Anti-Inflammatory and Neuroprotective Constituents from the Peels of Citrus grandis

A series of chromatographic separations performed on the ethanol extracts of the peels of Citrus grandis has led to the characterization of forty compounds, including seventeen coumarins, eight flavonoids, two triterpenoids, four benzenoids, two steroids, one lignan, one amide, and five other compounds, respectively. The chemical structures of the purified constituents were identified on the basis of spectroscopic elucidation, including 1D- and 2D-NMR, UV, IR, and mass spectrometric analysis. Most of the isolated compounds were examined for their inhibition of superoxide anion generation and elastase release by human neutrophils. Among the isolates, isomeranzin (3), 17,18-dihydroxybergamottin (12), epoxybergamottin (13), rhoifolin (19), vitexicarpin (22) and 4-hydroxybenzaldehyde (29) displayed the most significant inhibition of superoxide anion generation and elastase release with IC50 values ranged from 0.54 to 7.57 μM, and 0.43 to 4.33 μM, respectively. In addition, 7-hydroxy-8-(2′-hydroxy-3′-methylbut-3′-enyl)coumarin (8) and 17,18-dihydroxybergamottin (12) also exhibited the protection of neurons against Aβ-mediated neurotoxicity at 50 μM.


Introduction
The genus Citrus is composed of various species grown all over the tropical and subtropical regions. Each species has its own characteristic aroma and some Citrus peel oils have been used
In the primary screening of the neuroprotective activity of the isolated compounds, 50 µM of the compound was used. Alternatively, 10 µM of the compound was used as this compound possessed neurotoxicity at 20 µM (Figure 2A) [68]. The results showed that 7-hydroxy-8-(2 -hydroxy-3methylbut-3 -enyl)coumarin (8) and 17,18-dihydroxybergamottin (12) (Figure 1) protected neurons against Aβ-mediated neurotoxicity. The concentration dependency of neurons protection was further examined for compounds 8 and 12 and the experimental results displayed a dose-dependent manner as shown in Figure 2B,C.   The present experimental data suggest that the extracts and purified compounds of the peel of C. grandis have the potential to be developed as novel anti-inflammatory lead drugs or health foods. In addition, some purified constituents also exhibited the protection of neurons against Aβ-mediated neurotoxicity. It merits further investigations of the anti-inflammatory and neuroprotective mechanism of these natural heterocyclic compounds.   50 ). Results are presented as means ± SD. (n = 3~5). *** p < 0.001 compared with the control value. b Sorafenib, a tyrosine kinase inhibitor, was used as a positive control.
The present experimental data suggest that the extracts and purified compounds of the peel of C. grandis have the potential to be developed as novel anti-inflammatory lead drugs or health foods. In addition, some purified constituents also exhibited the protection of neurons against Aβ-mediated neurotoxicity. It merits further investigations of the anti-inflammatory and neuroprotective mechanism of these natural heterocyclic compounds.

General Information
Melting points were determined using an MP-S3 apparatus (Yanaco, Tokyo, Japan). UV spectra were recorded at room temperature on a U-0080-D spectrophotometer (Hitachi, Tokyo, Japan). IR spectra were obtained with a FT-IR Spectrum RX I spectrophotometer (PerkinElmer, Waltham, MA, USA). Optical rotations were measured using a P-2000 digital polarimeter (JASCO, Tokyo, Japan). 1 H-and 13 C-NMR spectra were recorded on Avance III HD 700 and Avance III 400 NMR spectrometers (Bruker, Billerica, MA, USA). Chemical shifts are shown in δ values (ppm) with tetramethylsilane as an internal standard. The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer (positive-ion mode). Column chromatography (CC) was performed on silica (70-230 mesh and 230-400 mesh, Merck, Darmstadt, Germany), Diaion HP-20 (Mitsubishi, Tokyo, Japan), and C 18 (Sigma-Aldrich, St. Louis, MO, USA) gels, respectively, and preparative TLC (thin-layer chromatography) was conducted on Merck precoated silica gel 60 F254 plates, using UV light to visualize the spots. High-performance liquid chromatography (HPLC) was performed on an LC-20AT series pumping system (Shimadzu, Kyoto, Japan) equipped with a Shimadzu SPD-20A UV-vis detector, and a SIL-10AF auto-sampling system at ambient temperature.

Materials
The peels of C. grandis were bought from the market in Tainan, Taiwan in 2010 and identified by Prof. Chang-Sheng Kuoh, Department of Life Science, National Cheng Kung University (NCKU), Tainan, Taiwan. A voucher specimen (Wu-2010009) was stored in School of Pharmacy, NCKU.

Extraction and Isolation
The dried peels of C. grandis (1.0 kg) were pre-treated with EtOH for one day at room temperature and then extracted with 95% EtOH five times (10 L × 5 h) at room temperature. The resulting extract was then filtered and concentrated in vacuo to yield a crude extract. The EtOH extract (260 g) was suspended in distilled water and successively partitioned with EtOAc yielding an EtOAc layer (20 g) and water layer (240 g). The EtOAc-soluble fraction was subjected to silica gel CC using a gradient solvent system of n-hexane-acetone (8:1, 4:1, 1:1 and MeOH) to afford ten fractions (F1~F10) according to TLC monitoring. Recrystallization of water layer produced the solids myo-inositol (37) (10.3 mg) and scyllo-inositol (38) (8.2 mg). The mother liquid of water layer was applied to CC on Diaion HP-20 gel using a gradient of increasing MeOH in H 2 O to yield eight fractions (W1~W8).

Preparation of Human Neutrophils
A study involving human neutrophils was approved by the Institutional Review Board at Chang Gung Memorial Hospital, Taoyuan, Taiwan, and was conducted according to the Declaration of Helsinki (2013). The written informed consent was obtained from each healthy donor before blood was drawn. Blood was drawn from healthy human donors (20-30 years old) by venipuncture into heparin-coated vacutainer tubes, using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. Blood samples were mixed gently with an equal volume of 3% dextran solution. Neutrophils were isolated with a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. The leukocyte-rich plasma was collected after sedimentation of the red cells for 30 min at room temperature, and was transferred to 20 mL Ficoll solution (1.077 g/mL) and spun down at 400 g for 40 min at 20 • C. The granulocyte/erythrocyte pellets were resuspended in ice-cold 0.2% NaCl to lyse erythrocytes. After 30 s, the same volume of 1.6% NaCl solution was added to reconstitute the isotonic condition. Purified neutrophils were pelleted and then resuspended in a calcium (Ca 2+ )-free Hank's balanced salt solution (HBSS) buffer at pH 7.4, and were maintained at 4 • C before use.

Inhibition of Superoxide Anion Generation
The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c [65,66]. 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, negative control) for 5 min. Cells were activated with 100 nM fMLP during the preincubation of 1 µg/mL cytochalasin B (fMLP/CB) for 3 min. Changes in the absorbance with a reduction in 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 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 at the concentration of 1 mM in cuvette with 1-cm optical path length).

Inhibition of Elastase Release
Degranulation of azurophilic granules was determined by elastase release as described previously [65,66]. 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 test compounds or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated by 100 nM fMLP and 0.5 µg/mL cytochalasin B, and changes in absorbance at 405 nm were continuously monitored 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
Normal distribution with Shapiro-Wilk was performed. The results are expressed as the mean ± SD. and were analyzed by analysis of variance (ANOVA) with post-hoc Bonferroni multiple comparisons tests. Calculations of 50% inhibitory concentrations (IC 50 ) were 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.

Neuroprotective Activity
Primary cultures of neonatal cortical neurons were prepared from the cerebral cortex of Harlan Sprague-Dawley rat pups at postnatal day 1 [67]. Briefly, each pup was decapitated and the cortex was digested in 0.5 mg/mL papain at 37 • C for 15 min. The tissue was dissociated in Hypernate A medium (containing B27 supplement) by aspirating trituration. Cells were plated (5 × 10 4 cells/cm 2 ) onto poly-D-lysine-coated dishes and maintained in neurobasal medium containing B27 supplement, 10 units/mL penicillin, 10 mg/mL streptomycin, and 0.5 mg/mL glutamine (5% CO 2 /9% O 2 ) for 3 days. Cells were then exposed to cytosine-β-D-arabinofuranoside (5 mM) for 1 day to inhibit proliferation of non-neuronal cells. The cells were used for the experiment on the fifth day. The reduction of MTT was used to evaluate the cell viability. Cells were incubated with minimum essential medium containing 0.5 mg/mL MTT for 1 h. The medium was aspirated, and the formazan particle was dissolved with lysis buffer (10% sodium dodecyl sulfate, 3.3 mM HCl, 50% dimethylformamide). A 600 nm absorbance was measured by using enzyme-linked immunosorbent assay reader. The results are expressed as the mean ± SD and were analyzed by analysis of variance (ANOVA) with post-hoc Bonferroni multiple comparisons tests.

Conflicts of Interest:
The authors declare no conflict of interest.