Chemical constituents from the roots of Ampelopsis delavayana and their antibacterial activities

Abstract Two new aromatic glycosides, 2-methylphenyl O-β-d-xylopyranosyl-(1→6)-O-β-d-glucopyranoside (1) and 2-methylphenyl O-α-arabinofuranosyl-(1→6)-O-β-glucopyranoside (2), together with eight known compounds were isolated from the roots of Ampelopsis delavayana. Their structures were elucidated on the basis of extensive spectroscopic analysis. Furthermore, the in vitro antibacterial activities of 1 and 2 were investigated using serial twofold dilution in three bacteria including Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.


Results and discussion
Compound 2 has the same molecular formula C 18 H 26 O 10 as 1 according to its HREIMS at m/z 402.1532 [M] + . The 1 H and 13 C NMR spectra of 2 (Table S1) were very similar to those of 1, except that the chemical shift values of methines moiety (C-2″ and C-4″) in 1 were shifted to a lower field in 2, indicating the absence of xylose. By comparing 13 C NMR data of 1 and 2, we infered that the xylose group in 1 was substituted by an arabinofuranose unit in 2. This assumption was further supported by the HMBC correlations of δ H 4.92 (d, J = 1.5 Hz, H-1″) with δ C 68.2 (C-6′) and δ C 83.3 (C-2″) ( Figure S1). The configurations for anomeric H-atoms of glucose and arabinose were determined to be β and α, respectively, based on coupling constants (7.4 and 1.5 Hz, respectively). unfortunately, the absolute configuration of glucose and arabinose cannot be determined because the sample amount of 2 is too little. Therefore, the structure of 2 was determined as Compounds 1 and 2 were evaluated for their antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using serial twofold dilution. However, compounds 1 and 2 had the minimal inhibitory concentrations (MICs) exceeding 100 μg/mL.

Plant material
The roots of A. delavayana was collected from Chuxiong in Yunnan Province, China, in June 2011 and were identified by Professor Bin Qiu of Yunnan Institute of Materia Medica. A voucher specimen (2011-YPTG-001) was deposited at herbarium of Yunnan Institute of Materia Medica.

Acid hydrolysis and HPLC analysis
The absolute configuration of the sugar moieties in compound 1 were determined by the method of Tu et al (Tu et al. 2011). Compound 1 (5 mg) was hydrolyzed with 2 M HCl for 2 h at 90 °C. The mixture was evaporated to dryness under a vacuum, and then the residue was dissolved in H 2 O and extracted with CHCl 3 . The aqueous layer was collected. After drying in vacuum, the residue was dissolved in pyridine (1 mL) containing L-cysteine methyl ester (1 mg) and heated at 60 °C for 1 h. Then, o-tolylisothiocyanate (5 mL) was added to the mixture, which was heated at 60 °C for 1 h. The reaction mixture was directly analysed by reversed-phase HPLC. Analytical HPLC was performed on a Thermo C18 column (250 × 4.6 mm, 5 μm) at 30 °C with 20% CH 3 CN at a flow rate 1.0 mL/min. Peaks were detected by a uV detector at 254 nm. The mixture of standard monosaccharides, d-glucose and d-xylose, were subjected to the same method. The peaks of the standard monosaccharide derivatives were recorded at t R 15.8 (d-Glc), 17.0 (d-Xyl) min. Following the above procedure, the derivatives of 1 gave two peaks at t R 15.9 (d-Glc) and 17.1 (d-Xyl) min, respectively.

Bacterial culture condition
Single Staphylococcus aureus, Escherichia Coli and Pseudomonas aeruginosa colony from viable, growing agar plates were transferred to 1 L of sterile liquid Mueller-Hinton (MH) broth containing 600 mL of veal infusion broth, 17.5 g of casein hydrolysate, 1.5 g of soluble starch, and 400 mL of Distillated water or 1 L of sterile liquid Luria-Bertani (LB) broth containing 10 g of tryptone, 10 g of NaCl, and 5 g of yeast and then cultivated aerobically in 50 mL volumes at 35 °C or 37 °C in a heated, shaking environmental chamber overnight. Then the activated bacteria were transferred to a 50 mL fresh MH or LB broth for another 8 h at 35 or 37 °C.

Drug susceptibility assay
Cells in log phase (1 × 10 5 CFu/mL) were cultivated in 96-well plates. Respectively, the MICs were determined by serial twofold dilutions in MH or LB broth containing various agents in accordance with National Committee for Clinical Laboratory Standards 2006.