Biflavans, Flavonoids, and a Dihydrochalcone from the Stem Wood of Muntingia calabura and Their Inhibitory Activities on Neutrophil Pro-Inflammatory Responses

Muntingia calabura (Tiliaceae) is commercially used in healthcare for the improvement of hypertension, myocardial infarction, spasm, and inflammatory conditions. Its fruits can be processed into jam and the leaves can be used for making tea. In the work reported herein a new biflavan, (M),(2S),(2''S)-,(P),(2S),(2''S)-7,8,3',4',5',7'',8'',3''',4''',5'''-decamethoxy-5,5'' biflavan (1), a new flavone, 4'-hydroxy-7,8,3',5'-tetramethoxyflavone (2), and a new dihydrochalcone, (R)-2',β-dihydroxy-3',4'-dimethoxydihydrochalcone (3), have been isolated from the stem wood of M. calabura, together with 12 known compounds (4–15). The structures of these new compounds were elucidated by the interpretations of extensive spectroscopic data. Among the isolated compounds, 5-hydroxy-7-methoxyflavone (5), quercetin (6), and (2S)-7-hydroxyflavanone (10) exhibited potent inhibition of fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 1.77 ± 0.70, 3.82 ± 0.46, and 4.92 ± 1.71 μM, respectively.


Introduction
Muntingia calabura L. (Tiliaceae) is an evergreen tree originally distributed in tropical America [1].In Mexico, the fruits of M. calabura are eaten and sold in markets.The fruits can be processed into jams and the leaves can be used for making tea.Past studies have revealed flavones, flavanones, flavans, and biflavans to be the major constituents of this species, some of which have displayed anti-platelet aggregation [2] and cytotoxic [3][4][5][6] activities.

Results
Chromatographic purification of the CH2Cl2-soluble fraction of a MeOH extract of stem wood of M. calabura on a silica gel column and preparative thin-layer chromatography (TLC) afforded three new compounds 1-3 and twelve known compounds 4-15.

Discussion
A new biflavan 1, a new flavone 2, a new dihydrochalcone 3, and twelve known compounds 4-15 were isolated from the stem wood of M. calabura.The structures of new compounds 1-3 were determined by NMR and MS analyses.Among the known isolates, compound 12 has been found for the first time in this plant species.More discovery of new compounds from the genus Muntingia may not only provide more structure-activity data of these isolates, but also contribute to enhancing our understanding of the taxonomy and evolution of the genus Muntingia.
Human neutrophils are known to play a significant role in the host defense against microorganisms and in the pathogenesis of various diseases such as ischemia-reperfusion injury, asthma, rheumatoid arthritis, and chronic obstructive pulmonary disease [21,22].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 [21,23,24].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 4), 5-hydroxy-7-methoxyflavone ( 5), quercetin (6), and (2S)-7-hydroxyflavanone (10) exhibited potent inhibition with IC50 values of 1.77 ± 0.70, 3.82 ± 0.46, and 4.92 ± 1.71 μM, respectively, against fMLP-induced superoxide anion generation.These findings indicated that the promising inhibitory activity against fMLP-induced O2 •− generation of M. calabura and its isolates (especially 5, 6, and 10) could stimulate future development of new anti-inflammatory agents.

Plant Material
The stem wood of M. calabura was collected from Changjhih Township, Pingtung County, Taiwan, in August 2011 and identified by Dr. J. J. Chen.A voucher specimen (MC-201108) 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 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) obtained by venipuncture were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes [25].Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclusion method [26], were re-suspended in a calcium (Ca 2+ )-free HBSS buffer at pH 7.4 and were maintained at 4 °C prior to use.The protocol was 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.

Measurement of Superoxide Anion Generation
The assay for measurement of O2 •− generation was based on the SOD-inhibitable reduction of ferricytochrome c [27].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 ferricytochrome 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.

Statistical Analysis
Results were 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
Fifteen compounds, including three new compounds 1-3, were isolated from the stem wood of M. calabura.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/CB-induced O2 •−

2 a
The IC 50 values were calculated from the slope of the dose-response curves (SigmaPlot).Values are expressed as average ± SEM (n = 4); b Ibuprofen was used as a positive control.
a Recorded in CDCl 3 at 100 MHz.b Recorded in DMSO-d 6 at 75.6 MHz.Values in ppm (δ).