A New Canthinone-Type Alkaloid Isolated from Ailanthus altissima Swingle

The present investigation of the chemical constituents of the stem barks of Ailanthus altissima has resulted in the isolation of six canthinone-type alkaloids, including a new compound, (R)-5-(1-hydroxyethyl)-canthine-6-one (1), and five known compounds (2–6). Moreover, four phenyl propanoids (7–10), two lignans (11 and 12), two triterpenoids (13 and 14) and a fatty acid (15) having previously known chemical structures were isolated during the same course of this study. The structure of the new compound was elucidated by physical (m.p., [α]D) and spectroscopic data (1H-NMR, 13C-NMR, 2D NMR, and HR-DART-MS) interpretation and its absolute configuration was determined by electronic circular dichroism (ECD) data and quantum chemical calculations. The inflammatory activities of the isolates were screened on lipopolysaccharide (LPS)-induced nitric oxide (NO), a proinflammatory mediator, in RAW 264.7 cells. Among these isolated compounds, six compounds exhibited significant inhibition of NO production, with IC50 values in the range of 5.92 ± 0.9 to 15.09 ± 1.8 μM.


Introduction
Ailanthus altissima Swingle (Simaroubaceae), the tree-of-heaven, has been used to treat diarrhea, dysentery, heat ailments, epilepsy, asthma, ophthalmic diseases, and it has also been used as an astringent.The stem bark of A. altissima has exhibited various biological activities, such as anti-proliferative, cytotoxic, anti-plasmodial, anti-malarial, anti-viral, antibacterial, anti-fungal, and analgesic activity [1,2].Additionally, it was reported that a decoction of A. altissima decreased the production of inflammatory cytokines, TNF, IL-6 and IL-8 as well as NF-κB activation on the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated human mast cell line, HMC-1 [3].Furthermore, the EtOH extract of A. altissima inhibited the generation of the cyclooxygenase-2 (COX2)-dependent phases of prostaglandin D2 in bone marrow-derived mast cells (BMMC) [4].Previous phytochemical investigations of A. altissima revealed the presence of alkaloids, terpenoids, steroids, and flavonoids [5].Among these compounds, quassinoids and indole and β-carboline alkaloids are common major constituents of A. altissima [6][7][8].Alkaloids from A. altissima are reported for their anti-herpes [9] and anti-mycotic properties [10] and for their action on the rate of intestinal blood flow in rabbits [11].Indole and β-carboline alkaloids have shown inhibitory activity on cyclic adenosine monophosphate (cAMP) phosphodiesterase [12].Moreover, it was demonstrated that canthin-6-one and its derivatives have anti-proliferative and cytotoxic activity [13], leishmanicidal activity [14], and gastro-protective effects [15].Although most β-carboline and canthin-6-one alkaloids have been reported to show diverse pharmacological effects, there are few studies regarding their anti-inflammatory effects.
Recently it was reported that some quassinoids from the stem barks of A. altissima inhibited the production of nitric oxide in RAW 264.7 cells [16].In our continuing study to search for anti-inflammatory agents from this plant, a new canthinone-type alkaloid (1), as well as 14 known compounds (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), was isolated further from the EtOAc-and BuOH-soluble fractions of the EtOH extract of the stem barks of A. altissima (Figure 1).Herein, this paper describes the isolation and structural elucidation of the isolates and their inhibitory effects on NO production in RAW 264.7 cells.
Recently it was reported that some quassinoids from the stem barks of A. altissima inhibited the production of nitric oxide in RAW 264.7 cells [16].In our continuing study to search for antiinflammatory agents from this plant, a new canthinone-type alkaloid (1), as well as 14 known compounds (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), was isolated further from the EtOAc-and BuOH-soluble fractions of the EtOH extract of the stem barks of A. altissima (Figure 1).Herein, this paper describes the isolation and structural elucidation of the isolates and their inhibitory effects on NO production in RAW 264.7 cells.

Anti-Inflammatory Activity of Isolated Compounds 1-15 from the Stem Bark of A. altissima
All the isolates 1-15 from the stem bark of A. altissima were evaluated for thei inhibitory effects on LPS-induced NO production in RAW 264.7 cells at non-toxic concentrations.As shown Table 2 and Figure 4, six compounds showed potent inhibitory effects on NO production (IC 50 values ď 50 µM) and were assessed using IC 50 values.Among the six canthin-6-one alkaloids isolated, 11-hydroxycanthin-6-one (6) was inactive in this assay system, while (R)-5-(1-hydroxyethyl)-canthin-6-one (1), canthin-6-one (2), 9-hydroxycanthin-6-one (4) and 10-hydroxycanthin-6-one (5) exhibited inhibitory activity with IC 50 values ranging from 7.73 to 15.09 µM.The compound 9-Hydroxycanthin-6-one (4) showed more a potent inhibitory effect than canthin-6-one (2, IC 50 value = 9.09 µM) with an IC 50 value of 7.73 µM, indicating the hydroxylation at position 9 seems to increase the inhibitory activity.However, canthin-6-one (2) showed a more potent inhibitory effect with an IC 50 value of 9.09 µM than (R)-5-(1-hydroxyethyl)-canthin-6-one (1, IC 50 value = 15.09µM), which has a 1-hydroxyethyl group at C-5.It was also observed that other canthin-6-one alkaloids possessing an additional hydroxyl group (5 and 6) exhibited weaker inhibitory effects than canthin-6-one (2).It seems that a hydroxyl group in canthin-6-one weakens the inhibitory effect with the exception of a hydroxyl group at C-9. However a further study is needed to ensure a structure-activity relationship for the canthin-6-one alkaloids.In a previously study, 9-hydroxycanthin-6-one (4) was reported to inhibit the NF-κB pathway in TNF-a-stimulated HEK-293/NF-kB-luc cells [31].Among the isolates, sinapaldehyde (9) was found to have the most potent inhibitory effect with an observed IC 50 value of 5.92 µM, and erythro-guaiacylglycerol-β-O-4 1 -coniferyl ether (11) also showed a significant inhibitory effect with an observed IC 50 value of 10.69 µM.By comparison with our previous study, two methoxy groups at C-3 and C-5 might be the bioactive groups for sinapaldehyde (9) [16].Morever, sinapaldehyde (9) has shown significant inhibitory activity on TNF-α-induced NF-κB transcriptional activity in HepG2 cells, ascribing the symmetrical methoxyl group a more important role in NF-κB inhibition [32].However, the roles of these functional groups in the activity are not clear due to a limited number of isolates in the present study.observed IC50 value of 10.69 μM.By comparison with our previous study, two methoxy groups at C-3 and C-5 might be the bioactive groups for sinapaldehyde (9) [16].Morever, sinapaldehyde ( 9) has shown significant inhibitory activity on TNF-α-induced NF-κB transcriptional activity in HepG2 cells, ascribing the symmetrical methoxyl group a more important role in NF-κB inhibition [32].However, the roles of these functional groups in the activity are not clear due to a limited number of isolates in the present study.

General Procedures
Optical rotations were determined with a JASCO P-2000 polarimeter (Jasco Inc., Easton, MD,

Plant Meterial
The stem barks of Ailanthus altissima Swingle were purchased at humanherb Co. Gyeongsangbuk-do, Gyeong-san, Korea, in November 2011.A voucher specimen (No. 2012-AIAL01) has been deposited in the Lab of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Seoul, Korea.

Computational Methods
The conformational analysis was performed with Spartan'14 software package [33]and geometry optimizations were operated by the Gaussian 09 package [34].TDDFT CD calculations for the optimized conformers were performed at the CAM-B3LYP/SVP level with a CPCM solvent model in MeCN.The CD spectra were generated by SpecDis 1.62 software [35].

Cell Culture and Sample
RAW 264.7 macrophages were obtained from the Korea Cell Line Bank (Seoul, Korea).Cells were grown at 37 ˝C in DMEM medium supplemented with 10% FBS, penicillin sulfates in a humidified atmosphere of 5% CO 2 .Cells were pretreated with isolated compounds from A. altissima (7.5 µM/mL) or positive controls (L-NIL for iNOS inhibitor) for 1 h, and then stimulated with LPS (1 µg/mL) for the indicated time.

Cell Viability Assay
Cell viability studies were performed using the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-dipheyl tetrazoliumbromide; Sigma-Aldrich, St. Louis, MO, USA) assay.Raw 264.7 cells were plated at a density of 0.9 ˆ10 5 cells/mL in 96-well.Cells were pretreated with isolated compounds from A. altissima (50 µM) for 1 h and then stimulated with LPS (1 µg/mL) for 24 h.Then 50 µL of MTT solution (5 mg/mL in PBS) was added to the medium and the cells were incubated at 37 ˝C for 4 h.The MTT-containing medium was removed and the cells were solubilized in DMSO (100 µL) for 10 min.The optical density at 540 nm was determined using a microplate spectrophotometer (Molecular Devices Inc., Sunnyvale, CA, USA) to determine the cell viability.
3.9.Measurment of Nitric Oxide Production RAW264.7 cells were plated at 2 ˆ10 5 cells/well in 60 mm dishes and incubated with or without LPS (1 µg/mL) in the absence or presence of indicate concentration of the samples for 24 h.The nitrite which accumulated in culture medium was measured as an indicator of NO production according to the Griess reagent.The culture supernatant (100 µL) was mixed with 100 µL of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthyl ethylenediamine-HCl] for 10 min, and then the absorbance at 540 nm was measured in a microplate reader.Fresh culture medium was used as the blank in all experiments.The amount of nitrite in the samples was determined with reference to a sodium nitrite standard curve.

Conclusions
The new canthin-6-one alkaloid (1) and 14 known compounds (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were isolated from the stem bark of A. altissima.The structure of the new compound (1) was elucidated by its physical and spectroscopic data, and its absolute configuration was determined by comparison of its experimental and calculated ECD spectra.Compounds 3-9 and 12 were isolated from this plant for the first time.The isolates were screened for inhibitory activity against LPS-induced NO production in RAW 264.7 cells.Compounds 1, 2, 4, 5, 9 and 11 exhibited a significant inhibitory activity, with IC 50 values in the range of 5.92 ˘0.9 to 15.09 ˘1.8 µM.Thus, the active isolates seem to be worthy of additional biological tests to more fully evaluate the plant's potential as a therapeutic agent for anti-inflammatory diseases with an excess production of NO.

Figure 1 .
Figure 1.Structures of 1-15 isolated from the stem barks of A. altissima.

Figure 1 .
Figure 1.Structures of 1-15 isolated from the stem barks of A. altissima.

2. 1 .
Identification of Isolated Compounds 1-15 from the Stem Bark of A. altissima Compound 1 was isolated as a yellow amorphous powder, in which the molecular formula was established as C 16 H 13 N 2 O 2 by HR-DART-MS (m/z 265.0992 [M + H] + ; calcd.for C 16 H 13 N 2 O 2 , 265.0977) (Figure C-NMR signals at δ C 66.1 (C-17) and δ C 23.4 (C-18) of 1 were shifted downfield due to the influence of the hydroxyl group.

Figure 3 .
Figure 3. Experimental and calculated CD spectra of 1.

Figure 3 .
Figure 3. Experimental and calculated CD spectra of 1.

Figure 4 .
Figure 4. Effects of compounds 1 (a), 2 (b), 4 (c), 5 (d), 9 (e) and 11 (f) isolated from the stem barks of A. altissima on LPS-induced NO production in RAW 264.7 cells.Based on MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay data, concentrations of isolates that would not affect cell viability were used for the following experiments.Cells were pretreated with the indicated concentrations of isolates for 1 h and then stimulated with LPS (1 μg/mL) for 24 h.NO level in culture media was measured by Griess assay.NIL (iNOS inhibitor, 10 μM) was used as a positive control to inhibit NO production.Values represent the means ± SD of three independent experiments.# p < 0.05 vs. the control group; * p < 0.05 vs. LPS-stimulated group.CON, control; NIL, L-N 6 -(1-iminoethyl) lysine.

Figure 4 .
Figure 4. Effects of compounds 1 (a), 2 (b), 4 (c), 5 (d), 9 (e) and 11 (f) isolated from the stem barks of A. altissima on LPS-induced NO production in RAW 264.7 cells.Based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay data, concentrations of isolates that would not affect cell viability were used for the following experiments.Cells were pretreated with the indicated concentrations of isolates for 1 h and then stimulated with LPS (1 µg/mL) for 24 h.NO level in culture media was measured by Griess assay.NIL (iNOS inhibitor, 10 µM) was used as a positive control to inhibit NO production.Values represent the means ˘SD of three independent experiments.# p < 0.05 vs. the control group; * p < 0.05 vs. LPS-stimulated group.CON, control; NIL, L-N 6 -(1-iminoethyl) lysine.

Table 2 .
Inhibitory effects of 1-15 isolated from the stem bark of A. altissima on nitric oxide production in LPS-induced RAW 264.7 cells.
* IC 50 value is defined as the concentration that results in a 50% decreased production of nitric oxide.The values represent the means of the results from three independent experiments with similar patterns.L-N 6 -(1-iminoethyl)lysine (L-NIL) was used as assay positive control for NO production (IC 50 value: 15.8 µM).** ND: not determined due to a limited amount of the sample.

Table 2 .
Inhibitory effects of 1-15 isolated from the stem bark of A. altissima on nitric oxide production in LPS-induced RAW 264.7 cells.
* IC50 value is defined as the concentration that results in a 50% decreased production of nitric oxide.The values represent the means of the results from three independent experiments with similar patterns.L-N 6 -(1-iminoethyl)lysine (L-NIL) was used as assay positive control for NO production (IC50 value: 15.8 μM).** ND: not determined due to a limited amount of the sample.