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NF-κB Inhibitory Activities of Glycosides and Alkaloids from Zanthoxylum schinifolium Stems

Zanthoxylum schinifolium is an aromatic shrub, the pericarp and leaves of which are widely used in culinary applications in East Asian countries. In the present study, one new neolignan glycoside, zanthoxyloside A (1) together with 16 known glycosides (2–12) and alkaloids (13–17), were isolated from methanol extract of the stems of Z. schinifolium. The absolute configuration of one known monoterpenoid glycoside (2) was determined. The structures of the isolated compounds were established by one dimensional (1D), 2D NMR and mass spectrometry. The nuclear factor-κB (NF-κB) inhibitory activities of the isolated compounds stimulated with tumor necrosis factor alpha (TNFα) were measured using a luciferase reporter system. Compounds 1, 5, 16, and 17 exhibited significant inhibition of NF-κB activation in a dose-dependent manner. Furthermore, compounds 1, 5, 16, and 17 inhibited TNFα-induced expression of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) mRNA and dose-dependent inhibition of iNOS promoter activity.

Zanthoxylum schinifolium SIEB. & ZUCC., which belongs to the Rutaceae family, is an aromatic plant, widely used as a pungent condiment and seasoning in Korea, China, Japan and other East Asian countries because of its unique aroma and taste. 1) The leaves and seeds of Z. schinifolium have been used primarily for treatment of various types of pain, the common cold, diarrhea, and jaundice in traditional Chinese medicine. Pharmacological research on Z. schinifolium has focused on its inhibitory effect on monoamine oxidase and its anti-platelet, anti-diabetes, and anti-inflammatory activities. [2][3][4] Phytochemical studies on Z. schinifolium indicated the main constituents to be coumarins, essential oils, triterpenoids, monoterpenoids, steroids, and flavonoids. [5][6][7] Coumarins were shown to have significant platelet aggregation activity. Essential oils were found to have antioxidant activity and are used in skin care products. Alkaloids have various bioactivities such as antimicrobial effects. 8) Although there have been many investigations of the active constituents of Z. schinifolium, there have been no detailed reports of the isolation and structural elucidation of glycosides and alkaloids from the stems of this plant or their bioactivities.
Nuclear factor-κB (NF-κB) represents a family of Rel domain-containing proteins, which includes five NF-κB units that can form 15 transcription factors through homo-and heterodimerization. NF-κB plays an important role in the transcriptional regulation of numerous cytokines and adhesion molecules. It is the most extensively studied transcription factor in the immune system. Known inducers of NF-κB activity are highly variable and include reactive oxygen species (ROS), tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, bacterial lipopolysaccharides (LPS), isoproterenol, cocaine, and ionizing radiation. The activation of NF-κB causes transcription at the κB site, which is involved in many diseases, includ-ing inflammatory disorders and cancer. Hence, the inhibition of NF-κB signaling has become a therapeutic target for the treatment of inflammatory diseases and cancer. [9][10][11][12] In the present study, the effects of isolated compounds 1-17 from Z. schinifolium on TNFα-induced NF-κB transcriptional activity in human embryonic kidney (293T) cells were evaluated by NF-κB-luciferase assay. To confirm the inhibitory effects of these compounds on NF-κB transcriptional activity, the effects of compounds 1, 5, 16, and 17 on upregulation of the proinflammatory protein inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), as well as their iNOS promoter activities.
Glycohaplopine (15) previously isolated from Haplophyllum perforatum, 24) but the spectroscopic data were not given. Here, we reported the 1 H-and 13 C-NMR data for glycohaplopine for the first time ( Table 2).
The NF-κB inhibitory activity of the isolated compounds was evaluated by inhibition of TNFα-induced NF-κB luciferase reporter. Cell viability was measured using a Cell-Counting Kit (CCK)-8. Compounds 1-12, 16, and 17 did not exhibit significant cytotoxicity against 293T cells at the concentrations tested, whereas compounds 13-15 showed moderate cytotoxicity (data not shown). 293T cells were treated with 10 ng/mL TNFα and showed increased transcriptional activity compared to untreated cells. The compounds were pretreated with transfected 293T cells at various concentrations (0.01, 0.1, 1, 10 µM), followed by stimulation with TNFα. Apigenin was used as a positive control (Fig. 3). The results showed that compounds 1, 5, 16, and 17 exhibited significant inhibition  of NF-κB activation in a dose-dependent manner, with IC 50 values ranging from 0.67 to 37.5 µM, respectively. Compounds 2 and 6-8 inhibited moderate NF-κB transcriptional activity (data not shown). Especially compound 16, which showed significantly activity more potent than positive control. However, other compounds were inactive at the indicated concentrations (IC 50 >100 µM, data not shown).
The activation of NF-κB is involved in immunity, inflammation, cell proliferation and negative feedback of the NF-κB signal. NF-κB targets the expression of genes such as iNOS and ICAM-1, which play important roles in the inflammatory response. [31][32][33] Compounds 1, 5, 16, and 17 were investigated for their transcriptional inhibitory effects on iNOS and ICAM-1 gene expression. Compound 16 significantly inhibited the induction of iNOS and ICAM-1 mRNA in a concentrationdependent manner; compounds 1, 5, and 17 also showed moderate effects, indicating that these compounds reduced the transcription of these genes (Fig. 4). The housekeeping protein, β-actin was unchanged by the presence of compounds 1, 5, 16, and 17 at the same concentration. Moreover, compounds 1, 5, 16, and 17 also decreased TNFα-induced iNOS promoter activity in a dose-dependent manner, with IC 50 values ranging from 0.58 to 82.8 µM, respectively (Table 3). These data suggested that compounds 1, 5, 16, and 17 isolated from Z. schinifolium suppressed TNFα-induced NF-κB transcriptional activity via inhibition of iNOS gene transcription.
In this study, 12 glycosides (1-12) and 5 alkaloids (13-17) were isolated from the stems of Z. schinifolium. To our knowledge, this is the first report regarding the effects on NF-κB inhibition of glycosides and alkaloids from Z. schinifolium. These results may provide a scientific basis for the active components in Z. schinifolium, as well as for the development of novel anti-inflammatory agents.

Experimental
General Experimental Procedures Optical rotations were determined using a Jasco DIP-370 automatic polarimeter. The FT-IR spectra were measured using a Nicolet 380 FTIR spectrometer. GC was carried out on a Shimadzu-2010 spectrometer: detector, FID; detection temperature, 300°C; column, SPB-1 (0.25 mm i.d.×30 m); column temperature, 230°C; carrier gas, He (2 mL/min) injection temperature, 250°C; injection volume, 0.5 µL. The NMR spectra were recorded using a JEOL ECA 600 spectrometer ( 1 H, 600 MHz; 13     Enzymatic Hydrolysis Compounds 1 and 2 (3.0 mg each) were mixed with β-glucosidase (3.0 mg) in water (1.0 mL) respectively and were shaken in a water bath at 37°C for 12 h. After this, the reaction mixture of 1 was concentrated and then subjected to silica gel (1.0×15 cm, 40-63 µm) column chromatography with CHCl 3 -MeOH (15 : 1, 70 mL) and CHCl 3 -MeOH-H 2 O (7 : 3 : 0.5, 60 mL) to afford an aglycone 1a (1.2 mg) and a sugar fraction. The reaction mixture of 2 was concentrated and then subjected to silica gel (1.0×15 cm, 40-63 µm) column chromatography with CHCl 3 -MeOH (20 : 1, 50 mL) and CHCl 3 -MeOH-H 2 O (7 : 3 : 0.5, 60 mL) to afford an aglycone 2a (1.1 mg) and a sugar fraction. The sugar fractions were concentrated to dryness using N 2 gas. The resulting residues were dissolved in dry pyridine (0.1 mL), and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solution. After heating the reaction mixtures at 60°C for 2 h, 0.1 mL of trimethylsilylimidazole solution was added. Heating at 60°C was continued for a further 1.5 h. The dried products were partitioned with n-hexane and H 2 O (0.1 mL each), and the organic layers were analyzed using gas liquid chromatography (GC): DB-5 capillary column (0.32 mm×30 m); hydrogen flame ionization detector (FID) detector; column temp., 210°C; injector temp., 270°C; detector temp., 300°C; carrier gas He (3 mL/min). Under these conditions, standard sugars gave peaks at t R (min)=12. Cell Toxicity Assay CCK-8 (Dojindo, Kumamoto, Japan) was used to analyze the effect of compounds on cell toxicity according to the manufacturer's instructions. Cells were cultured overnight in 96-well plate (ca. 1×10 4 cells/well). Cell toxicity was assessed after the addition of compounds on dose-dependent manner. After 24 h of treatment, 10 µL of the CCK-8 solution was added to triplicate wells, and incubated for 1 h. Absorbance was measured at 450 nm to determine viable cell numbers in wells.
NF-κB and iNOS-Luciferase Assay 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, U.S.A.) containing 10% heatinactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 10 µg/mL streptomycin at 37°C and 5% CO 2 . The luciferase vector was first transfected into 293T cells. After a limited amount of time, the cells were lysed, and luciferin, the substrate of luciferase, was introduced into the cellular extract along with Mg 2+ and an excess of ATP. Under these conditions, luciferase enzymes expressed by the reporter vector could catalyze the oxidative carboxylation of luciferin. Cells were seeded at 2×10 5 cells per well in a 12-well plate and grown. After 24 h, cells were transfected with inducible NF-κB or iNOS firefly luciferase reporter and constitutively expressing Renilla reporter. After 24 h of transfection, medium was changed to assay medium (Opti-MEM+ 0.5% FBS+ 0.1 mM NEAA+ 1 mM sodium pyruvate +100 units/mL peni-cillin+ 10 µg/mL streptomycin) and cells were pretreated for 1 h with either vehicle (1% DMSO in water) and compounds, followed by 1 h of treatment with 20 ng/mL TNFα for 23 h. Unstimulated 293T cells were used as a negative control (−), apigenin was used as a positive control. Dual luciferase assay was performed 48 h after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization.
RNA Preparation and RT-PCR Total RNA was extracted using Easy-blue reagent (Intron Biotechnology, Seoul, Korea). Approximately 2 µg total RNA was subjected to reverse transcription using Moloney murine leukemia virus (MMLV) reverse transcriptase and oligo-dT primers (Promega, Madison, WI, U.S.A.) for 1 h at 42°C. PCR for synthetic cDNA was performed using a Taq polymerase premixture (TaKaRa, Japan). The PCR products were separated by electrophoresis on 1% agarose gels and stained with EtBr.
PCR was conducted with the following primer pairs: iNOS sense 5′-TCA TCC GCT ATG CTG GCT AC-3′, iNOS antisense  5′-CTC AGG GTC ACG GCC ATT G-3′, ICAM-1 sense 5′-CTG  CAG ACA GTG ACC ATC-3′, ICAM-1 antisense 5′-GTC CAG  TTT CCC GGA CAA-3′, β-actin sense 5′-TCA CCC ACA CTG  TGC CCA TCT ACG-3′, and β-actin antisense 5′-CAG CGG AAC CGC TCA TTG CCA ATG-3′. The specificity of products generated by each set of primers was examined using gel electrophoresis and further confirmed by a melting curve analysis. HepG2 cells were pretreated in the absence and presence of compounds for 1 h, then exposed to 20 ng/mL TNFα for 6 h. Total mRNA was prepared from the cell pellets using Easyblue. The levels of mRNA were assessed by RT-PCR.
Statistical Analysis All data represent the mean± standard deviation (S.D.) of at least three independent experiments performed in triplicate. Statistical significance is indicated as * p<0.05 and ** p<0.01 as determined by Dunnett's multiple comparison test.