Fitoterapia 95 (2014) 175–181

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Indole alkaloids from the roots of Isatis indigotica and their inhibitory effects on nitric oxide production Liguo Yang a,b, Guan Wang a,b, Meng Wang a,b, Hongmei Jiang a,b, Lixia Chen a,b, Feng Zhao d,⁎, Feng Qiu a,b,c,⁎⁎ a b c d

Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, PR China Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China School of Pharmacy, Yantai University, Yantai 264005, PR China

a r t i c l e

i n f o

Article history: Received 8 February 2014 Accepted in revised form 14 March 2014 Available online 29 March 2014 Keywords: Isatis indigotica Indole alkaloids Indole-2-S-glycosides Anti-inflammatory activity

a b s t r a c t Three rare indole-2-S-glycosides, indole-3-acetonitrile-2-S-β-D-glucopyranoside (1), indole-3acetonitrile-4-methoxy-2-S-β-D-glucopyranoside (2) and N-methoxy-indole-3-acetonitrile2-S-β-D-glucopyranoside (3), together with 11 known indole alkaloids were isolated from the roots of Isatis indigotica Fort. (Cruciferae). The structures of 1–3 were elucidated on the basis of mass spectrometry and extensive 1D and 2D NMR spectroscopy. All of the isolated compounds were tested for inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages. A plausible biosynthesis pathway of 1–3 is also proposed. © 2014 Elsevier B.V. All rights reserved.

Chemical compounds studied in this article: Indole-3-acetonitrile (PubChem CID: 351795) Arvelexin (PubChem CID: 119406) 1-Methoxy-indole-3-acetonitrile (PubChem CID: 11954881) 3-Indoleformic acid (PubChem CID: 69867) 3-Indoleacetic acid (PubChem CID: 802)

1. Introduction Isatis indigotica Fort. (Cruciferae) is a biennial herbaceous plant widely distributed and cultivated in China. Its dried roots, named “Ban-Lan-Gen” in Chinese, are one of the most frequently used traditional Chinese medicine for treatment of influenza, ⁎ Correspondence to: F. Zhao, School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai 264005, PR China. Tel.: +86 535 6706021. ⁎⁎ Correspondence to: F. Qiu, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, PR China. Tel./fax: +86 22 59596163. E-mail addresses: [email protected] (F. Zhao), [email protected] (F. Qiu).

http://dx.doi.org/10.1016/j.fitote.2014.03.019 0367-326X/© 2014 Elsevier B.V. All rights reserved.

fever, bacterial infection and epidemic hepatitis [1]. Previous chemical investigation of this plant has led to the isolation of alkaloids [2–6], organic acids [7,8], lignans [9,10] and ceramides [11]. Indole alkaloids are the main active constituents of this plant. The blue dye indigo (indigotin) is one of the oldest natural dyestuffs known to human beings [12]. Indirubin, a 3,2′-bisindole isomer of indigotin, is effective against chronic granulocytic leukemia [13]. Arvelexin, a crucifer phytoalexin, is known to possess antifungal activity [14]. Indole glycosides are an important class of secondary metabolites often found in plants of the genus Isatis. About 20 indole glycosides, including indole-O-glycosides, indole-Cglycosides, indole-S-glycoside and indole glucosinolates, have been isolated from Isatis up to now [4,5,15–19]. During the

176

L. Yang et al. / Fitoterapia 95 (2014) 175–181

course of our studies on bioactive constituents from 70% EtOH extracts of the roots of I. indigotica, three rare indole-2-Sglycosides along with 11 known indole alkaloids were isolated (Fig. 1). In this paper, the isolation, structure elucidation, inhibitory effects on nitric oxide production and plausible biosynthesis pathway of these alkaloids were reported. 2. Experimental 2.1. General UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer. IR spectra were conducted on a Bruker IFS 55 spectrometer. NMR experiments were performed on Bruker ARX-300 and AV-600 spectrometers. The chemical shifts are stated relative to TMS and expressed in δ values (ppm), with coupling constants reported in Hz. HRESIMS was obtained on a Bruker APEX-II mass spectrometer, and ESIMS was recorded on an Agilent 1100-LC/MSD TrapSL mass spectrometer. Silica gel GF254 (10–40 μm) prepared for TLC and silica gel (200–300 mesh) for column chromatography were obtained from Qingdao Marine Chemical Factory (Qingdao, China). Octadecyl silica gel was purchased from Merck Chemical Company Ltd. Macroporous resin D101 was a product of Chemical Plant of Nankai University (Tianjin, China). Preparative HPLC separations were conducted using a Shimadzu HPLC system equipped with a LC-6AD pump and a SPD-20A detector using a C18 column (250 mm × 20 mm, 5 μm; YMC Co. Ltd.). GC was carried out on an Agilent GC-series system and performed with an HP-5 column (30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA). All the reagents were HPLC grade or analytical grade and purchased from Tianjin Damao Chemical Company. 2.2. Plant material The roots of I. indigotica Fort. were collected from Hebei Province, China, and identified by Professor Qishi Sun of the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (II-20101115) has been deposited in the herbarium of the Department

of Natural Products Chemistry, Shenyang Pharmaceutical University.

2.3. Extraction and isolation The roots of I. indigotica Fort. (8 kg) were cut into small pieces and extracted with 70% EtOH (72 L × 2 h × 2). The combined extracts were concentrated under vacuum. The residue (2.7 kg) was suspended in H2O (10 L) and partitioned successively with cyclohexane, EtOAc and n-BuOH (10 L × 3). The EtOAc extract (51 g) was subjected to silica gel CC (10 × 60 cm) with CH2Cl2/MeOH (100:1 to 0:100) to obtain seven fractions (E1–E7), which were combined according to TLC analysis. Fraction E1 (3.5 g) was chromatographed on silica gel CC (2.5 × 30 cm) with a petroleum ether/EtOAc gradient solvent system (100:0 to 0:100) to obtain three fractions (E11– E13). E12 (700 mg) was chromatographed over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give 7 (179 mg) and 14 (18 mg). Fraction E2 (8 g) was chromatographed on silica gel CC (2.5 × 30 cm) eluted with petroleum ether/EtOAc (100:0 to 0:100) to yield six fractions (E21–E26). E25 (120 mg) was purified by preparative TLC (petroleum ether/EtOAc, 2:1) to obtain 6 (16 mg). E26 (170 mg) was purified by preparative TLC (cyclohexane/acetone, 2:1) to obtain 5 (23 mg). Fraction E3 (13 g) was subjected to silica gel CC (6 × 50 cm) with a petroleum ether/EtOAc gradient solvent system (100:0 to 0:100) to obtain five fractions (E31–E35). E33 (190 mg) was chromatographed over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give 9 (9 mg) and 10 (17 mg). Fraction E4 (9 g) was chromatographed on silica gel CC (2.5 × 30 cm) eluted with CH2Cl2/Me2CO (100:0 to 0:100) to obtain six fractions (E41– E46). E44 (75 mg) was fractioned by CC over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to yield 8 (20 mg). E45 (260 mg) was purified by preparative TLC (CH2Cl2/Me2CO, 2:1) to obtain 13 (119 mg). E5 (6.5 g) was subjected to silica gel CC (2.5 × 30 cm) eluted with CH2Cl2/Me2CO (100:0 to 0:100) to give three fractions (E51–E53). E51 (220 mg) was chromatographed over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to give three fractions (E511–E513). E511 (53 mg) was separated by preparative HPLC eluted with MeOH–H2O (40:60) to afford 11 (12 mg, tR 35.6 min) and 12 (17 mg, tR 41.3 min). The

Fig. 1. Chemical structures of compounds 1–14 isolated from the roots of Isatis indigotica.

L. Yang et al. / Fitoterapia 95 (2014) 175–181

n-BuOH extract (110 g) was subjected to a D101 macroporous resin column (10 × 120 cm) eluted with a gradually increasing amount of ethanol in water and got seven fractions (N1–N7). Fraction N3 (19 g) was purified by ODS open column (5 × 35 cm) chromatography eluting with MeOH–H2O (1:9 to 8:2) to give six fractions (N31–N36). Fractions N34 (1.6 g) and N35 (2.1 g) were applied to a Sephadex LH-20 column eluting with CH2Cl2/MeOH (1:1), and then purified by repeated RP-HPLC to give 1 (MeOH/H2O, 40:60, tR = 55.2 min, 13 mg) and 2 (MeOH/H2O, 50:50, tR = 30.4 min, 16 mg). Fraction N4 (7 g) was chromatographed on a Sephadex LH-20 column with CH2Cl2/MeOH (1:1) to give three subfractions (N41–N43). Fraction N42 (75 mg) was purified by repeated RP-HPLC to give 3 (MeOH/H2O, 40:60, tR = 55.1 min, 12 mg) and 4 (MeOH/H2O, 50:50, tR = 30.5 min, 54 mg). Indole-3-acetonitrile-2-S-β-D-glucopyranoside (1): yellow, amorphous powder (MeOH); UV (MeOH) λmax (log ε) 219 (4.55), 282 (4.12) nm; IR (KBr) vmax 3333, 2251, 1619, 1024, 749 cm−1; 1H NMR (300 MHz, DMSO-d6) and 13C NMR (75 MHz, DMSO-d6) data, see Table 1; HRESIMS m/z 373.0832 [M + Na]+ (calcd for C16H18N2O5SNa+: 373.0834); ESIMS m/z 723 [2 M + Na]+, 368 [M + NH4]+, 189 [M + H-Glc]+; ESIMS–MS MS2 (189) m/z 162 [M + H-Glc-HCN]+, 155 [M + H-Glc-H2S]+. Indole-3-acetonitrile-4-methoxy-2-S-β-D-glucopyranoside (2): colorless, amorphous powder (MeOH); UV (MeOH) λmax (log ε) 225 (4.58), 278 (4.26) nm; IR (KBr) vmax 3409, 2257, 1585, 1033, 724 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6) data, see Table 1; HRESIMS m/z 403.0932 [M + Na]+ (calcd for C17H20N2O6SNa+: 403.0940); ESIMS m/z 398 [M + NH4]+, 219 [M + H-Glc]+; ESIMS-MS MS2 (219) m/z 185 [M + H-Glc-H2S]+. N-methoxy-indole-3-acetonitrile-2-S-β-D-glucopyranoside (3): colorless, amorphous powder (MeOH); UV (MeOH) λmax (log ε) 220 (4.56), 282 (4.18); IR (KBr) vmax 3428, 2256, 1632, 1030, 737 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data, see Table 1; HRESIMS m/z

177

403.0935 [M + Na]+ (calcd for C17H20N2O6SNa+: 403.0940); ESIMS m/z 419 [M + K]+, 219 [M + H-Glc]+; ESIMS-MS MS2 (219) m/z 192 [M + H-Glc-HCN]+, 185 [M + H-Glc-H2S]+. 2.4. Acid hydrolysis of 1 and sugar analysis A solution of compound 1 (2.0 mg) in 2 M HCl (2 mL) was stirred at 80 °C in a stoppered vial for 2 h. The solution after cooling was evaporated under a stream of N2. Anhydrous pyridine solutions (1.0 mL) of the residue and L-cysteine methyl ester hydrochloride (1.5 mg) were mixed and warmed at 60 °C for 2 h. After drying the solution, trimethylsilyl imidazole (150 μL) was added to the mixture, which was warmed at 60 °C for another 1 h and then partitioned between H2O (500 μL) and cyclohexane (500 μL). The cyclohexane layer was concentrated and analyzed by GC using a HP-5 column. Temperatures of the injector and detector were 260 and 280 °C, respectively. A temperature gradient system was used for the oven, starting at 100 °C and increasing up to 140 °C at a rate of 4 °C/min, and then increasing up to 170 °C for 8 min at a rate of 13 °C/min, and finally, increasing up to 200 °C at a rate of 5 °C/min. The peaks of authentic samples of D-glucose and L-glucose after treatment in the same manner were detected at 20.25 and 21.48 min. 2.5. Acid hydrolysis of 1 with 5% aqueous HCl—1,4-dioxane A solution of compound 1 (5 mg) in 5% aqueous HCl— 1,4-dioxane (1:1, v/v, 1 mL) was stirred under reflux in a stoppered vial for 1 h. The reaction mixture was evaporated to dryness and then applied to an open ODS column (2 × 5 cm, 50 μm). The column was eluted with 50 mL H2O. The H2O eluate was concentrated to obtain 1-thio-glucose (0.6 mg), which was identified by ESI–MS and NMR analysis. 1-thio-glucose: ESI–MS m/z 195 [1-thio-Glc-H]−, δH (400 MHz, CD3OD) 2.65 (s, \SH), 3.30–3.40 (3H, o, H-2, 3, 5), 3.49 (1H, t, J = 8.7 Hz, H-4), 3.67 (1H, dd, J = 12.0, 5.4 Hz, H-6), 3.86 (1H,

Table 1 1 H and 13C NMR data for compounds 1–3a. Position

1 δC

1 2 3 4 5 6 7 8 9 10 11 1' 2' 3' 4' 5' 6' -OCH3 a

2 δH (J in Hz)

δC

11.26 brs 123.4 110.8 118.3 119.5 122.8 111.5 136.5 125.9 13.2 119.1 87.5 72.3 77.7 69.6 81.0 61.1

7.64 7.09 7.20 7.38

d (7.9) dd (8.1, 7.1) dd (7.9, 7.1) d (8.1)

4.13 s 4.38 2.81 3.17 2.97 3.14 3.68 3.47

d (9.5) m m m m m m

3 δH (J in Hz)

δC

δH (J in Hz)

11.26 brs 121.8 110.9 153.2 99.7 123.9 104.8 137.9 116.4 14.9 119.6 87.5 72.2 77.8 69.6 81.1 61.1 55.3

6.54 d (7.7) 7.09 dd (8.1, 7.7) 6.95 d (8.1)

4.11 s 4.34 2.78 3.15 2.97 3.13 3.67 3.47 3.89

d (9.6) m m m m m m s

124.5 111.5 120.0 122.1 125.7 110.0 134.9 123.5 14.7 119.9 90.3 74.4 79.5 71.2 82.4 62.8 67.1

7.72 7.21 7.37 7.51

d (8.0) dd (8.0, 7.7) dd (8.2, 7.7) d (8.2)

4.20 s 4.51 3.17 3.38 3.29 3.25 3.85 3.70 4.22

d (9.7) m m m m m m s

The 1H NMR data of 1 were obtained at 300 MHz, while the 1H NMR data of 2–3 were obtained at 600 MHz. The 13C NMR data of 1 were obtained at 75 MHz, while the 13C NMR of 2–3 was obtained at 150 MHz. The spectrum of compounds 1–2 was obtained in DMSO-d6, and the spectrum of 3 was obtained in CD3OD.

178

L. Yang et al. / Fitoterapia 95 (2014) 175–181

Fig. 2. Key HMBC correlations of compounds 1–3.

dd, J = 12.0, 1.6 Hz, H-6′), 4.40 (1H, d, J = 9.6 Hz, H-1); δC (150 MHz, CD3OD) 62.9, 71.3, 73.3, 79.7, 82.6, 91.6. 2.6. NO production bioassay The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction [20]. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 × 105 cells/well, and stimulated with 1 μg/mL of LPS in the presence or absence of test compounds. After incubation at 37 °C for 24 h, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent (mixture of equal volumes of reagent A and reagent B, A: 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B: 0.1% (w/v) of N-(1-naphthyl) ethylenediamine). Absorbance was measured in a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated by a calibration curve prepared with sodium nitrite standards. 3. Results and discussion Compound 1 was obtained as a yellow amorphous powder (MeOH). The IR spectrum showed absorption bands indicating the presence of aromatic function (1619 and 749 cm−1), nitrile group (2251 cm− 1) and OH group (3333 cm− 1). The UV spectrum of 1 showed the absorption maximum at 219 and 282 nm, which suggested the presence of an indole skeleton [21]. The HRESIMS analysis (m/z 373.0832 [M + Na]+) and the NMR data (Table 1) revealed the molecular formula C16H18N2O5S. The 13C NMR spectrum exhibited 16 carbon signals, consisting of nine unsaturated carbon atoms (δC 136.5, 125.9, 123.4, 122.8, 119.5, 119.1, 118.3, 111.5, 110.8) and seven aliphatic carbon atoms (δC 87.5, 81.0, 77.7, 72.3, 69.6, 61.1, 13.2). The 1H NMR spectrum showed signals of an exchangeable proton at δH 11.26 (brs, NH); an ortho-disubstituted benzene ring at δH 7.64 (1H, d, J = 7.9 Hz), 7.38 (1H, d, J = 8.1 Hz), 7.20 (1H, dd, J = 7.9, 7.1 Hz) and 7.09 (1H, dd,

J = 8.1, 7.1 Hz); an isolated methylene at δH 4.13 (2H, s); and some protons belonging to the sugar moiety. These protons and protonated carbon resonances in the NMR spectra were unambiguously assigned by the HSQC experiment. The HMBC spectrum (Fig. 2) showed the signal of H-4 (δH 7.64, 1H, d, J = 7.9 Hz) correlated with C-3 (δC 110.8), C-6 (δC 122.8) and C-8 (δC 136.5), H-5 (δH 7.09, 1H, dd, J = 8.1, 7.1 Hz) correlated with C-7 (δC 111.5) and C-9 (δC 125.9), H-6 (δH 7.20, 1H, dd, J = 7.9, 7.1 Hz) correlated with C-4 (δC 118.3), H-7 (δH 7.38, 1H, d, J = 8.1 Hz) correlated with C-5 (δC 119.5) and C-9 (δC 125.9), and H-10 (δH 4.13, 2H, s) correlated with C-2 (δC 123.4), C-3 (δC 110.8) and C-9 (δC 125.9). These spectroscopic data suggested that 1 possesses a 2-substituted indole3-acetonitril skeleton. Acid hydrolysis of 1 [22] with 2 M HCl afforded D-glucose by GC analysis following conversion to the trimethylsilyl thiazolidine derivatives. Moreover, acid hydrolysis of 1 with 5% aqueous HCl—1,4-dioxane [21] afforded 1-thio-glucose, which were identified by ESI–MS analysis and comparison of 1H and 13C NMR data with reported values [23]. This suggested that the S-D-glucopyranosyl moiety was located at C-2, which was confirmed by the HMBC correlation from the anomeric proton H-1′ (δH 4.38) to C-2 (δC 123.4). The β-configuration of the glycosidic linkage was established due to the coupling constant of the anomeric proton signal at δH 4.38 (1H, d, J = 9.5 Hz) [5]. Furthermore, the indole-2-Sglucopyranoside skeleton was supported by a series of characteristic mass spectral fragment ion peaks at m/z 351 [M + H]+, 189 [M + H-Glc]+, 162 [M + H-Glc-HCN]+ and 155 [M + H-Glc-H2S]+ in ESIMS and MS2 spectra (Fig. 3). Based on the above analysis, the structure of compound 1 was characterized as indole-3-acetonitrile-2-S-β-D-glucopyranoside. Compound 2 was isolated as colorless amorphous powder (MeOH). The ESI–MS exhibited an ion peak [M + NH4]+ at m/z 398 indicating a mass 380 compatible with the molecular formula of C17H20N2O6S. Its molecular formula was confirmed by HRESIMS, which showed an ion peak [M + Na]+ at m/z 403.0932 (calcd for C17H20N2O6SNa+: 403.0940). The 1H

Fig. 3. Characteristic mass spectral fragment ions of compound 1.

L. Yang et al. / Fitoterapia 95 (2014) 175–181 Table 2 Inhibitory effects of compounds 1–14 on NO production induced by LPS in RAW264.7 macrophages. Compound

IC50 ± SD (μM)

Compound

IC50 ± SD (μM)

1 2 3 4 5 6 7 8

91.43 57.89 78.95 5.87 8.19 5.34 8.80 12.48

9 10 11 12 13 14 Indomethacina

13.45 7.71 7.14 6.83 6.72 9.29 14.10

a

± ± ± ± ± ± ± ±

9.01 4.82 5.15 0.27 0.69 0.45 0.63 1.06

± ± ± ± ± ± ±

0.86 0.51 0.55 0.52 0.43 0.84 0.92

Positive control.

NMR data of 2 were closely similar to those of 1, except for the absence of an aromatic proton at δH 7.64 (1H, d, J = 7.9 Hz), the presence of an extra methoxy signal at δH 3.89 (3H, s) and a significant upfield shift of H-5 (δH 6.54). Furthermore, the 13C NMR spectrum showed an extra methoxy carbon signal at δC 55.3, a significant downfield shift of C-4 (δC 153.2) and the upfield shifts of C-5 (δC 99.7) and C-9 (δC 116.4) compared to that of 1. Therefore, it suggested that the aromatic proton (δ 7.64, 1H, d, J = 7.9 Hz, H-4) was substituted by the methoxy (δH 3.89, 3H, s). The HMBC correlation between the methoxy protons (δH 3.89) and C-4 (δC 153.2) confirmed that the methoxy group was located at C-4. The S-β-D-glucopyranosyl moiety was determined by using the same method as 1. Therefore, the structure of compound 2 was identified to be indole-3-acetonitrile-4-methoxy-2-S-β-D-glucopyranoside.

179

Compound 3 was also obtained as colorless amorphous powder (MeOH). The ESI–MS showed an ion peak [M + K]+ at m/z 419 indicating a mass 380 compatible with the molecular formula of C17H20N2O6S. Its molecular formula was confirmed by HRESIMS, which exhibited an ion peak [M + Na]+ at m/z 403.0935 (calcd for C17H20N2O6SNa+: 403.0940). The 1H NMR data of 3 were similar to those of 1, except for the presence of an extra methoxy proton signal at δH 4.22 (3H, s), and the 13C NMR spectrum showed an extra carbon signal at δC 67.1, indicating the presence of a N\OCH3 group in 3 [5]. The S-β-Dglucopyranosyl moiety was determined by using the same method as 1. On the basis of these evidences, the structure of compound 3 was determined to be N-methoxy-indole-3acetonitrile-2-S-β-D-glucopyranoside. In addition, the known compounds were identified as indole-3-acetonitrile-6-O-β-D-glucopyranoside (4) [4], indole3-acetonitrile (5) [24], arvelexin (6) [25], 1-methoxy-indole-3acetonitrile (7) [24], 3-indoleformic acid (8) [26], 3-indoleformic acid methyl ester (9) [27], 1-methoxy-3-indoleformic acid (10) [28], 3-indoleacetic acid (11) [29], 4-methoxy-3-indoleacetic acid (12) [30], 1-methoxy-3-indoleacetic acid (13) [31] and 1-methoxy-3-indolecarbaldehyde (14) [10] by comparing their measured spectroscopic data with the reported data in literature. Indole alkaloids are the main type of alkaloids in I. indigotica, which exhibited anti-inflammatory, antivirus and anticancer activities [5,6]. These pharmacologic actions, together with the traditional use for treatment of epidemic hepatitis, promoted us to test inhibitory effects on nitric oxide production of all the isolates. Nitric oxide (NO) is a short-lived free radical produced

Fig. 4. The proposed biosynthesis pathway of compounds 1–3.

180

L. Yang et al. / Fitoterapia 95 (2014) 175–181

from L-arginine by nitric oxide synthase (NOS) and affects every step of the development of inflammation. The effect of NO is exerted through multiple mechanisms including interaction with cell signaling systems like cGMP, cAMP, G-protein, JAK/ STAT or MAPK dependent signal transduction pathways. It may also lead to modification of transcription factors activity and in this way modulate the expression of multiple other mediators of inflammation [32]. In the present study, all isolated compounds were tested for their inhibitory effects on NO production induced by LPS in macrophages [20]. The IC50 values (Table 2) suggested that compounds 5–14, all indole aglycones, exhibited potent inhibitory activities against NO production, with IC50 values of 5.34 to 13.45 μM (the positive control, indomethacin, gave an IC50 value of 14.10 μM). This suggested that different substituents at C-3 (\CH2CN in 5–7, \COOH in 8–10, \CH2COOH in 11–13 and \CHO in 14) did not produce a significant effect on NO inhibitory activity, and the N1 or C4 methoxy-substituted derivatives (6, 7, 10, 12, 13 and 14) maintain the activity. Compounds 1–3, three indole-2-Sglucopyranosides, displayed weak inhibitory activities (IC50 91.43, 57.89 and 78.95 μM, respectively). However, compound 4, an indole-6-O-glucopyranoside, showed a strong inhibitory activity on nitric oxide production (IC50 5.87 μM). This suggested that having no substituent at C-2 is essential for the NO inhibitory activity, which can explain the higher NO inhibitory activity of compound 4, an indole-6-glycoside, compared to three indole-2-glycosides (1–3). Further studies on a detailed structure–activity relationship remain to be explored. There are nine indole glucosinolates (with a -S-β-D-glucose group linked to indole side chain) [15–18] and two indole-2C-glycosides [5] isolated from Isatis up to now. However, indole-2-S-glycosides are not common and compounds 1–3 are the first reported indole-2-S-glycosides from Isatis. Therefore, a plausible biosynthesis pathway is proposed in Fig. 4. Firstly, tryptophan is converted to indole-3-acetaldoxime, which is conjugated with a sulfur donor (presumably cysteine) to form S-(indolylacetohydroximoyl)-L-cysteine [33]. Secondly, S-(indolylacetohydroximoyl)-L-cysteine is converted to indole-3-thiohydroximic acid catalysed by a C-S lyase [33]. Thirdly, indole-3-thiohydroximic acid is isomerized to form indolyl-3-acetothiohydroxamic acid [34,35], which is then oxidatively cyclized to generate intermediate a [36]. Finally, ring-opening of intermediate a would produce intermediate b [37], which then undergoes glycosylation and dehydration to generate compounds 1–3 [33]. Appendix A. Supplementary data 1D NMR, 2D NMR, ESIMS and MS2, HRESIMS, IR and UV spectra for new compounds 1–3. Supplementary related to this article can be found online at http://dx.doi.org/10.1016/ j.fitote.2014.03.019. References [1] Jiangsu New Medical College. Dictionary of traditional Chinese medicine. Shanghai: Shanghai Science and Technology Publishing House; 1985 1250–2. [2] Wu XY, Qin GW, Cheung KK, Cheng KF. New alkaloids from Isatis indigotica. Tetrahedron 1997;53:13323–8. [3] Wu XY, Liu YH, Sheng WY, Sun J, Qin GW. Chemical constituents of Isatis indigotica. Planta Med 1997;63:55–7.

[4] Li B, Chen WS, Zheng SQ, Yang GJ, Qiao CZ. Two new alkaloids isolated from tetraploidy banlangen. Acta Pharm Sin 2000;35:508–10. [5] WuY,ZhangZX,HuH,LiDM,QiuGF,HuXM,etal.NovelindoleC-glycosides from Isatis indigotica and their potential cytotoxic activity. Fitoterapia 2011;82:288–92. [6] Chen MH, Gan LS, Lin S, Wang XL, Li L, Li YH, et al. Alkaloids from the root of Isatis indigotica. J Nat Prod 2012;75:1167–76. [7] Li B, Chen WS, Yang GJ, Zhang WD, Qiao CZ. Organic acids of tetraploidy Isatis indigotica. Acad J Sec Mil Med Univ 2000;21:207–8. [8] Liu HL, Wu LJ, Li H, Wang J. Study on the chemical constituents of Isatis indigotica Fort. J Shenyang Pharm Univ 2002;19:93–5. [9] He LW, Li X, Chen JW, Sun DD, Ju WZ, Wang KC. Chemical constituents from water extract of Radix isatidis. Acta Pharm Sin 2006;41:1193–6. [10] Zuo L, Li JB, Xu J, Yang JZ, Zhang DM, Tong YL. Studies on chemical constituents in root of Isatis indigotica. Chin J Chin Mater Med 2007;32:688–91. [11] Sun DD, Dong WW, Zhang HQ, Huang XF. A new ceramide from the root of Isatis indigotica and its cytotoxic activity. Chem Nat Comp 2010;46:180–3. [12] Ensley BD, Ratzkin BJ, Osslund TD, Simon MJ, Wackett LP, Gibson DT. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 1983;222:167–9. [13] Alex D, Lam IK, Lin ZX, Lee SMY. Indirubin shows anti-angiogenic activity in an in vivo zebrafish model and an in vitro HUVEC model. J Ethnopharmacol 2010;131:242–7. [14] Pedras MSC, Chumala PB, Suchy M. Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity. Phytochemistry 2003;64:949–56. [15] Elliott MC, Stowe BB. A novel sulphonated natural indole. Phytochemistry 1970;9:1629–32. [16] Goetz JK, Schraudolf H. Two natural indole glucosinolates from Brassicaceae. Phytochemistry 1983;22:905–7. [17] Frechard A, Fabre N, Pean C, Montaut S, Fauvel MT, Rollin P, et al. Novel indole-type glucosinolates from woad (Isatis tinctoria L.). Tetrahedron Lett 2001;42:9015–7. [18] Mohn T, Hamburger M. Glucosinolate pattern in Isatis tinctoria and I. indigotica seeds. Planta Med 2008;74:885–8. [19] Yoshikawa M, Murakami T, Kishi A, Sakurama T, Matsuda H, Nomura M, et al. Novel indole S, O-bisdesmoside, calanthoside, the precursor glycoside of tryptanthrin, indirubin, and isatin, with increasing skin blood flow promoting effects, from two calanthe species (Orchidaceae). Chem Pharm Bull 1998;46:886–8. [20] Dirsch VM, Stuppner H, Vollmar AM. The Griess assay: suitable for a bio-guided fractionation of anti-inflammatory plant extracts? Planta Med 1998;64:423–6. [21] Murakami T, Kishi A, Sakurama T, Matsuda H, Yoshikawa M. Chemical constituents of two oriental orchids, Calanthe discolor and C. liukiuensis: precursor indole glycoside of tryptanthrin and indirubin. Heterocycles 2001;54:957–66. [22] Dai Y, Zhou GX, Kurihara H, Ye WC, Yao XS. Biphenyl glycosides from the fruit of Pyracantha fortuneana. J Nat Prod 2006;69:1022–4. [23] Bellostas N, Sørensen AD, Sørensen JC, Sørensen H. Fe2+-catalyzed formation of nitriles and thionamides from intact glucosinolates. J Nat Prod 2008;71:76–80. [24] Kim JS, Choi YH, Seo JH, Lee JW, Kim YS, Ryu SY, et al. Chemical constituents from the root of Brassica campestris ssp rape. Kor J Pharmacogn 2004;35:259–63. [25] Choi YH, Kim JS, Seo JH, Lee JW, Kim YS, Ryu SY, et al. Chemical constituents of Brassica campestris ssp pekinensis. Kor J Pharmacogn 2004;35:255–8. [26] Hagemeier J, Schneider B, Oldham NJ, Hahlbrock K. Accumulation of soluble and wall-bound indolic metabolites in Arabidopsis thaliana leaves infected with virulent or avirulent Pseudomonas syringae pathovar tomato strains. Proc Natl Acad Sci U S A 2001;98:753–8. [27] Bano S, Ahmad VU, Perveen S, Bano N, Shafiuddin Shameel M. Chemical constituents of red alga Botryocladia leptopoda. Planta Med 1987;53:117–8. [28] Somei M, Tanimoto A, Orita H, Yamada F, Ohta T. Syntheses of Wasabi phytoalexin (methyl 1-methoxyindole-3-carboxylate) and its 5-iodo derivative, and their nucleophilic substitution reactions. Heterocycles 2001;54:425–32. [29] Wang YY, Li X, Chen JW, Cheng Y, Wang S. Study on chemical constituents from water extract of Radix isatidis. Res Pract Chin Med 2009;23:54–6. [30] Kai K, Horita J, Wakasa K, Miyagawa H. Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana. Phytochemistry 2007;68:1651–63. [31] Luo DQ, Chen YP, Zhang J, Shi BZ, Yang ZQ, Chen C. A new glycine derivative and a new indole alkaloid from the fermentation broth of the plant endophytic fungus Pestalotiopsis podocarpi isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. Helv Chim Acta 2013;96:309–12.

L. Yang et al. / Fitoterapia 95 (2014) 175–181 [32] Guzik TJ, Korbut R, Adamek GT. Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Pharmacol 2003;54:469–87. [33] Redovnikovic IR, Glivetic T, Delonga K, Furac JV. Glucosinolates and their potential role in plant. Period Biol 2008;110:297–309. [34] Pedras MSC, Hossain S. Interaction of cruciferous phytoanticipins with plant fungal pathogens: indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are. Phytochemistry 2011;72:2308–16.

181

[35] Pedras MSC, Okinyo DPO. Remarkable incorporation of the first sulfur containing indole derivative: another piece in the biosynthetic puzzle of crucifer phytoalexins. Org Biomol Chem 2008;6:51–4. [36] Monde K, Takasugi M, Ohnishi T. Biosynthesis of cruciferous phytoalexins. J Am Chem Soc 1994;116:6650–7. [37] Pedras MSC, Okanga FI. Strategies of cruciferous pathogenic fungi: detoxification of the phytoalexin cyclobrassinin by mimicry. J Agric Food Chem 1999;47:1196–202.

Indole alkaloids from the roots of Isatis indigotica and their inhibitory effects on nitric oxide production.

Three rare indole-2-S-glycosides, indole-3-acetonitrile-2-S-β-D-glucopyranoside (1), indole-3-acetonitrile-4-methoxy-2-S-β-D-glucopyranoside (2) and N...
559KB Sizes 0 Downloads 4 Views