Isobutylhydroxyamides from Zanthoxylum bungeanum and Their Suppression of NO Production

Four new unsaturated aliphatic acid amides, named zanthoamides A–D (1–4), and eight known ones—tetrahydrobungeanool (5), ZP-amide A (6), ZP-amide B (7), ZP-amide C (8), ZP-amide D (9), ZP-amide E (10), bugeanumamide A (11), and (2E,7E,9E)-N-(2-hydroxy-2-methylpropyl)-6,11-dioxo-2,7,9-dodecatrienamide (12)—were isolated from the pericarps of Zanthoxylum bungeanum. The structures of these compounds were elucidated by extensive use of spectroscopic methods, including HRESIMS, 1D and 2D NMR analyses and comparison with previously reported data. Compound 4 contained a rare C6 fatty acid unit with an acetal group. Results revealed that compounds 1, 5, 6, and 12 showed inhibitory effects on nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages, with IC50 values of 48.7 ± 0.32, 27.1 ± 1.15, 49.8 ± 0.38, and 39.4 ± 0.63 µM, respectively, while the other compounds were inactive (IC50 > 60 μM). They could contribute to the anti-inflammatory effects of Z. bungeanum by suppression of NO production.


Introduction
The genus Zanthoxylum (family Rutaceae), commonly called "prickly ash", comprises more than 200 species distributed worldwide. Phytochemical studies of this genus have revealed a variety of biologically active secondary metabolites, including alkaloids, aromatic and aliphatic amides, lignans and coumarins with antitumor, anti-inflammation, and anesthetic properties [1][2][3]. Zanthoxylum bungeanum Maxim is an aromatic tree and shrub, which is native to the provinces of Sichuan, Shaanxi, Yunnan, Guizhou, Guanxi, and Guandong in southwestern China. The fruits of this species, called "da hong pao" (big red robe), are the most popular red huajiao commercial product. Red huajiao, the pericarps of the fruits of Z. bungeanum have been utilized as a pungent foodstuff and also as a kind of traditional Chinese medicine for the treatment of vomiting, toothache, stomachache, abdominal pain, and diarrhea [4]. Previous phytochemical research on Z. bungeanum has focused on the essential oils, coumarins, flavonoids, aliphatic amides (classified as sanshools), and alkaloids of the fruits and leaves [5][6][7][8][9].The huajiao extracts have great potential for applications in savory and sweet goods and beverages. Some of the aliphatic acid amides display antioxidant activities, modulatory effects on relaxing the circle muscle of the gastric body (β-and γ-sanshool), In addition, three aliphatic acid amides (β-, γ-and hydroxy-β-sanshool) exhibited human acyl-CoA: cholesterol acyltransferase inhibitory activities [15], and tumuramide C, ZP-amide A, and ZP-amide D exerted significant effects on PPAR transactivational activity [16]. Most recently, Hofmann et al. [1] have shown that the all-trans-configured amides hydroxy-β-sanshool and hydroxy-γ-isosanshool from Z. piperitum induced a numbing and anesthetic sensation. Our recent studies have showed that several alkylamides from cultivated Z. bungeanum pericarps possessed nerve growth factor-potentiating activity [17].
As part of our further search for bioactive substances from medicinal plants [18][19][20][21], our bioassays indicated that the CHCl3-soluble material of a crude EtOH extract of Z. bungeanum pericarps displayed inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. Further phytochemical investigation of the CHCl3-soluble material of Z. bungeanum led to the isolation of four new alkylamides, named zanthoamides A-D (1-4), and eight known ones 5-12 ( Figure 1). Here, we describe the isolation and structure elucidation of the compounds 1-12 and their NO inhibitory activity in LPS-stimulated RAW 264.7 cells.
Compound 1 was obtained as a pale yellow syrup. Its molecular formula was determined as C18H27NO4 by HRESIMS at m/z 344.1810 [M + Na] + (calcd. for C18H27NO4Na, 344.1838), indicating six degrees of unsaturation. The IR spectrum displayed characteristic absorptions of hydroxyl and amide NH (3370 cm −1 ) and amide (1665 cm −1 ) groups. The 13 C-NMR and DEPT spectra (Table 1)
Compound 1 was obtained as a pale yellow syrup. Its molecular formula was determined as C 18  , an amide carbon (δ C 169.4) and a ketonic carbon (δ C 201.5), and one oxygenated quaternary carbon (δ C 71.7). These data were consistent with the resonances observed in the 1 H-NMR spectrum. The 1 H-NMR spectrum of 1 revealed three tertiary methyl signals at δ H 1.18, 1.18, and 2.28 (each 3H of singlet). Meanwhile, eight olefinic protons at δ H 6.01, 7.14, 6.18, 6.14, 6.27, 6.44, 7.29, and 6.15, and an oxygenated methine proton at δ H 4.22 (m), were clearly visible. Other overlapping proton resonances occurred between δ H 1.66 and 2.28, resulting from either methyl or methylene protons. Comparison of the 1 H-and 13 C-NMR data ( Table 1) of 1 with two known compounds (5 and 6) indicated that 1 was an unsaturated fatty acid amide bearing an N-hydroxylisobutyl moiety. Detailed analysis of the 1 H and 13 C-NMR data, along with the COSY, HSQC, and HMBC spectra ( Figure 2) led to the conclusion that 1 was structurally similar to ZP-amide A (6). Meanwhile, eight olefinic protons at δH 6.01, 7.14, 6.18, 6.14, 6.27, 6.44, 7.29, and 6.15, and an oxygenated methine proton at δH 4.22 (m), were clearly visible. Other overlapping proton resonances occurred between δH 1.66 and 2.28, resulting from either methyl or methylene protons. Comparison of the 1 H-and 13 C-NMR data ( Table 1) of 1 with two known compounds (5 and 6) indicated that 1 was an unsaturated fatty acid amide bearing an N-hydroxylisobutyl moiety. Detailed analysis of the 1 H and 13 C-NMR data, along with the COSY, HSQC, and HMBC spectra ( Figure 2) led to the conclusion that 1 was structurally similar to ZP-amide A (6).  The major difference was one more olefinic bond between the C-3 and C-6 positions in the aliphatic chain of 1 than in ZP-amide A (6), which was consistent with its molecular formula and degree of unsaturation. The 1 H- 1 Figure 2), thus suggesting that the locations of the amide, hydroxyl, ketonic carbons to be at C-1, C-8 and C-13, respectively, and the connectivity between the aliphatic acid and the amine moieties. Additionally, the geometry of the C 4 /C 5 olefinic bond was deduced to be trans-configured, like those of C 2 /C 3 , C 9 /C 10 , and C 11 /C 12 olefinic bond, from the coupling constant (J H-4/H-5 = 15.7 Hz). The optical inactivity of this compound indicated that 1 was a racemic mixture. Consequently, compound 1 was identified as (2E,4E,9E,11E)-N-(2-hydroxy-2-methypropyl)-8-hydroxy-13-oxo-2,4,9,11-tetradecatetraenamide, and named zanthoamide A.
Compound 2 had a molecular formula C 18 H 29 NO 4 by HRESIMS at m/z 346.1961 [M + Na] + , indicating five degrees of unsaturation. Comparison of the 1D NMR ( Table 1) spectroscopic data of 2 with those of 1 clearly disclosed that 2 was also an aliphatic acid amide. The obvious difference between the two compounds was that the ketone carbonyl at C-13 (δ C 201.5) in 1 was replaced by a hydroxyl group in 2, which was confirmed by the upfield shift of C-13 (δ C 68.8) in 2, in combination of the 1 H-1 H COSY correlations of H 3 -14/H-13/H-12 and the HMBC correlations from H-13 to C-11 (δ C 145.4), and H-12 (δ H 6.18) to C-13 in 2. Moreover, the HSQC spectrum suggested the hydroxyl group to be ascribable to C-6 in 2 rather than to C-8 in 1, as evident from the large downfield chemical shift of C-6 (δ C 72.4) and upfield shift of C-8 (δ C 29.9) observed in the 13 C-NMR spectrum of 2. This was further confirmed by the COSY correlations of H-2/H-3/H-4/H-5/H-6. The assignments of all proton and carbon signals were fully made by 2D ( 1 H-1 H COSY, HSQC, and HMBC) NMR data. The optical inactivity of this compound indicated that 1 was a racemic mixture. Thus, 2 was identified as (2E,4E,9E,11E)-N-(hydroxy-2-methypropyl)-8,13-dihydroxy-2,4,9,11-tetradeca-tetraenamide, and named zanthoamide B.
Compound 3 was obtained as a colorless syrup. Its molecular formula C 16 H 27 NO 4 was determined by HRESIMS at m/z 320.1813 [M + Na] + (calcd. for C 16 H 27 NO 4 Na, 320.1838). The 1 H and 13 C-NMR data of compound 3 were very similar to those of ZP-amide C (8). The only differences between the two compounds were that the cis-configured olefin at C-6 and C-7 at δ H 5.40 (dt, J = 11, 7 Hz, H-6) in 8 was replaced by a trans-configured one at δ H 5.70 (dt, J = 15.3, 7.0 Hz, H-6) in 3. The structure of this compound was confirmed by detailed analysis of the 2D NMR data including its HSQC, HMBC, and 1 H-1 H COSY spectra. The optical inactivity of this compound indicated that it was a racemic mixture.
The relative configuration of the two asymmetric carbons still remains to be determined. Consequently, 3 was established to be (10*,11*)-(2E,6E,8E)-10,11-dihydroxy-N-(2-hydroxy-2,6,8-dodecatrienamide, and named zanthoamide C. Compound 4 was also obtained as a colorless syrup having a molecular formula C 12 H 23 NO 4 , as deduced from HRESIMS at m/z 268.1505 [M + Na] + (calcd. for C 12 H 23 NO 4 Na, 268.1525). The 1 H and 13 C-NMR spectra of 4 (Table 2), compared with those of 3, clearly showed that 4 was also an aliphatic acid amide, which contained a rare C 6 fatty acid with an acetal group, as evident from the 1 H-1 H COSY correlations of H-2 (δ H 6.02)/H-3(δ H 6.80)/H 2 -4(δ H 2.25)/H 2 -5(δ H 1.74)/H-6(δ H 4.39) as well as from the 1 H and 13 C-NMR data of an acetal group (δ H 4.39, J = 5.7 Hz, H-6; δ C 105.5, C-6; δ H 3.33 and δ C 53.6, 2 × OCH 3 ). The presence of the acetal moiety at C-6 was supported by the HMBC correlation of OCH 3 (δ C 53.6) with H-6 (δ H 4.39) obtained in MeOH-d 4 solvent. However, due to overlapping between the methoxyl and MeOH-d 4 signals, consequently, we measured the 1 H-NMR spectrum (see Figure S25 in Supplementary Materials) with DMSO-d 6 as solvent, in which the six protons of the two methoxyl groups as a strong singlet were observed at δ H 3.22. Therefore, compound 4 was identified as (2E)-6,6-dimethoxy-N-(2-hydroxy-2-methylpropyl)-2-hexenamide, and named zanthoamide D. Unsaturated fatty acid amides are characteristic constituents of the genus Zanthoxylum, which contains more than 50 such compounds [16,[22][23][24][25]. Nearly all aliphatic acid amides from the Zanthoxylum species have been isolated as racemates, and their absolute configurations have yet to be determined [16]. To the best of our knowledge, compound 4 is the first example of a fatty acid amide containing a C 6 fatty acid unit found in the genus Zanthoxylum. It should be noted that 4 possesses an acetal group in its structure. Owing to contact with methanol during the extraction and purification processes, it may be a new fatty acid amide artifact presumably formed during these processes.

Biological Activity Assays
Compounds 1-12 were evaluated for their inhibitory activities against nitric oxide (NO) production in LPS-activated RAW264.7 cells and their cytotoxicities against two human cancer cell lines [26,27]. This assay indicated that compounds 1, 5, 6, and 12 exhibited inhibitory activities against NO production in LPS-activated RAW264.7 macrophages, with IC 50 values of 48.7 ± 0.32, 27.1 ± 1.15, 49.8 ± 0.38, and 39.4 ± 0.63 µM, respectively, while the other compounds were inactive (IC 50 > 60 µM) ( Table 3). Among them, 5 exhibited the highest inhibitory activity. The observations suggested that the unsaturated longer aliphatic chain without oxygen functions appears to play an important role in inhibiting NO production (5 vs . 1 and 2). Additionally, the cytotoxicity of each compound was examined. Cell viability was measured by the MTT colorimetric assay. Compounds 1-12 had no significant cytotoxicity in LPS-stimulated RAW 264.7 cells at concentrations up to 50.0 µM (data not shown). At 50 µM, the cytotoxic activities against human colon cancer (HCT116) and human prostate cancer (PC-3) cells were also tested, and none of them exhibited detectable cytotoxicity.
It has been reported that four amides-ZP-amide A (6), ZP-amide C (7), ZP-amide D (9), and ZP-amide E (10)-inhibited the growth of a neurofibromatosis type 1 (NF1)-and p53-deficient mouse glioma cell line at non-cytotoxic concentrations [14]. In addition, ZP-amide A (6) and ZP-amide D (9) exerted significant anti-inflammatory effects through enhancing the peroxisome proliferator-activated receptor (PPAR) transactivational activity with EC 50 values of 19.1 and 12.0 µM, respectively [16]. The findings further suggest that alkylamides are the major anti-inflammatory components of the edible spice, Z. bungeanum, which was also substantiated by our observations that some compounds (i.e., 5 and 12) display NO-inhibitory activity. It is well known that NO is a crucial cellular-signaling molecule associated with several physiological and pathological processes [28]. It is therefore a fundamental component in the fields of neuroscience, physiology, and immunology [29]. NO has been shown to activate NF-κB in peripheral blood mononuclear cells, an important transcription factor in iNOS gene expression in response to inflammation [30]. So suppression of NO production was a direct indicator of those compounds to resist inflammation.

General Procedures
Optical rotations were measured using an Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectrum were obtained on an Evolution-300 UV-visible spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a Tensor 27 FT-IR spectrometer (Bruker Optics, Germany) in KBr pellets, respectively. NMR spectra were measured on an Avance III 500 instruments (Bruker Daltonics Inc., Bremen, Germany), with tetramethylsilane (TMS) as an internal standard at room temperature. ESI-MS were performed on a LTQ Fleet instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA), and HRESIMS were obtained on a Thermo Fisher Scientific Q-TOF mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Semipreparative HPLC was performed on a Waters 1100

Plant Materials
The pericarps of Z. bungeanum were collected from Feng County (Shaanxi, China), in July 2013 and authenticated by Prof. Zai-Min Jiang (Plant Laboratory, College of Life Sciences, Northwest A&F University, Shaanxi, China). A voucher specimen (LXY-0156) was deposited in our laboratory.

Nitric Oxide Production Inhibition Assay
RAW 264.7 cells were seeded at a density of 5 × 10 5 -cells/well in 24 well plates and incubated for 12 h at 37 • C and 5% CO 2 [26]. Then media of each well were aspirated and fresh FBS-free DMEM media were replaced. Compounds 1-12 (20 µM) were prepared in FBS-free DMEM to give a total volume of 500 µL in each well of a microtiter plate. After 1 h treatment, cells were stimulated with 100 ng/mL of LPS for 24 h. Nitrite concentrations were determined from a standard curve using sodium nitrite at concentrations ranging from 0 to 120 µM. The absorbance was measured at 540 nm in a microplatereader (Biotek, Winooski, VT, USA). The amount of nitrite inthe media was calculated from sodium nitritestandard curve. The data show the mean ± S.D. of three independent experiments. p < 0.05, and p < 0.001. The values expressed are means of three replicate determinations ± standard deviation. The data were evaluated with SPSS 20.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
In conclusion, we have identified 12 lipophilic alkyamides of the sandshool class from the pericarps of Z. bungeanum, including four new ones 1-4, which we have named zanthoamides A-D. NO inhibitory activity was observed for these alkyamides (compounds 1, 5, 6, and 12) in LPS-stimulated RAW 264.7 cells, of which 5 was the best inhibitor. The results demonstrated that these fatty acid amides might play important roles in anti-inflammatory activity of the pericarps of Z. bungeanum.