PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5168
Antiinflammatory Effects and Chemical Constituents of Veronicastrum axillare Cheng-Jian Zheng, 1† Xue-Hong Deng, 1,2† Yu Wu, 1 Yi-Ping Jiang, 1 Jian-Yong Zhu 1 and Lu-Ping Qin1,3* 1
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Department of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Huatuo Road, Fuzhou 350108, China 3 Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai 200433, China 2
Our study aims to ascertain the antiinflammatory activity of Veronicastrum axillare and characterize the bioactive constituents. Antiinflammatory activity of the total extract and different fractions from V. axillare was investigated by employing the xylene-induced mouse ear edema model. As a result, the ethyl acetate (EtOAc) fraction showed the highest antiinflammatory activity in vivo. From the EtOAc fraction and the inactive dichloromethane fraction, a total of five new compounds, axillasides A–C and axillactones A and B, together with four known compounds, procumboside A, buergeriside C1, indole-3-carboxylic acid and apigenin, were isolated and identified. Their structures were elucidated on the basis of spectroscopic analysis and by comparison of their nuclear magnetic resonance data with those reported in the literature. Procumboside A, a major constituent in EtOAc fraction, showed significant antiinflammatory activity in vivo. Further studies revealed that procumboside A was a potent COX-2 inhibitor, significantly reducing the COX-2 protein level in lipopolysaccharide-stimulated RAW 264.7 macrophages. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Veronicastrum axillare; axillaside; axillactone; antiinflammatory activity.
INTRODUCTION Veronicastrum axillare (Sieb. et Zucc.) Yamazaki (Scrophulariaceae) is a perennial herb with bent stems, hairless paper-like leaves and axillary inflorescence. This plant is mainly distributed in Jiangsu, Anhui, Jiangxi, Fujian, Guangdong and Taiwan in China, and it also spreads in Japan (Chinese Academy of Sciences, 1979). Traditional use of V. axillare focused on relieving edema through diuresis, eliminating blood stasis, relieving pain and expelling retained water in the treatment of ascites in folk medicine. Up to the present, there has been little phytochemical data and none biological activity reported on this plant, whereas other Veronicastrum plants have been shown versatile pharmacological properties, including antiinflammatory, analgesic, antibiosis and immunosuppressive activities (Gao et al., 2004; Huang et al., 2009; Zhou and Meng, 1992). In our recent study, we investigated the antiinflammatory activity of V. axillare using the xyleneinduced mouse ear edema model and found the ethyl acetate (EtOAc) fraction to be the most active. Further phytochemical study led to the isolation of three new phenylpropanoid glycosides, axillasides A–C (1–3); a new aryl coumarin, axillactone A (4); and a new macrolide, axillactone B (5), along with four known compounds, procumboside A (6), buergeriside C1 (7), indole-
* Correspondence to: Lu-Ping Qin, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. E-mail: [email protected] † Both authors contributed equally to this work.
Copyright © 2014 John Wiley & Sons, Ltd.
3-carboxylic acid (8) and apigenin (9) (Arunendra et al., 2005; Hagemeier et al., 2001; Kim and Kim, 2000; Luo et al., 2011). Furthermore, procumboside A (6), a major constituent of the EtOAc fraction, showed significant antiinflammatory activity both in vitro and in vivo. Herein, we present the antiinflammatory assay on V. axillare as well as the isolation and structural elucidation for new compounds 1–5.
MATERIALS AND METHODS General. Optical rotations were acquired with a PerkinElmer 341 polarimeter (PerkinElmer, Norwalk, CA, USA) at room temperature. Infrared (IR) spectra were recorded on a Bruker Vector 22 spectrometer (Bruker, Billerica, MA, USA) with KBr pellets. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 NMR spectrometer (Bruker, Billerica, MA, USA) with tetramethylsilane as an internal standard. Electrospray ionisation mass spectrometry (ESI-MS) were measured on an Agilent LC/MSD Trap XCT mass spectrometer, whereas high resolution (HR)-ESI-MS were measured using an Agilent 6538 UHD AccurateMass Q-TOF LC/MS spectrometer (Agilent Technologies Inc., Santa Clara, CA). All solvents used were of analytical grade (Shanghai Chemical Plant). Materials for column chromatography were silica gel (200–300 mesh; Huiyou Silica Gel Development Co., Ltd. Yantai, China), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), silica gel H (10–40 μm; Yantai), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and YMC-Gel Octadecylsilyl (ODS)-A (50 μm; YMC, Milford, MA, USA). Medium Received 1 March 2014 Revised 8 April 2014 Accepted 16 April 2014
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pressure liquid chromatography (MPLC) was carried on Dr Flash II system (Lisure Science (Suzhou) Co., Ltd). Semipreparative HPLC was performed on an Agilent 1200 series (XDB-C18, 9.4 250 mm). HSGF254 silica gel thin layer chromatography (TLC) plates (Yantai) were used for analytical TLC. Preparative TLC (0.4–0.5 mm) was conducted on glass plates precoated with silica gel GF254 (Yantai). Plant material. The whole plants of V. axillare (Sieb. et Zucc.) Yamazaki were collected in Jiande, Zhejiang Province, China, in July 2011 and identified by Prof. Lu-Ping Qin, School of Pharmacy, Second Military Medical University. A voucher specimen (#201107) has been deposited at the Herbarium of Materia Medica, School of Pharmacy, Second Military Medical University, Shanghai, China. Extraction and isolation. The air-dried and powdered whole plants (8.5 kg) of V. axillare were extracted with hot aqueous ethanol (80% v/v) under reflux three times for 2 h each time. After removal of the solvent under reduced pressure, the total extract (1700 g) was partitioned sequentially with petroleum ether, CH2Cl2, EtOAc and n-BuOH, obtaining four fractions with yields of 91.5, 55, 132 and 500 g, respectively. The CH2Cl2 fraction was subjected to silica gel chromatography using petroleum ether-EtOAc mixtures (20:1, 10:1, 5:1, 3:1, 1:1 v/v) to afford fractions A–E. Fr.D (4.5 g) was rechromatographed on a silica gel column chromatography (CC) with gradient petroleum ether-EtOAc (10:1, 8:1, 5:1, 3:1, 1:1) to yield four fractions Fr. D-IFr.D-IV. Fr.D-II (1.7 g) was subjected to silica gel CC eluted with petroleum ether-EtOAc mixtures (3:1, 2:1, 1:1) to obtain two fractions, Fr.D-II-1 and Fr. D-II-2. Fr.D-II-1 (750 mg) was loaded onto a Sephadex LH-20 column (CH2Cl2-MeOH, 1:4) to give two subfractions, the former (88.4 mg) of which was further purified by preparative TLC (CH2Cl2-MeOH, 30:1) to afford 5 (42.2 mg), and the latter was (457.3 mg) purified by HPLC on a semi-preparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 75% MeOH) to afford 3 (36.6 mg). Fr.D-II-2 (104.9 mg) was subjected to a Sephadex LH-20 column (CH2Cl2-MeOH, 1:4) to yield apigenin (9, 3.0 mg). Fr.D-III (622.9 mg) was refractionated by silica gel CC eluted with gradient petroleum ether-EtOAc (4:1, 3:1, 2:1, 1:1) to give two fractions, Fr.D-III-1 (340.5 mg) and Fr.D-III-2 (238.4 mg), which was further purified by preparative TLC (CH 2 Cl 2 -MeOH, 20:1) to yield 2 (180.0 mg) and 1 (52.5 mg), respectively. Fr.E (1.5 g) was rechromatographed on a silica gel CC with gradient petroleum ether-EtOAc (2:1, 1:1, 1:2) to give Fr.E-IFr.E-III. Fr.E-III (502 mg) was loaded onto a Sephadex LH-20 column (CH2Cl2-MeOH, 1:1), followed by HPLC on a semi-preparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 50% MeOH) to yield buergeriside C1 (7, 3.4 mg). The EtOAc extract (110 g) was subjected to silica gel CC using CH2Cl2-MeOH mixtures (30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 0:1) to yield fractions F–K. Fr.F (5.9 g) was chromatographed by MPLC over a ODS-C18 column, eluted with gradient MeOH-H2O (3:7→9:1) at 15 mL/min for 50 min and further purified on a Sephadex LH-20 column (MeOHCopyright © 2014 John Wiley & Sons, Ltd.
H2O, 4:1) to yield indole-3-carboxylic acid (8, 1.5 mg). Fr.H (1.8 g) was also chromatographed by MPLC over a ODS-C18 column, eluted with 20% MeOH at 15 mL/min for 30 min, followed by HPLC on a semipreparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 50% MeOH) to give 4 (24.4 mg). Fr.I (13.5 g) was chromatographed by MPLC over a silica gel CC with gradient CH2Cl2-MeOH (100:0→90:10) to yield procumboside A (6, 5.0 g). Axillaside A (1): White amorphous powder; [α] 25 D +23.7 (c 0.10, MeOH); ultraviolet (UV) (MeOH) λmax 291 nm; IR (KBr) νmax 599, 711, 766, 1207, 1282, 1334, 1387, 1449, 1496, 1636, 1708, 2935, 2968, 3442 and 3482 cm1; HR-ESI-MS m/z 317.0998 [M + Na]+ (calculated for C15H18O6Na, 317.1003); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillaside B (2): Yellowish oil; [α]25 D +50.0 (c 0.10, MeOH); UV (MeOH) λmax 290 nm; IR (KBr) νmax 684, 768, 1058, 1184, 1450, 1496, 1635, 1698, 2933, 2976 and 3421 cm1; HR-ESI-MS m/z 293.1103 [M H] (calculated for C15H17O6, 293.1036); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillaside C (3): Yellowish oil; [α] 25 D +236.4 (c 0.11, MeOH); UV (MeOH) λmax 275, 337 nm; IR (KBr) νmax 683, 767, 1062, 1120, 1162, 1204, 1282, 1312, 1449, 1496, 1635, 1716, 2933, 2976, 3028, 3061 and 3412 cm1; HRESI-MS m/z 447.1414 [M + Na]+ (calculated for C24H24O7Na, 447.1419 ); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillactone A (4): White amorphous powder; UV (MeOH) λmax 288, 345 nm; IR (KBr) νmax 782, 819, 866, 1131, 1188, 1384, 1447, 1563, 1625, 1689, 2850, 2919 and 3423 cm1; HR-ESI-MS m/z 271.0601 [M + H]+ (calculated for C15H11O5, 271.0606); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 2). Axillactone B (5): Yellowish oil; [α] 25 D +1.2 (c 0.10, MeOH); UV (MeOH) λmax 288 nm; IR (KBr) νmax 1187, 1221, 1362, 1383, 1412, 1464, 1595, 1634, 1713, 2853 and 2925 cm1; HR-ESI-MS m/z 293.2096 [M + H]+ (calculated for C18H29O3, 293.2102); 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data (Table 2).
Antiinflammatory assays. The antiinflammatory activity was investigated by employing the xylene-induced mouse ear edema model as previously described (Tang et al., 1984). Institute of Cancer Research (ICR) mice were randomly divided into 15 groups (n = 10) and were acclimated to condition for 4 days before the experiment. On the fifth day, positive drug dexamethasone (1 mg/kg), total extract (225 and 900 mg/kg), petroleum ether fraction (12 and 48 mg/kg), dichloromethane fraction (7.5 and 30 mg/kg), ethyl acetate fraction (17.5 and 70 mg/kg), n-BuOH fraction (66 and 264 mg/kg), procumboside A (2.5, 5 and 10 mg/kg) and 5% carboxymethyl cellulose–Na solution were intragastrically given in a single dose. One hour later, all animals were treated with xylene on the anterior and posterior surfaces of the right ear (15 μL on each side of the ear), and the left ear was used as a control. Mice were sacrificed through cervical dislocation 0.5 h after xylene application. Ear biopsies of 6.0 mm in diameter were punched out and Phytother. Res. (2014)
ANTIINFLAMMATORY CONSTITUENTS OF VERONICASTRUM AXILLARE
Table 1. 1H and
C NMR spectroscopic data of compounds 1–3 (δ in ppm, J in Hz)
13
1 No. 1 2 3 4 5 6 1' 2' 3' 4' 5' 6' α' β' C═O 1" 2" 3" 4" 5" 6" α" β" C=O
2
3
δH
δC
δH
δC
δH
δC
5.03, d (1.8) 3.85, dd (3.6, 1.8) 3.96, dd (9.6, 3.6) 5.06, brt (9.6) 4.03, dq (9.6, 6.6) 1.16, d (6.6) — 7.59, dd (7.2, 1.8) 7.39, m 7.39, m 7.39, m 7.59, dd (7.2, 1.8) 7.70, d (16.2) 6.55, d (16.2) — — — — — — — — — —
95.8 73.2 70.2 76.0 67.3 18.1 135.8 129.3 130.1 131.6 130.1 129.3 146.5 118.9 168.4 — — — — — — — — —
5.03, d (1.8) 4.02, dd (3.6, 1.8) 5.17, dd (9.6, 3.0) 3.66, brt (9.6) 3.96, dq (9.6, 6.6) 1.29, d (6.6) — 7.58, dd (7.8, 1.8) 7.38, m 7.38, m 7.38, m 7.58, dd (7.8, 1.8) 7.75, d (16.2) 6.59, d (16.2) — — — — — — — — — —
95.8 71.0 75.7 71.7 69.5 18.2 135.9 129.2 130.0 131.5 130.0 129.2 146.4 119.2 168.4 — — — — — — — — —
5.11, d (1.8) 5.40, dd (3.6, 1.8) 5.36, dd (9.6, 3.6) 3.72, brt (9.6) 4.06, dq (9.6, 6.6) 1.35, d (6.6) — 7.55, dd (7.8, 1.2) 7.38, m 7.38, m 7.38, m 7.55, dd (7.8, 1.2) 7.68, d (16.2) 6.57, d (16.2) — — 7.48, m 7.31, m 7.31, m 7.31, m 7.48, m 7.64, d (15.6) 6.46, d (15.6) —
93.4 72.6 73.4 72.0 69.4 18.3 135.5 129.4 129.9 131.8 129.9 129.4 147.3 118.4 167.6 135.6 129.3 130.1 131.5 130.1 129.3 146.7 118.7 167.9
Table 2. 1H and 13C NMR spectroscopic data of compounds 4 and 5 (δ in ppm, J in Hz) 4
5
No.
δH
δC
No.
δH
δC
1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 17 18
— —
— 163.7 116.5 156.2 113.1 148.5 118.6 121.1 155.2 121.3 124.2 117.0 151.6 148.2 118.1 118.5 — —
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
— 2.33, t (7.2) 1.61, m 1.32, m 1.32, m 1.32, m 1.61, m 2.53, t (7.2) — 6.15, d (15.0) 7.13, dd (15.0, 10.8) 6.35, dd (15.0, 10.8) 6.15, d (15.0) 4.24, dd (12.0, 6.0) 1.60, m 1.32, m 1.32, m 0.90, t (6.6)
178.6 33.8 24.4 28.6 28.7 28.8 24.0 40.6 200.9 129.8 141.7 127.9 145.7 72.1 36.7 27.4 22.4 13.9
6.30, s — 6.74, d (3.0) — 7.03, dd (9.0, 3.0) 7.23, d (9.0) — — — 6.64, d (3.0) — — 6.81, d (9.0) 6.80, dd (9.0, 3.0) — —
weighed. The extent of ear edema was determined by the weight difference (⊿W) between the right and left ear biopsies of the same animal. The inhibition percentage was expressed as the reduction in ear weight compared with the vehicle group. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Animal Experimentation Ethics Committee of the Second Military Copyright © 2014 John Wiley & Sons, Ltd.
Medical University (Date: 2013-03-14; Approval number: 2013LY014). Edema inhibition ð%Þ ¼ ð⊿Wvehicle –⊿Wtreated Þ=⊿Wvehicle 100%: Western blot analysis. The murine RAW 264.7 cell line was seeded at an initial density of 2 106 cells/well in Phytother. Res. (2014)
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6-well tissue culture plates overnight. Cells were exposed to Escherichia coli lipopolysaccharide (LPS) (1 μg/mL; Sigma) for 24 h in the presence or absence of the tested compounds. Indomethacin, a COX-2 inhibitor, was used as a positive control in this experiment. Protein samples were collected and prepared as described previously (Zheng et al., 2010; Wang et al., 2012). Briefly, cells were washed with phosphate buffered saline and harvested by scraping in appropriate volume of cell lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were then incubated on ice for 20 min with intermittent vortexing followed by centrifugation at 40 000 g for 10 min at 4 °C. Samples containing equal quantities of protein (40 μg) were subjected to SDS/20%–polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes. The resultant nitrocellulose membranes were incubated with blocking solution and probed using antibodies specific to COX-2 (1:1000 dilution; cell signalling) protein and visualized using an ECL detection kit (Perkin-Elmer, Western Lightning Chemiluminescence Reagent Plus). Acid hydrolysis and sugar analysis. Compounds 1–3 (each 1.5 mg) were hydrolyzed with 1 mL of 2 M aqueous CF3CO2H (trifluoroacetic acid) in a 10 mL round bottomed flask at 120 °C for 2 h, after which the solvent was evaporated with a stream of N2. Then, the following solutions were added: (a) 1:8 (S)-1-amino-2-propanolMeOH (25 μL); (b) 1:4 glacial acetic acid-MeOH (25 μL); and (c) 3% NaBH3CN in MeOH (25 μL). The vial was capped, and the mixture was allowed to react for 1.5 h at 65 °C. After cooling, the mixture was evaporated and co-evaporated with MeOH (5 0.5 mL). The residue was dried overnight in a desiccator and treated with 1:1 pyridine-acetic anhydride (0.4 mL) for 45 min at 100 °C. After cooling, the derivatives were extracted with CHCl3 and washed with water (3 1 mL). The organic phase was dried with anhydrous Na2SO4 and subjected to gas chromatography (GC)-MS using an Agilent 7000 QQQ GC/MS (Agilent, CA, USA) system equipped with a HP-5ms column (30 m 0.25 mm 0.25 μm) with He as a carrier gas at a flow rate of 1.0 mL/min to identify the monosaccharide. The oven temperature started at 140 °C and was increased to 198 °C at a rate of 3 °C/min, and then increased to 220 °C at a rate of 1 °C/min, keeping it at 220 °C for 10 min, and then increased to 280 °C at a rate of 3 °C/min. Derivatives of D-rhamnose and L-rhamnose (reference standards) eluted at 23.953 and 23.515 min, and the tested samples eluted at 23.513 min (L-rhamnose).
dexamethasone (1 mg/kg, 29.82%). Among several fractions with different polarity from the total extract of V. axillare, only the EtOAc fraction showed potent antiinflammatory activity at both doses of 17.5 and 70 mg/ kg, with oedema inhibition of 42.11% and 43.86%, respectively (Fig. 1). Further bioactivity-guided isolation of the EtOAc fraction yielded a major constituent, procumboside A (6), which exhibited notable antiinflammatory effects at doses of 2.5, 5 and 10 mg/kg, with inhibition of 29.82%, 48.25% and 37.72%, respectively. Further studies revealed that procumboside A (6) (40 μM) and indomethacin (20 μM) significantly reduced the COX-2 protein level to 38.31 ± 4.19% and 29.94 ± 1.43% (Fig. 2), respectively, in LPS-stimulated RAW 264.7 cells, which indicated that the antiinflammatory of procumboside A (6) was related to its down-regulation COX-2 protein expression, thus validating the antiinflammatory properties of V. axillare extract and supporting its use in traditional medicine.
Characterization of new compounds In addition to procumboside A (6), three new phenylpropanoid glycosides, axillasides A–C (1–3); a new aryl coumarin, axillactone A (4); and a new macrolide, axillactone B (5), along with other three known compounds (Fig. 3) were isolated from the dichloromethane and EtOAc fractions from the extract of V. axillare, using successive silica gel, Chromatorex ODS and Sephadex LH20 column chromatography, semi-preparative HPLC and preparative TLC. Axillaside A (1) was obtained as an amorphous white powder and gave the molecular formula, C15H18O6, by positive-ion HR-ESI-MS. The 1H and 13C NMR spectra of 1 showed close similarities to those of buergeriside C1 (7), an analogue reported from Scrophularia buergeriana [6]. The major differences were the disappearance of the signals corresponding to an O-methyl group (δH 3.62 and δC 54.8 in 7) and the splitting pattern of the aromatic protons (A2B2C in 1; AABB in 7), which led to the assumption that 1 was a 4'-demethoxyl derivative of 7. The sugar moiety was recognized as α-L-rhamnopyranose based on its NMR
RESULTS AND DISCUSSION Biological evaluation The antiinflammatory activity of the total extract and different fractions from V. axillare was investigated by employing the xylene-induced mouse ear edema model. Dexamethasone (1 mg/kg) was used as a positive drug in our assay. As a result, the total extract of V. axillare, at high dose of 900 mg/kg, produced significant inhibition (45.61%) of oedema comparable with that of Copyright © 2014 John Wiley & Sons, Ltd.
Figure 1. Antiinflammatory activity of Veronicastrum axillare on xylene-induced ear edema in mice (DA: dexamethasone; TF: total extract; PEF: petroleum ether fraction; DF: dichloromethane fraction; AF: ethyl acetate fraction; BF: n-BuOH fraction; *p < 0.05 or **p < 0.01 compared with vehicle group). Phytother. Res. (2014)
ANTIINFLAMMATORY CONSTITUENTS OF VERONICASTRUM AXILLARE
Figure 2. Western blot analysis of procumboside A (6) on the COX-2 protein expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Data were expressed as mean ±SD (n = 3). **p 0.01 compared with LPS-treated group. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr
data (Table 1) and confirmed by GC analysis after alkali hydrolysis of 1. The esterification site of the cinnamoyl group was determined to be the C-4 position of the rhamnose unit by heteronuclear multiple bond correlation (HMBC; Fig. 4) observed from H-4 at δH 5.06 (1H, brt, J = 9.6 Hz) to the carbonyl signal at δC 168.4. Thus, the structure of 1 was elucidated as 4-O-(E)cinnamoyl-α-L-rhamnopyranose. Axillaside B (2) was obtained as a yellowish oil and analysed for the molecular formula, C15H18O6, by negative-ion HR-ESI-MS. According to its molecular formula and NMR data, 2 was determined to possess the same carbon framework as 1, differing only in the splitting pattern and chemical shift of an oxygenated methine group (δH 5.17, dd, J = 9.6, 3.0 Hz; δC 75.7 in 2; δH 5.06, brt, J = 9.6 Hz; δC 76.0 in 1), indicating that 2 is a regioisomer with a cinnamoyl group at C-3 instead of C-4. This was confirmed by the HMBC correlation from H-3 at δH 5.17 (1H, dd, J = 9.6, 3.0 Hz) to the ester carbonyl signal at δC 168.4 (Fig. 4). The structure of 2
was therefore determined as 3-O-(E)-cinnamoyl-α-Lrhamnopyranose. Axillaside C (3), obtained as a yellowish oil, gave the molecular formula, C24H24O7, based on its positive-ion HR-ESI-MS. Its 1H and 13C NMR spectra were similar to those of 1 and 2, apart from the appearance of two (E)-cinnamic acid moieties in the molecule, as represented by ten protons of two monosubstituted aromatic rings and four trans olefinic protons (Table 1). Its sugar moiety was also confirmed as α-L-rhamnopyranose with a free hydroxyl group at C-1, evidenced by the up-field shifted signal of C-1 at δC 93.4 [6]. The signals of H-2 and H-3 of rhamnose were shifted 1.4 ppm downfield (5.40 and 5.36, respectively), indicating esterification of two separate (E)-cinnamoyl groups at the C-2 and C-3 positions of the rhamnose unit, which was confirmed by HMBC correlations (Fig. 4) from H-2 at δH 5.40 to a carbonyl carbon at δC 167.6 and from H-3 at δH 5.36 to another carbonyl carbon at δC 167.9. Therefore, the structure of 3 was verified as 2, 3-di-O-(E)-cinnamoylα-L-rhamnopyranose. Axillactone A (4) was obtained as an amorphous white power, and the molecular formula was established as C15H10O5 deduced from the positive-ion HR-ESI-MS. Its NMR data were characterized by the presence of two phenyl moieties of ABX (a spin pattern of three nuclei constituted of AB part and X part) spin system, which were analogous to those of 4-(3',4'-dimethoxyphenyl)-6-methoxycoumarin, a synthesized 4-arylcoumarin with antiprotozoal activity, except for the disappearance of the signals corresponding to three O-methyl groups (Pierson et al., 2010). All data indicated that 4 was a 3',4',6-O-tridemethyl derivative of 4-(3',4'-dimethoxyphenyl)-6-methoxycoumarin. In addition, the linkage position of the aryl group was determined to be the C-4 position of the coumarin moiety, evidenced by HMBC correlations from H-3 at δH 6.30 (1H, s) to C-1' at δC 124.2 and from H-2’ at δH 6.64 (1H, d, J = 3.0 Hz) to C-4 at δC 156.2, as well as supported by the nuclear overhauser effect spectroscopy correlations between H-3/H-2' and H-5/H-2' (Fig. 4). Finally, the structure of 4 was deduced to be 4-(3',4'-dihydroxyphenyl)-6-hydroxy-coumarin. Axillactone B (5) was obtained as a yellowish oil and gave the molecular formula, C18H28O3, by positive-ion
Figure 3. Chemical structures of compounds 1–9. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. (2014)
C.-J. ZHENG ET AL.
1
1
Figure 4. H– H COSY (▬) and key heteronuclear multiple bond correlations (→) of compounds 3–5 and selected nuclear overhauser effect spectroscopy correlations (↔) of compound 4. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr
Figure 5. Main electron ionization/mass spectrometry fragments of compound 5.
HR-ESI-MS. Its NMR spectral features (Table 2) along with five unsaturation degrees in the molecular formula suggested 5 to be a C18 unsaturated oxo fatty acid with cyclic ester structure, characterized by the presence of a conjugated E,E-form dienone system from C-9, a carbonyl carbon at δC 200.9, to C-13, and an oxygenated methine carbon at C-14 (δC 72.1), identified from the HMBC correlations of H-10/C-9, C-12; H-11/C-9, C-13; H-12/C-10, C-11, C-14; H-13/C-11, C-14; H-14/C-12, C13, C-15, C-16; and H-18/C-16, C-17 (Fig. 4), as well as electron ionization–MS analysis (Fig. 5). Detailed analysis of electron ionization–MS data indicated that notable fragment ion peaks were observed at m/z 57, 107, and 149. The 1H–1H COSY spectrum revealed the existence of fragment –CH═CH–CH═CH–CH–CH2– CH2–CH2–CH3–, from C-10 to C-18 (Fig. 4). All the aforementioned data of 5 showed close similarities to
those of porrigenic acid, a conjugated ketonic fatty acid isolated from Pleurocybella porrigens, apart from one more degree of unsaturation and the up-field shifted signals of C-2 and C-13 (δC 33.8 and δC 145.7 in 5; δC 38.0 and δC 148.5 in porrigenic acid), which led to the assumption that the cyclic ester moiety in 5 was from C-1 to C-14, forming a 15-membered macrolide. In addition, the opposite optical activities between 5 ([α] 25 D +1.2, c 0.10, in MeOH) and porrigenic acid ([α]25 10.5, D c 0.088, in MeOH) revealed the R configuration of C-14 in 5 (Amakura et al., 2006; Hasegawa et al., 2007). Finally, the structure of 5 was thus elucidated as (+)-(rel-14R)(10E,12E)-9-oxo-octadeca-10,12-dien-13-olide.
Acknowledgements The authors are grateful to the financial support from the National Natural Science Fund of China (no. 81102773) and Outstanding Youth Program of Shanghai Medical System (XYQ2013100).
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Amakura Y, Kondo K, Akiyama H, et al. 2006. Conjugated ketonic fatty acids from Pleurocybella porrigens. Chem Pharm Bull 54: 1213–1215. Arunendra P, Kulshreshtha DK, Maurya R. 2005. Chemical constituents of Bacopa Procumbens. Nat Prod Res 19: 131–136. Chinese Academy of Sciences. 1979. Flora of China, Vol. 67, Part 2. Science Press: China; 239–241. Gao WY, Zhang R, Jia W, Takaishi Y, Duan HQ. 2004. Immunosuppressive diterpenes from Veronicastrum sibiricum. Chem Pharm Bull 52: 136–137. Hagemeier J, Schneider B, Oldham NJ, Hahlbrock K. 2001. 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 98: 753–758. Hasegawa T, Ishibashi M, Takata T, Takano F, Ohta T. 2007. Cytotoxic fatty acid from Pleurocybella porrigens. Chem Pharm Bull 55: 1748–1749. Huang KN, Dai H, Hou XT, Ning XQ, Wu G. 2009. Antibacterial experiment on aqueous extract of Veronicastrum cauloptera (Hance) Yamazaka in vitro. J Guangxi Tradit Chin Med Univ 12: 48–49. Kim SR, Kim YC. 2000. Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana. Phytochemistry 54: 503–509.
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Luo C, Chen Z, Zhang WF, Li XR, Xu QM, Yang S. 2011. Chemical constituents of Huperzia serrate. Chin Tradit Herb Drugs 42: 2407–2409. PiersonJT,DumètreA,HutterS,etal.2010.Synthesisandantiprotozoal activity of 4-arylcoumarins. Eur J Med Chem 45: 864–869. Tang XC, Lin ZG, Cai W, Chen N, Shen L. 1984. Anti-inflammatory effect of 3-acetylaconitine. Acta Pharmacol Sin 5: 85–89. Wang JY, Guo J, Dong CS, Zheng Z, Wang Q. 2012. Effects of Feiyanning decoction, a compound traditional Chinese medicine, on iNOS and COX-2 expressions induced by tumor necrosis factor-α in lung adenocarcinoma cell line. J Chin Integr Med 10: 695–700. Zheng CJ, Huang BK, Wang Y, et al. 2010. Anti-inflammatory diterpenes from the seeds of Vitex negundo. Bioorg Med Chem 18: 175–181. Zhou BX, Meng XS. 1992. Determination of the active constituent in Veronicastrum sibiricun (L.) Pennell. China J Chin Mater Med 17: 102–103.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
Phytother. Res. (2014)
Antiinflammatory Effects and Chemical Constituents of Veronicastrum axillare Cheng-Jian Zheng, 1† Xue-Hong Deng, 1,2† Yu Wu, 1 Yi-Ping Jiang, 1 Jian-Yong Zhu 1 and Lu-Ping Qin1,3* 1
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Department of Pharmacy, Fujian University of Traditional Chinese Medicine, 1 Huatuo Road, Fuzhou 350108, China 3 Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai 200433, China 2
Our study aims to ascertain the antiinflammatory activity of Veronicastrum axillare and characterize the bioactive constituents. Antiinflammatory activity of the total extract and different fractions from V. axillare was investigated by employing the xylene-induced mouse ear edema model. As a result, the ethyl acetate (EtOAc) fraction showed the highest antiinflammatory activity in vivo. From the EtOAc fraction and the inactive dichloromethane fraction, a total of five new compounds, axillasides A–C and axillactones A and B, together with four known compounds, procumboside A, buergeriside C1, indole-3-carboxylic acid and apigenin, were isolated and identified. Their structures were elucidated on the basis of spectroscopic analysis and by comparison of their nuclear magnetic resonance data with those reported in the literature. Procumboside A, a major constituent in EtOAc fraction, showed significant antiinflammatory activity in vivo. Further studies revealed that procumboside A was a potent COX-2 inhibitor, significantly reducing the COX-2 protein level in lipopolysaccharide-stimulated RAW 264.7 macrophages. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Veronicastrum axillare; axillaside; axillactone; antiinflammatory activity.
INTRODUCTION Veronicastrum axillare (Sieb. et Zucc.) Yamazaki (Scrophulariaceae) is a perennial herb with bent stems, hairless paper-like leaves and axillary inflorescence. This plant is mainly distributed in Jiangsu, Anhui, Jiangxi, Fujian, Guangdong and Taiwan in China, and it also spreads in Japan (Chinese Academy of Sciences, 1979). Traditional use of V. axillare focused on relieving edema through diuresis, eliminating blood stasis, relieving pain and expelling retained water in the treatment of ascites in folk medicine. Up to the present, there has been little phytochemical data and none biological activity reported on this plant, whereas other Veronicastrum plants have been shown versatile pharmacological properties, including antiinflammatory, analgesic, antibiosis and immunosuppressive activities (Gao et al., 2004; Huang et al., 2009; Zhou and Meng, 1992). In our recent study, we investigated the antiinflammatory activity of V. axillare using the xyleneinduced mouse ear edema model and found the ethyl acetate (EtOAc) fraction to be the most active. Further phytochemical study led to the isolation of three new phenylpropanoid glycosides, axillasides A–C (1–3); a new aryl coumarin, axillactone A (4); and a new macrolide, axillactone B (5), along with four known compounds, procumboside A (6), buergeriside C1 (7), indole-
* Correspondence to: Lu-Ping Qin, Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China. E-mail: [email protected] † Both authors contributed equally to this work.
Copyright © 2014 John Wiley & Sons, Ltd.
3-carboxylic acid (8) and apigenin (9) (Arunendra et al., 2005; Hagemeier et al., 2001; Kim and Kim, 2000; Luo et al., 2011). Furthermore, procumboside A (6), a major constituent of the EtOAc fraction, showed significant antiinflammatory activity both in vitro and in vivo. Herein, we present the antiinflammatory assay on V. axillare as well as the isolation and structural elucidation for new compounds 1–5.
MATERIALS AND METHODS General. Optical rotations were acquired with a PerkinElmer 341 polarimeter (PerkinElmer, Norwalk, CA, USA) at room temperature. Infrared (IR) spectra were recorded on a Bruker Vector 22 spectrometer (Bruker, Billerica, MA, USA) with KBr pellets. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 NMR spectrometer (Bruker, Billerica, MA, USA) with tetramethylsilane as an internal standard. Electrospray ionisation mass spectrometry (ESI-MS) were measured on an Agilent LC/MSD Trap XCT mass spectrometer, whereas high resolution (HR)-ESI-MS were measured using an Agilent 6538 UHD AccurateMass Q-TOF LC/MS spectrometer (Agilent Technologies Inc., Santa Clara, CA). All solvents used were of analytical grade (Shanghai Chemical Plant). Materials for column chromatography were silica gel (200–300 mesh; Huiyou Silica Gel Development Co., Ltd. Yantai, China), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), silica gel H (10–40 μm; Yantai), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and YMC-Gel Octadecylsilyl (ODS)-A (50 μm; YMC, Milford, MA, USA). Medium Received 1 March 2014 Revised 8 April 2014 Accepted 16 April 2014
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pressure liquid chromatography (MPLC) was carried on Dr Flash II system (Lisure Science (Suzhou) Co., Ltd). Semipreparative HPLC was performed on an Agilent 1200 series (XDB-C18, 9.4 250 mm). HSGF254 silica gel thin layer chromatography (TLC) plates (Yantai) were used for analytical TLC. Preparative TLC (0.4–0.5 mm) was conducted on glass plates precoated with silica gel GF254 (Yantai). Plant material. The whole plants of V. axillare (Sieb. et Zucc.) Yamazaki were collected in Jiande, Zhejiang Province, China, in July 2011 and identified by Prof. Lu-Ping Qin, School of Pharmacy, Second Military Medical University. A voucher specimen (#201107) has been deposited at the Herbarium of Materia Medica, School of Pharmacy, Second Military Medical University, Shanghai, China. Extraction and isolation. The air-dried and powdered whole plants (8.5 kg) of V. axillare were extracted with hot aqueous ethanol (80% v/v) under reflux three times for 2 h each time. After removal of the solvent under reduced pressure, the total extract (1700 g) was partitioned sequentially with petroleum ether, CH2Cl2, EtOAc and n-BuOH, obtaining four fractions with yields of 91.5, 55, 132 and 500 g, respectively. The CH2Cl2 fraction was subjected to silica gel chromatography using petroleum ether-EtOAc mixtures (20:1, 10:1, 5:1, 3:1, 1:1 v/v) to afford fractions A–E. Fr.D (4.5 g) was rechromatographed on a silica gel column chromatography (CC) with gradient petroleum ether-EtOAc (10:1, 8:1, 5:1, 3:1, 1:1) to yield four fractions Fr. D-IFr.D-IV. Fr.D-II (1.7 g) was subjected to silica gel CC eluted with petroleum ether-EtOAc mixtures (3:1, 2:1, 1:1) to obtain two fractions, Fr.D-II-1 and Fr. D-II-2. Fr.D-II-1 (750 mg) was loaded onto a Sephadex LH-20 column (CH2Cl2-MeOH, 1:4) to give two subfractions, the former (88.4 mg) of which was further purified by preparative TLC (CH2Cl2-MeOH, 30:1) to afford 5 (42.2 mg), and the latter was (457.3 mg) purified by HPLC on a semi-preparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 75% MeOH) to afford 3 (36.6 mg). Fr.D-II-2 (104.9 mg) was subjected to a Sephadex LH-20 column (CH2Cl2-MeOH, 1:4) to yield apigenin (9, 3.0 mg). Fr.D-III (622.9 mg) was refractionated by silica gel CC eluted with gradient petroleum ether-EtOAc (4:1, 3:1, 2:1, 1:1) to give two fractions, Fr.D-III-1 (340.5 mg) and Fr.D-III-2 (238.4 mg), which was further purified by preparative TLC (CH 2 Cl 2 -MeOH, 20:1) to yield 2 (180.0 mg) and 1 (52.5 mg), respectively. Fr.E (1.5 g) was rechromatographed on a silica gel CC with gradient petroleum ether-EtOAc (2:1, 1:1, 1:2) to give Fr.E-IFr.E-III. Fr.E-III (502 mg) was loaded onto a Sephadex LH-20 column (CH2Cl2-MeOH, 1:1), followed by HPLC on a semi-preparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 50% MeOH) to yield buergeriside C1 (7, 3.4 mg). The EtOAc extract (110 g) was subjected to silica gel CC using CH2Cl2-MeOH mixtures (30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 0:1) to yield fractions F–K. Fr.F (5.9 g) was chromatographed by MPLC over a ODS-C18 column, eluted with gradient MeOH-H2O (3:7→9:1) at 15 mL/min for 50 min and further purified on a Sephadex LH-20 column (MeOHCopyright © 2014 John Wiley & Sons, Ltd.
H2O, 4:1) to yield indole-3-carboxylic acid (8, 1.5 mg). Fr.H (1.8 g) was also chromatographed by MPLC over a ODS-C18 column, eluted with 20% MeOH at 15 mL/min for 30 min, followed by HPLC on a semipreparative XDB-C18 column (9.4 250 mm, flow rate: 3 mL/min, 50% MeOH) to give 4 (24.4 mg). Fr.I (13.5 g) was chromatographed by MPLC over a silica gel CC with gradient CH2Cl2-MeOH (100:0→90:10) to yield procumboside A (6, 5.0 g). Axillaside A (1): White amorphous powder; [α] 25 D +23.7 (c 0.10, MeOH); ultraviolet (UV) (MeOH) λmax 291 nm; IR (KBr) νmax 599, 711, 766, 1207, 1282, 1334, 1387, 1449, 1496, 1636, 1708, 2935, 2968, 3442 and 3482 cm1; HR-ESI-MS m/z 317.0998 [M + Na]+ (calculated for C15H18O6Na, 317.1003); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillaside B (2): Yellowish oil; [α]25 D +50.0 (c 0.10, MeOH); UV (MeOH) λmax 290 nm; IR (KBr) νmax 684, 768, 1058, 1184, 1450, 1496, 1635, 1698, 2933, 2976 and 3421 cm1; HR-ESI-MS m/z 293.1103 [M H] (calculated for C15H17O6, 293.1036); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillaside C (3): Yellowish oil; [α] 25 D +236.4 (c 0.11, MeOH); UV (MeOH) λmax 275, 337 nm; IR (KBr) νmax 683, 767, 1062, 1120, 1162, 1204, 1282, 1312, 1449, 1496, 1635, 1716, 2933, 2976, 3028, 3061 and 3412 cm1; HRESI-MS m/z 447.1414 [M + Na]+ (calculated for C24H24O7Na, 447.1419 ); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 1). Axillactone A (4): White amorphous powder; UV (MeOH) λmax 288, 345 nm; IR (KBr) νmax 782, 819, 866, 1131, 1188, 1384, 1447, 1563, 1625, 1689, 2850, 2919 and 3423 cm1; HR-ESI-MS m/z 271.0601 [M + H]+ (calculated for C15H11O5, 271.0606); 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data (Table 2). Axillactone B (5): Yellowish oil; [α] 25 D +1.2 (c 0.10, MeOH); UV (MeOH) λmax 288 nm; IR (KBr) νmax 1187, 1221, 1362, 1383, 1412, 1464, 1595, 1634, 1713, 2853 and 2925 cm1; HR-ESI-MS m/z 293.2096 [M + H]+ (calculated for C18H29O3, 293.2102); 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data (Table 2).
Antiinflammatory assays. The antiinflammatory activity was investigated by employing the xylene-induced mouse ear edema model as previously described (Tang et al., 1984). Institute of Cancer Research (ICR) mice were randomly divided into 15 groups (n = 10) and were acclimated to condition for 4 days before the experiment. On the fifth day, positive drug dexamethasone (1 mg/kg), total extract (225 and 900 mg/kg), petroleum ether fraction (12 and 48 mg/kg), dichloromethane fraction (7.5 and 30 mg/kg), ethyl acetate fraction (17.5 and 70 mg/kg), n-BuOH fraction (66 and 264 mg/kg), procumboside A (2.5, 5 and 10 mg/kg) and 5% carboxymethyl cellulose–Na solution were intragastrically given in a single dose. One hour later, all animals were treated with xylene on the anterior and posterior surfaces of the right ear (15 μL on each side of the ear), and the left ear was used as a control. Mice were sacrificed through cervical dislocation 0.5 h after xylene application. Ear biopsies of 6.0 mm in diameter were punched out and Phytother. Res. (2014)
ANTIINFLAMMATORY CONSTITUENTS OF VERONICASTRUM AXILLARE
Table 1. 1H and
C NMR spectroscopic data of compounds 1–3 (δ in ppm, J in Hz)
13
1 No. 1 2 3 4 5 6 1' 2' 3' 4' 5' 6' α' β' C═O 1" 2" 3" 4" 5" 6" α" β" C=O
2
3
δH
δC
δH
δC
δH
δC
5.03, d (1.8) 3.85, dd (3.6, 1.8) 3.96, dd (9.6, 3.6) 5.06, brt (9.6) 4.03, dq (9.6, 6.6) 1.16, d (6.6) — 7.59, dd (7.2, 1.8) 7.39, m 7.39, m 7.39, m 7.59, dd (7.2, 1.8) 7.70, d (16.2) 6.55, d (16.2) — — — — — — — — — —
95.8 73.2 70.2 76.0 67.3 18.1 135.8 129.3 130.1 131.6 130.1 129.3 146.5 118.9 168.4 — — — — — — — — —
5.03, d (1.8) 4.02, dd (3.6, 1.8) 5.17, dd (9.6, 3.0) 3.66, brt (9.6) 3.96, dq (9.6, 6.6) 1.29, d (6.6) — 7.58, dd (7.8, 1.8) 7.38, m 7.38, m 7.38, m 7.58, dd (7.8, 1.8) 7.75, d (16.2) 6.59, d (16.2) — — — — — — — — — —
95.8 71.0 75.7 71.7 69.5 18.2 135.9 129.2 130.0 131.5 130.0 129.2 146.4 119.2 168.4 — — — — — — — — —
5.11, d (1.8) 5.40, dd (3.6, 1.8) 5.36, dd (9.6, 3.6) 3.72, brt (9.6) 4.06, dq (9.6, 6.6) 1.35, d (6.6) — 7.55, dd (7.8, 1.2) 7.38, m 7.38, m 7.38, m 7.55, dd (7.8, 1.2) 7.68, d (16.2) 6.57, d (16.2) — — 7.48, m 7.31, m 7.31, m 7.31, m 7.48, m 7.64, d (15.6) 6.46, d (15.6) —
93.4 72.6 73.4 72.0 69.4 18.3 135.5 129.4 129.9 131.8 129.9 129.4 147.3 118.4 167.6 135.6 129.3 130.1 131.5 130.1 129.3 146.7 118.7 167.9
Table 2. 1H and 13C NMR spectroscopic data of compounds 4 and 5 (δ in ppm, J in Hz) 4
5
No.
δH
δC
No.
δH
δC
1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 17 18
— —
— 163.7 116.5 156.2 113.1 148.5 118.6 121.1 155.2 121.3 124.2 117.0 151.6 148.2 118.1 118.5 — —
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
— 2.33, t (7.2) 1.61, m 1.32, m 1.32, m 1.32, m 1.61, m 2.53, t (7.2) — 6.15, d (15.0) 7.13, dd (15.0, 10.8) 6.35, dd (15.0, 10.8) 6.15, d (15.0) 4.24, dd (12.0, 6.0) 1.60, m 1.32, m 1.32, m 0.90, t (6.6)
178.6 33.8 24.4 28.6 28.7 28.8 24.0 40.6 200.9 129.8 141.7 127.9 145.7 72.1 36.7 27.4 22.4 13.9
6.30, s — 6.74, d (3.0) — 7.03, dd (9.0, 3.0) 7.23, d (9.0) — — — 6.64, d (3.0) — — 6.81, d (9.0) 6.80, dd (9.0, 3.0) — —
weighed. The extent of ear edema was determined by the weight difference (⊿W) between the right and left ear biopsies of the same animal. The inhibition percentage was expressed as the reduction in ear weight compared with the vehicle group. All animal treatments were strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals, and the experiments were carried out with the approval of the Animal Experimentation Ethics Committee of the Second Military Copyright © 2014 John Wiley & Sons, Ltd.
Medical University (Date: 2013-03-14; Approval number: 2013LY014). Edema inhibition ð%Þ ¼ ð⊿Wvehicle –⊿Wtreated Þ=⊿Wvehicle 100%: Western blot analysis. The murine RAW 264.7 cell line was seeded at an initial density of 2 106 cells/well in Phytother. Res. (2014)
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6-well tissue culture plates overnight. Cells were exposed to Escherichia coli lipopolysaccharide (LPS) (1 μg/mL; Sigma) for 24 h in the presence or absence of the tested compounds. Indomethacin, a COX-2 inhibitor, was used as a positive control in this experiment. Protein samples were collected and prepared as described previously (Zheng et al., 2010; Wang et al., 2012). Briefly, cells were washed with phosphate buffered saline and harvested by scraping in appropriate volume of cell lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were then incubated on ice for 20 min with intermittent vortexing followed by centrifugation at 40 000 g for 10 min at 4 °C. Samples containing equal quantities of protein (40 μg) were subjected to SDS/20%–polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes. The resultant nitrocellulose membranes were incubated with blocking solution and probed using antibodies specific to COX-2 (1:1000 dilution; cell signalling) protein and visualized using an ECL detection kit (Perkin-Elmer, Western Lightning Chemiluminescence Reagent Plus). Acid hydrolysis and sugar analysis. Compounds 1–3 (each 1.5 mg) were hydrolyzed with 1 mL of 2 M aqueous CF3CO2H (trifluoroacetic acid) in a 10 mL round bottomed flask at 120 °C for 2 h, after which the solvent was evaporated with a stream of N2. Then, the following solutions were added: (a) 1:8 (S)-1-amino-2-propanolMeOH (25 μL); (b) 1:4 glacial acetic acid-MeOH (25 μL); and (c) 3% NaBH3CN in MeOH (25 μL). The vial was capped, and the mixture was allowed to react for 1.5 h at 65 °C. After cooling, the mixture was evaporated and co-evaporated with MeOH (5 0.5 mL). The residue was dried overnight in a desiccator and treated with 1:1 pyridine-acetic anhydride (0.4 mL) for 45 min at 100 °C. After cooling, the derivatives were extracted with CHCl3 and washed with water (3 1 mL). The organic phase was dried with anhydrous Na2SO4 and subjected to gas chromatography (GC)-MS using an Agilent 7000 QQQ GC/MS (Agilent, CA, USA) system equipped with a HP-5ms column (30 m 0.25 mm 0.25 μm) with He as a carrier gas at a flow rate of 1.0 mL/min to identify the monosaccharide. The oven temperature started at 140 °C and was increased to 198 °C at a rate of 3 °C/min, and then increased to 220 °C at a rate of 1 °C/min, keeping it at 220 °C for 10 min, and then increased to 280 °C at a rate of 3 °C/min. Derivatives of D-rhamnose and L-rhamnose (reference standards) eluted at 23.953 and 23.515 min, and the tested samples eluted at 23.513 min (L-rhamnose).
dexamethasone (1 mg/kg, 29.82%). Among several fractions with different polarity from the total extract of V. axillare, only the EtOAc fraction showed potent antiinflammatory activity at both doses of 17.5 and 70 mg/ kg, with oedema inhibition of 42.11% and 43.86%, respectively (Fig. 1). Further bioactivity-guided isolation of the EtOAc fraction yielded a major constituent, procumboside A (6), which exhibited notable antiinflammatory effects at doses of 2.5, 5 and 10 mg/kg, with inhibition of 29.82%, 48.25% and 37.72%, respectively. Further studies revealed that procumboside A (6) (40 μM) and indomethacin (20 μM) significantly reduced the COX-2 protein level to 38.31 ± 4.19% and 29.94 ± 1.43% (Fig. 2), respectively, in LPS-stimulated RAW 264.7 cells, which indicated that the antiinflammatory of procumboside A (6) was related to its down-regulation COX-2 protein expression, thus validating the antiinflammatory properties of V. axillare extract and supporting its use in traditional medicine.
Characterization of new compounds In addition to procumboside A (6), three new phenylpropanoid glycosides, axillasides A–C (1–3); a new aryl coumarin, axillactone A (4); and a new macrolide, axillactone B (5), along with other three known compounds (Fig. 3) were isolated from the dichloromethane and EtOAc fractions from the extract of V. axillare, using successive silica gel, Chromatorex ODS and Sephadex LH20 column chromatography, semi-preparative HPLC and preparative TLC. Axillaside A (1) was obtained as an amorphous white powder and gave the molecular formula, C15H18O6, by positive-ion HR-ESI-MS. The 1H and 13C NMR spectra of 1 showed close similarities to those of buergeriside C1 (7), an analogue reported from Scrophularia buergeriana [6]. The major differences were the disappearance of the signals corresponding to an O-methyl group (δH 3.62 and δC 54.8 in 7) and the splitting pattern of the aromatic protons (A2B2C in 1; AABB in 7), which led to the assumption that 1 was a 4'-demethoxyl derivative of 7. The sugar moiety was recognized as α-L-rhamnopyranose based on its NMR
RESULTS AND DISCUSSION Biological evaluation The antiinflammatory activity of the total extract and different fractions from V. axillare was investigated by employing the xylene-induced mouse ear edema model. Dexamethasone (1 mg/kg) was used as a positive drug in our assay. As a result, the total extract of V. axillare, at high dose of 900 mg/kg, produced significant inhibition (45.61%) of oedema comparable with that of Copyright © 2014 John Wiley & Sons, Ltd.
Figure 1. Antiinflammatory activity of Veronicastrum axillare on xylene-induced ear edema in mice (DA: dexamethasone; TF: total extract; PEF: petroleum ether fraction; DF: dichloromethane fraction; AF: ethyl acetate fraction; BF: n-BuOH fraction; *p < 0.05 or **p < 0.01 compared with vehicle group). Phytother. Res. (2014)
ANTIINFLAMMATORY CONSTITUENTS OF VERONICASTRUM AXILLARE
Figure 2. Western blot analysis of procumboside A (6) on the COX-2 protein expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Data were expressed as mean ±SD (n = 3). **p 0.01 compared with LPS-treated group. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr
data (Table 1) and confirmed by GC analysis after alkali hydrolysis of 1. The esterification site of the cinnamoyl group was determined to be the C-4 position of the rhamnose unit by heteronuclear multiple bond correlation (HMBC; Fig. 4) observed from H-4 at δH 5.06 (1H, brt, J = 9.6 Hz) to the carbonyl signal at δC 168.4. Thus, the structure of 1 was elucidated as 4-O-(E)cinnamoyl-α-L-rhamnopyranose. Axillaside B (2) was obtained as a yellowish oil and analysed for the molecular formula, C15H18O6, by negative-ion HR-ESI-MS. According to its molecular formula and NMR data, 2 was determined to possess the same carbon framework as 1, differing only in the splitting pattern and chemical shift of an oxygenated methine group (δH 5.17, dd, J = 9.6, 3.0 Hz; δC 75.7 in 2; δH 5.06, brt, J = 9.6 Hz; δC 76.0 in 1), indicating that 2 is a regioisomer with a cinnamoyl group at C-3 instead of C-4. This was confirmed by the HMBC correlation from H-3 at δH 5.17 (1H, dd, J = 9.6, 3.0 Hz) to the ester carbonyl signal at δC 168.4 (Fig. 4). The structure of 2
was therefore determined as 3-O-(E)-cinnamoyl-α-Lrhamnopyranose. Axillaside C (3), obtained as a yellowish oil, gave the molecular formula, C24H24O7, based on its positive-ion HR-ESI-MS. Its 1H and 13C NMR spectra were similar to those of 1 and 2, apart from the appearance of two (E)-cinnamic acid moieties in the molecule, as represented by ten protons of two monosubstituted aromatic rings and four trans olefinic protons (Table 1). Its sugar moiety was also confirmed as α-L-rhamnopyranose with a free hydroxyl group at C-1, evidenced by the up-field shifted signal of C-1 at δC 93.4 [6]. The signals of H-2 and H-3 of rhamnose were shifted 1.4 ppm downfield (5.40 and 5.36, respectively), indicating esterification of two separate (E)-cinnamoyl groups at the C-2 and C-3 positions of the rhamnose unit, which was confirmed by HMBC correlations (Fig. 4) from H-2 at δH 5.40 to a carbonyl carbon at δC 167.6 and from H-3 at δH 5.36 to another carbonyl carbon at δC 167.9. Therefore, the structure of 3 was verified as 2, 3-di-O-(E)-cinnamoylα-L-rhamnopyranose. Axillactone A (4) was obtained as an amorphous white power, and the molecular formula was established as C15H10O5 deduced from the positive-ion HR-ESI-MS. Its NMR data were characterized by the presence of two phenyl moieties of ABX (a spin pattern of three nuclei constituted of AB part and X part) spin system, which were analogous to those of 4-(3',4'-dimethoxyphenyl)-6-methoxycoumarin, a synthesized 4-arylcoumarin with antiprotozoal activity, except for the disappearance of the signals corresponding to three O-methyl groups (Pierson et al., 2010). All data indicated that 4 was a 3',4',6-O-tridemethyl derivative of 4-(3',4'-dimethoxyphenyl)-6-methoxycoumarin. In addition, the linkage position of the aryl group was determined to be the C-4 position of the coumarin moiety, evidenced by HMBC correlations from H-3 at δH 6.30 (1H, s) to C-1' at δC 124.2 and from H-2’ at δH 6.64 (1H, d, J = 3.0 Hz) to C-4 at δC 156.2, as well as supported by the nuclear overhauser effect spectroscopy correlations between H-3/H-2' and H-5/H-2' (Fig. 4). Finally, the structure of 4 was deduced to be 4-(3',4'-dihydroxyphenyl)-6-hydroxy-coumarin. Axillactone B (5) was obtained as a yellowish oil and gave the molecular formula, C18H28O3, by positive-ion
Figure 3. Chemical structures of compounds 1–9. Copyright © 2014 John Wiley & Sons, Ltd.
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C.-J. ZHENG ET AL.
1
1
Figure 4. H– H COSY (▬) and key heteronuclear multiple bond correlations (→) of compounds 3–5 and selected nuclear overhauser effect spectroscopy correlations (↔) of compound 4. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr
Figure 5. Main electron ionization/mass spectrometry fragments of compound 5.
HR-ESI-MS. Its NMR spectral features (Table 2) along with five unsaturation degrees in the molecular formula suggested 5 to be a C18 unsaturated oxo fatty acid with cyclic ester structure, characterized by the presence of a conjugated E,E-form dienone system from C-9, a carbonyl carbon at δC 200.9, to C-13, and an oxygenated methine carbon at C-14 (δC 72.1), identified from the HMBC correlations of H-10/C-9, C-12; H-11/C-9, C-13; H-12/C-10, C-11, C-14; H-13/C-11, C-14; H-14/C-12, C13, C-15, C-16; and H-18/C-16, C-17 (Fig. 4), as well as electron ionization–MS analysis (Fig. 5). Detailed analysis of electron ionization–MS data indicated that notable fragment ion peaks were observed at m/z 57, 107, and 149. The 1H–1H COSY spectrum revealed the existence of fragment –CH═CH–CH═CH–CH–CH2– CH2–CH2–CH3–, from C-10 to C-18 (Fig. 4). All the aforementioned data of 5 showed close similarities to
those of porrigenic acid, a conjugated ketonic fatty acid isolated from Pleurocybella porrigens, apart from one more degree of unsaturation and the up-field shifted signals of C-2 and C-13 (δC 33.8 and δC 145.7 in 5; δC 38.0 and δC 148.5 in porrigenic acid), which led to the assumption that the cyclic ester moiety in 5 was from C-1 to C-14, forming a 15-membered macrolide. In addition, the opposite optical activities between 5 ([α] 25 D +1.2, c 0.10, in MeOH) and porrigenic acid ([α]25 10.5, D c 0.088, in MeOH) revealed the R configuration of C-14 in 5 (Amakura et al., 2006; Hasegawa et al., 2007). Finally, the structure of 5 was thus elucidated as (+)-(rel-14R)(10E,12E)-9-oxo-octadeca-10,12-dien-13-olide.
Acknowledgements The authors are grateful to the financial support from the National Natural Science Fund of China (no. 81102773) and Outstanding Youth Program of Shanghai Medical System (XYQ2013100).
Conflict of Interest The authors have declared that there is no conflict of interest.
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