2. 上海中医药大学中药学院中药化学教研室, 上海 201203
2. Department of Phytochemistry, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Petasites tricholobus Franch is a perennial plant of Compositae as a member of Senecioneae Cass. (Tussilaginae Dum),which is widely distributed in the southwest and northwest China. As a traditional Chinese medicine,the rhizomes of Petasites tricholobus have been used for the treatment of blood stasis,fracture,and snake-bite. Phytochemical investigation of this herb previously focused on its fat-soluble chemical constituents,and lots of sesquiterpenoids,especially bakkenane had been isolated from this plant. In addition,these compounds showed multiply pharmacological activities,such as anti-inflammatory,anti-tumor,antibacterial,and neuroprotective[5,6]. Although there are many natural sulfur sesquiterpenoids had been isolated from the genus Petasites in the past few decades,for example S-petasin,neo-S-petasin and S-isopetasin et al,the discovering of sulfated sesquiterpenoids from the genus Petasites was not reported.
Herein,we present the isolation and structure elucidation of two new sulfated sesquiterpenoids which are megastigman-7-ene-3,5,6,9-tetrol-3-O-β-D-6'- sulfonated-glucopyranoside (1) and 3-O-β-D-6'- sulfonated-glucopyranosyl-6-(3-oxo-2-butenylidenyl)- 1,1,5-trimethylcyclohexan-5-ol (2),and one known sesquitepenoid compound icariside B1 (3) which was isolated from the genus Petasites for the first time (Figure 1). And all obtained compounds were tested for their cytotoxicity against four cancer cell lines.
Results and discussion
Compound1 was isolated as a white powder,[α]22D -28.1 (c 0.16,MeOH). Its molecular formula was established as C19H34O12S by HR-ESI-MS. Compound 1 displayed a quasi-molecular ion peak at m/z 485.164 9 [M-H]- (calcd. 485.169 3). The IR spectrum showed 1 had absorption bands at 3 429,1 633 and 997 cm-1,which were assignable to hydroxyl and double bond groups,respectively. The 1H NMR (400 MHz,CD3OD) spectrum (Table 1) of 1 showed signals corresponding to four methyl protons at δH 0.84 (3H,s,CH3-11),1.14 (3H,s,CH3-13) ,1.21 (3H,s,CH3-12) and 1.26 (3H,d,J = 6.4 Hz,CH3-10); two olefinic protons at δH 6.05 (1H,dd,J = 16.0,1.2 Hz,H-7) and 5.77 (1H,dd,J = 16.0,6.4 Hz,H-8) for a disubstituted trans double bond; and an anomeric proton signal at δH 4.40 (1H,d,J = 8.0 Hz,H-1') corresponding to a β-glucopyranosyl moiety. 1H-1H COSY experiment revealed a spin system including signals of three methines [δH 6.05 (1H,dd,J = 16.0,1.2 Hz,H-7); 5.77 (1H,dd,J = 16.0,6.4 Hz,H-8); 4.31 (1H,quint. d,J= 6.4,1.2 Hz,H-9)] and a methyl [δH 1.26 (3H,d,J = 6.4 Hz,CH3-10)]. The 13C NMR (100 MHz,CD3OD) spectral data displayed 19 carbon resonances (Table 1),six of which were attributed to a β-glucopyranosyl moiety. The remainingthirteen signals consist of four methyls,two methylenes,four methines,and three quaternary carbons. The signals at δC 136.5 and 131.7 were assigned to a disubstituted double bond. These signal patterns indicated the presence of a C13-norisoprenoid. In addition,the downfield-shift displayed by the 6'-position of the β-glucopyranosyl moiety suggested the attachment of sulfonyl moiety at that position (δC 68.7),which was substantiated by the mass data. In the HMBC experiment (Figure 2),the long-range correlations from CH2-2 to C-3 and C-12,from H-3 to C-1',from CH2-4 to C-2 and C-3,from H-7 to C-6 and C-9,from H-9 to C-7 and C-8,from CH3-10 to C-8 and C-9,from CH3-11 to C-2 and C-6,from CH3-12 to C-2 and C-6,as well as from CH3-13 to C-4 and C-6 clearly supported the structure of 1. Based on the above data and comprehensive 2D NMR experiments (1H-1H COSY,HMQC,HMBC),the structure of 1 was established as shown in Figure 1. The relative configurations of 1 were deduced from analyses of the 1H-1H coupling constants and ROESY spectrum (Figure 2). The key ROESY correlations of H-3/H-1',H-3/CH3-12,and H-9/CH3-12 suggested that H-3,H-9,and CH3-12 were cofacial and α-orientation. Moreover,the 13C NMR spectrum displayed three important carbon signals at δC 27.9 (C-11),26.7 (C-12) and 27.7 (C-13) which could improve the relative configuration ofCH3-13 (α- orientation) according to literatures[7,8]. The 1H NMR spectrum signal at δH 4.31 (1H,quint. d,J = 6.3,1.9 Hz,H-9) which also could suggest the relative configuration of H-9 (α-orientation) according to literature. In addition,the relative configuration of 6-OH (β- orientation) has been determined by comparing the specific rotations of 1 ([α]22D -28.1) with a known compound (3S,5R,6R,7E,9S)-megastigman-7-ene-3,5,6,9-tetrol 3-O-D-glucopyranoside ([α]22D -38.0) at last. The sugar configuration was deduced as a β- glucopyranosyl moiety by its anomeric proton signal at δH 4.40 (1H,d,J = 8.0 Hz,H-1').
Compound 2 was obtained as a white powder,[α]22D -36.2 (c 0.34,MeOH),which was assigned a molecular formula of C19H30O11S by HR-ESI-MS. Compound 2 displayed a quasi-molecular ion peak at m/z 489.137 3 [M+Na]+ (calcd. 489.140 7). The IR spectrum of 2 indicated the presence of a hydroxyl (3 433 cm-1),an allenic (1 940 cm-1),and a conjugated
carbonyl (1655 cm-1) group. The 1H NMR (400 MHz,CD3OD) and 13C NMR (100 MHz,CD3OD) spectrum of 2 were very similar to 3 (Table 1),which were assigned a C13-norisoprenoid glucoside type,compared with that of the icariside B1 (3) isolated from peucedanum japonicum Thunb. And the only difference between 2 and 3 was the chemical shift of 6'-position. The downfield-shift displayed by the 6'-position of the β-glucopyranosyl moiety also suggested the attachment of sulfonyl moiety at that position,which was substantiatedby the measurement of the HR-ESI-MS (C19H30O11SNa [M+Na]+). The HMBC experiment (Figure 2) further accounted the presence of a C13-norisoprenoid glucoside. Based on the above data and comprehensive 2D NMR experiments (HMQC,HMBC),the structure of 2 was established as shown in Figure 1. The relative configurations of 2 were deduced from the analyses of the 1H-1H coupling constants and ROESY spectrum (Figure 2). The ROESY correlations of H-3/H-1',H-3/CH3-12,and H-8/CH3-12 indicated that H-3,H-1',H-8 and CH3-12 were cofacial and α-orientation,while the glycosidic bond was on the opposite side of the molecular plane and β-orientation. In addition,from the analysis of 1D NOESY spectrum of 2 (Figure 2),the signal at δH 4.30 (H-3) exhibited a NOE correlation of the signals at δH 4.43 (H-1') and δH 1.38 (CH3-12),which also suggested the configuration of the CH3-12 to be α-orientation,and those of the CH3-11 and CH3-13 to be β-orientation.
Compound 3 was identified as icariside B1,which was isolated from the genus Petasites for the first time. Experimental General experimental procedures
1H NMR and 13C NMR spectra were recorded on a Bruker AM-400 spectrometer with TMS as the internal standard. 2D NMR spectra were recorded on a Bruker DRX-500 spectrometer. ESI-MS and HR-ESI-MS were carried out on a Waters Q-TOF Premier instrument. Optical rotations were measured on a Perkin Elmer Model 341 polarimeter. The FT-IR spectra were recorded on an IR Perkin-Elmer 577 spectrophotometer with KBr pellets. An Agilent 1200 Series machine equipped withAgilent ZORBAX SB-C18 column (4.6 mm × 250 mm,5 μm) was used for HPLC analysis,and semi-preparative Agilent ZORBAX SB-C18 column (9.4 mm × 250 mm,5 μm) was used in sample preparation. Plant material
The whole plants of P. tricholobus Franch were collected from Chongqing,China in October 2009. The plants were identified by Prof. Li-Hong Wu,Shanghai R&D Center for Standardization of Chinese Medicines. A voucher specimen (No. FDC- 20091029) was deposited in Department of Phytochemistry,School of Pharmacy,Shanghai University of Traditional Chinese Medicine. Extraction and isolation
Air-dried whole plants of P. tricholobus (6.5 kg) were reflux-extracted with 95% ethanol for three times (1.5 hours each time) at 80 ℃,and then the solvent was evaporated under reduced pressure to give extracts (320 g). The extracts were suspended in hot water (60 ℃,1.5 L) and extracted successively with petroleum ether,EtOAc,and n-BuOH.
The n-BuOH soluble fraction was concentrated under reduced pressure to afford a residue (85 g). This residue was subjected to chromatographic separation on D101 macroporous adsorption resin using H2O,20% EtOH,40% EtOH,60% EtOH and 95% EtOH as the eluent. The 20% EtOH fraction (11.2 g) was separated by ODS-A column chromatography (10%-100% MeOH- H2O) to give fractions A-F. Fraction C (54.4 mg) was subjected to Sephadex LH-20 column using a 20% MeOH system to give compound 1 (2.5 mg). Fraction D (210 mg) was subjected to Sephadex LH-20 column using a 20% MeOH system to give fractions D1-D6. Fraction D1 (21.6 mg) was purified by semi-preparative HPLC using MeCN-H2O (15∶85,3 mL·min-1) as the mobile phase to obtain compound 2 (t = 2.326 min,7.2 mg). Fraction E (1.7 g) was subjected to silica gel column chromatography (300-400 mesh) eluted with CH2Cl2-MeOH (20∶1-0∶1) to give fractions E1-E11. Fraction E8 (68.9 mg) was separated by ODS-A column chromatography (20%-100% MeOH-H2O) to give fractions (E8 Fr.1-Fr.4). E8 Fr.2 (21.3 mg) was purified by semi-preparative HPLC using MeCN-H2O (15∶85,3 mL·min-1) as the mobile phase to afford compound 3 (t = 9.226 min,10.2 mg). Structure elucidation
Compound 1 White powder (MeOH); [α]22D -28.1 (c 0.16,MeOH); HR-ESI-MS m/z 485.164 9 [M-H]- (calcd. for C19H33O12S,485.169 3); ESI-MS m/z: 485 [M-H]-; IR (KBr) νmax 3 429,2 922,1 657,1 633,1 377,1 259,1 068,997,580 cm-1; 1H NMR (400 MHz,CD3OD) and 13C NMR (100 MHz,CD3OD) data see Table 1.
Compound 2 White powder (MeOH); [α]22D -36.2 (c 0.34,MeOH); HR-ESI-MS m/z 489.137 3 [M+Na]+ (calcd. for C19H30O11NaS,489.140 7); ESI-MS m/z: 465 [M-H]-,931 [2M-H]-; IR (KBr) νmax: 3 433,2 922,1 940,1 655,1 637,1 381,1 248,1 074,1 001,820,580 cm-1; 1H NMR (400 MHz,CD3OD) and 13C NMR (100 MHz,CD3OD) data see Table 1.
Compound 3 White powder (MeOH); ESI-MS m/z: 409 [M+Na]+,793 [2M+Na]+; 1H NMR and 13C NMR (CD3OD,100 MHz) data see Table 1. The above data were identical with icariside B1. Acid hydrolysis of compound 1 and 2
The acid hydrolysis and detection of sugars were conducted according to the method described in references[10,11]. Compounds 1 and 2 (each 2.0 mg) were separately hydrolyzed in 1.5 mL 2 mol·L-1 HCl at 80 ℃ for 3 h. After cooling to room temperature,each reaction mixture was washed with CHCl3 (1.5 mL×3) and the aqueous layer was neutralized with Ba(OH)2 caused white precipitates to form. Then each resulting mixture was centrifuged. The precipitates were separated and proved as BaSO4. It was indicated that 1 and 2 were sulfated compounds. The supernatant was concentrated under reduced pressure. D-glucose was observed in the supernatant by TLC analysis method (chloroform- MeOH-water,7∶3∶0.5,v/v/v) with standard D-glucosesample. Cytotoxic activity
All compounds were evaluated for cytotoxic activity against four human cancer cell lines,including HepG2 (human liver cancer),HCT 116 (human colon cancer),H460 (human lung cancer) and MCF7 (human breast cancer) by using the methyl thiazol tetrazolium (MTT) method. However,the three compounds did not exhibit significant cytotoxicity (IC50 values > 100 μmol·L-1) against the tested tumor cells.
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