflammatory compounds from Formosan plants [10][11][12][13][14], S. macrophylla has been found to be an active species. The MeOH extract of the seed of S. macrophylla showed potent inhibitory effects on superoxide anion generation by human neutrophils in response to fMLP and on NO generation by murine macrophages in response to LPS. The structures of new compound, swietemacrophin (1) and five known compounds 2-6 have been isolated from the seed of S. macrophylla and identified and their structures are depicted in Figure 1. This paper describes the structural elucidation of the compound 1, and the anti-inflammatory activities of all isolates.

General
Chromatographic purification of the EtOAc-soluble fraction of a MeOH extract of the seeds of S. macrophylla on a silica gel column and preparative thin-layer chromatography (TLC) afforded the new compound 1 and five known compounds 2-6.

Structure Identification of the Known Isolates
The known isolates were readily identified by comparison of their physical and spectroscopic data (UV, IR, 1 H-NMR, [α]D, and MS) with those of the corresponding authentic samples or literature values. They include five limonoids: [16], swietemahonin E (5) [17], and swietenine (6) [16]. Compound 2 was isolated from S. macrophylla for the first time.

Biological Studies
Reactive oxygen species (ROS) [e.g., superoxide anion (O2 •− ), hydrogen peroxide] and granule proteases (e.g., elastase, cathepsin G) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Inhibition of neutrophil O2 •− generation by drugs has been proposed as a way to ameliorate inflammatory diseases. The anti-inflammatory effects of the isolated compounds from the seed of S. macrophylla were evaluated by suppressing fMet-Leu-Phe (fMLP)-induced O2 •− generation by human neutrophils. The anti-inflammatory activity data are shown in Table 3. LY294002, a phosphatidylinositol-3-kinase inhibitior, was used as a positive control for superoxide anion generation. From the results of our biological tests, the following conclusions can be drawn: (a) Swietemacrophin (4), and swietemahonin E (5) exhibited inhibition (IC50 ≤ 45.44 μM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). (b) Among the limonoid analogues 1-6, compounds 1, 2, and 5, with the 8α,30α-epoxy group, and compounds 3 and 4, with the double bond between C-8 and C-14, exhibited more effective inhibition than analogue 6, with the double bond between C-8 and C-30, against fMLP-induced O2 •− generation. (c) Humilinolide F (2) is the most effective among the isolated compounds, with IC50 = 27.13 ± 1.82 μM, against fMLP-induced superoxide anion generation. Table 3. Inhibitory effects of compounds 1-6 from the seed of S. macrophylla on superoxide radical anion generation by human neutrophils in response to fMet-Leu-Phe.
Nitric oxide (NO) is a mediator in the inflammatory response involved in host defense. The anti-inflammatory effects of the compounds isolated from the seed of S. macrophylla were also evaluated by suppressing lipopolysaccharide (LPS)-induced NO generation in murine macrophage. The inhibitory activity data of the isolated compounds 1-6 against LPS-induced NO generation by macrophages are compiled in Table 4. Quercetin which was reported to inhibit NO production by LPS-stimulated macrophage [18,19] was used as the positive control. From the results of our anti-inflammatory tests, the following conclusions could be drawn: (a) Swietemacrophin (1), 3-O-tigloylswietenolide (4), swietemahonin E (5), and swietenine (6) exhibited inhibition (IC50 ≤ 36.32 μM) of NO generation by murine macrophages in response to LPS. (b) Among the limonoid analogues 1-6, compound 1, without any substituted group at C-6, and compounds 4-6, with the 6-hydroxy group, exhibited more effective inhibition than their analogues 2 and 3, with the 6-acetoxy group, against LPS-induced NO generation. (c) Swietemahonin E (5) is the most effective among the isolated compounds, with IC50 = 29.70 ± 2.11 μM, against LPS-induced NO generation. (d) Cytotoxic effects were determined using the MTT assay. The high cell viability (>92%) indicated that the inhibitory activities of compounds 1, 4, 5, and 6 against LPS-induced NO production did not resulted from their cytotoxicities.

Discussion
A new limonoid, swietemacrophin (1), and five known compounds 2-6 were isolated from the seeds of S. macrophylla. The structure of new compound 1 was determined by NMR and MS analyses. Among the known isolates, compound 2 has been found for the first time in this plant species. The discovery of more new compounds from the genus Swietenia may not only provide more structure-activity data of the isolates, but may also contribute to enhancing our understanding of the taxonomy and evolution of the genus Swietenia.
Human neutrophils are known to play a significant role in the host defense against microorganisms and in the pathogenesis of various diseases such as asthma, rheumatoid arthritis, ischemia-reperfusion injury, and chronic obstructive pulmonary disease [20,21]. In response to different stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anion (O2 •− ), a precursor of other reactive oxygen species (ROS), and bioactive lipids [20,22,23]. Suppression of the extensive or 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 3) NO is a physiological and a pathological mediator thought to be involved in inflammation [9]. Swietemacrophin (1), 3-O-tigloylswietenolide (4), swietemahonin E (5), and swietenine (6) showed potent inhibition with IC50 values of 33.45 ± 1.88, 32.62 ± 3.27, 29.70 ± 2.11, and 36.32 ± 2.84 μM, respectively, against LPS-induced NO generation.
The above findings indicated that the promising inhibitory activity against fMLP-induced O2 •− generation and LPS-induced NO release of S. macrophylla and its isolates could stimulate future development of new anti-inflammatory agents.

Ethics Statement
Blood was taken from healthy human donors (20-30 years old) by venipuncture, using a protocol 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 seeds of S. macrophylla were collected from Tajen University, Pintung County, Taiwan, in

Biological Assay
The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation in fMLP-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 (20-30 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes [24]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method [25], were resuspended 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 O2 •− generation was based on the SOD-inhibitable reduction of ferricytochrome c [26]. In brief, neutrophils (1 × 10 6 cells/mL) pretreated with the various test agents at 37 °C for 5 min were stimulated with fMLP (1 μmol/L) in the presence of ferryicytochrome c (0.5 mg/mL). Extracellular O2 •− production was assessed with a UV spectrophotometer at 550 nm (Hitachi U-3010, Tokyo, Japan). The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control -resting) -(compound -resting)}/(control -resting) × 100. The software, SigmaPlot was used for determining the IC50 values.

Determination of NO Production
The murine macrophage cell line RAW264.7 was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Life Technologies Inc., Burlington, ON, Canada) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37 °C in a humidified 5% CO2 atmosphere with a 96-well flat-bottomed culture plate. After 24 h, the condition medium was replaced with fresh DMEM; and FBS. Then, compounds 1-6 (0, 1, 2.5, 5, 10, and 20 μg/mL) were added, respectively, in the presence of lipopolysaccharide (LPS; 1 μg/mL; Cat No: L-2654, Sigma-Aldrich Co., St. Louis, MO, USA) and incubated under the same condition for 24 h. The cultured cells were then centrifuged, and the supernatants were used for NO-production measurement. The supernatant was mixed with an equal volume of the Griess reagent (1% sulfanilamide, 0.1% N-(naphthalen-1-yl)ethylenediamine dihydrochloride in 2.5% H2PO4 soln.) and incubated for 10 min at room temperature. Nitrite concentration was determined by measuring the absorbance at 540 nm using an ELISA plate reader (Anthos Labtec Instruments, Salzburg, Austria) [27]. The percentage of NO inhibition of the test compound was calculated as follows: inhibitory rate (%) = (1 − (LPS/sample − untreated)/(LPS − untreated) × 100. All tests were run in triplicate and averaged. The data were expressed as a mean of three experiments. The software SigmaPlot was used for determining the IC50 values.

Cell Viability Assay
A MTT colorimetric assay was used to determine cell viability. The assay was modified from that of Mosmann [28]. The test is based upon the selective ability of living cells to reduce the yellow soluble salt, MTT, to a purple-blue insoluble formazan. MTT (Merck; dissolved in phosphate-buffered saline at 5 mg/mL) soln. was added onto the attached cells mentioned above (10 μL per 100 μL culture) and incubated at 37 °C for 4 h. Then, DMSO was added, and amount of colored formazan metabolite formed was determined by absorbance at 550 nm. The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

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
Six compounds, including a new compound 1, were isolated from the seed of S. macrophylla. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) [e.g., superoxide anion (O2 •− ), hydrogen peroxide] 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-induced O2 •− generation by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 1-5 can significantly inhibit fMLP-induced O2 •− generation. Humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), and 3-Otigloylswietenolide (4) are the most effective among the isolated compounds, with IC50 values of 27.13 ± 1.82, 29.36 ± 1.75, and 5.58 ± 2.12 μM, respectively, against fMLP-induced superoxide anion generation. Furthermore, compounds 1, 4, 5, and 6 showed potent inhibition with IC50 values of 33.45 ± 1.88, 32.62 ± 3.27, 29.70 ± 2.11, and 36.32 ± 2.84 μM, respectively, against LPS-induced NO generation. Thus, our study suggests S. macrophylla and its isolates could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.