Indole Glycosides from Aqueous Fraction of Strychnos nitida

Three new indole glycosides 22-deoxystrictosamide (1), 22-deoxystrictosamide N b-oxide (2) and vincosamide 2′-O-β-D-xylopyranoside-11-O-β-D-glucopyranoside (3), together with four known analogues were isolated from aqueous fraction of Strychnos nitida. Their structures were elucidated on the basis of extensive analysis of spectroscopic data. All the alkaloids were tested for their cytotoxic activity, but they did not show any exciting result. Electronic supplementary material The online version of this article (doi:10.1007/s13659-016-0112-8) contains supplementary material, which is available to authorized users.

Compound 2 exhibited a molecular ion peak at m/z 500.2150 (calcd. for 500.2159) in its HREIMS spectrum, indicating the molecular formula of C 26 H 32 N 2 O 8 , sixteen mass units higher than that of 1.  . The ROESY correlations indicated that the relative configuration of 2 was the same as that of 1. Besides, other parts of 2 were identical to those of 1 as supported by detailed analysis of extensive 2D NMR spectral data of 2 (Fig. 2). Thus, the structure of 2 was elucidated as 22-deoxystrictosamide N boxide ( Fig. 1). Compound 3, Its molecular formula was deduced as C 37 H 48 N 2 O 18 based on 13 [22] as supported by intensive analysis of its 2D NMR spectral data (Fig. 2). Then, the structure of 3 was elucidated to vincosamide 2 0 -O-b-D-xylopyranoside-11-O-b-D-glucopyranoside (Fig. 1).

General Experimental Procedures
Optical rotations were obtained with a Jasco P-1020 Automatic Digital Polariscope. UV spectra were measured with a Shi madzu UV2401PC spectrometer. IR spectra were obtained on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr pellets. 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker DRX-400 NMR, Bruker DRX-500 NMR and Bruker DRX-600 spectrometer with

Extraction and Isolation
The air-dried and powdered twigs of S. nitida (7.0 kg) were extracted with MeOH under reflux conditions, and the solvent was evaporated in vacuo. The residue was dissolved in 0.37% HCl (pH 2-3) and the solution was subsequently basified using 10% ammonia to pH 9-10. The basic solution was partitioned with EtOAc, affording a two-phase mixture. The EtOAc fraction (40 g) and H 2 O fraction (100 g). Then H 2 O fraction (100 g) was subjected to a macroporous resin D101 and eluted with MeOH/H 2 O system to give MeOH fraction (28 g

Acid Hydrolysis of Compounds 1-3 and GC Analysis
Compounds 1-3 (each 3 mg) were refluxed with 2 M HCl (1, 4 dioxane/H 2 O 1:1, 2 mL) on water bath for 2 h. After cooling, the reaction mixture was neutralized with 1 M NaOH. The reaction mixture was extracted with CHCl 3 (3 9 5 mL). The aqueous layer was evaporated to dryness. The dried residue was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60°C for 1 h. Trimethylsilylimidazole (1.0 mL) was added to the reaction mixtures, and they were kept at 60°C for 30 min. The supernatants (4 lL) were analyzed by GC, respectively, under the following conditions: H 2 flame ionization detector. Column: 30QC2/AC-5 quartz capillary column (30 m 9 0.32 mm). Column temperature: 180-280°C with the rate of 3°C/min, and the carrier gas was N 2 (1 mL/min) injector temperature: 250°C; and split ratio: 1/50. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of D-glucose and D-xylose after treatment with trimethylchlorosilane (TMCS) in pyridine. The absolute configurations of the compounds 1-3 were determined by comparison of the retention times of the corresponding derivatives with those of standard D-glucose and D-xylose giving a single peak at 19.01 and 13.47 min, respectively.

Cytotoxic Activity Assay
The following human cancer cell lines were used: T98G, U87, A549, GITC-3#, and GITC-18#. All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum (Hyclone) at 37°C in a humidified atmosphere with 5% CO 2 . Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) [31]. Briefly, 100 lL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 9 10 5 cells/mL in 100 lL of medium. Each cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTT (100 lg) was added to each well, and the incubation continued for 4 h at 37°C. The cells were lysed with 100 lL of 20% SDS-50% DMF after removal of 100 lL of medium. The optical density of the lysate was measured at 595 nm in a 96-well Microtiter plate reader (Bio-Rad 680).