The Anticancer Activities Phenolic Amides from the Stem of Lycium barbarum

Four new phenolic amides, 4-O-methylgrossamide (1), (E)-2-(4,5-dihydroxy-2-{3-[(4-hydroxyphenethyl)amino]-3-oxopropyl}-phenyl)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-hydroxyphenethyl)acryl-amide (2), (Z)-lyciumamide C (3), (Z)-thoreliamide B (4), together with thirteen known phenolic amides were identified from the stem of Lycium barbarum. The structures of the new compounds were determined by spectroscopic methods. All compounds were evaluated for their anti-cancer activities against human glioma stem cell lines.

A literature survey shows that the E-isomers of this type of compounds are widespread in some genera and small amount of Z-isomers [12,18]. In this paper, three pairs of Z-E-isomers (3 and 6; 4 and 9; 7 and 8) were reported. Due to their different retention times on the Rp-C 18 column, every pair of isomers was separated by HPLC.
All the compounds were evaluated for their bioactivity against two human glioma stem cell lines (GSC-3# and GSC-12#), by the cell viability assay and phenotypic screening. The results showed that compound 5 exhibited the moderate cytotoxicity against GSC-3# and GSC-12# at the concentration of 10 lg/mL (Fig. 4a), and the IC 50 values were 6.40 and 5.85 lg/mL respectively (Fig. 5). Compound 1 showed the moderate cytotoxicity against GSC-3# and GSC-12# at the concentration of 25 lg/mL (Fig. 4b), and the IC 50 values were 28.51 and 19.67 lg/mL respectively (Fig. 5).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were detected on a SHMADZU Table 2 1 H (500 MHz) and 13 C NMR (125 MHz) Data for compounds 3 and 6 in DMSO-d 6 and CD 3 OD (d in ppm, J in Hz) No.

Extraction and Isolation
The air-dried and powdered sample (10.0 kg) was extracted with 85% aqueous EtOH (40 L 9 3) under reflux conditions for 3 h each time, and the solvent was evaporated in vacuum. The residue (607 g) was suspended in H 2 O and extracted with EtOAc (each 2 L 9 3). The EtOAc layer (224 g) was passed over a silica gel column, eluting with CHCl 3 -Me 2 CO (1:0 to 0:1) to give nine fractions.

Anticancer Activities
GSC-3# and GSC-12# were human glioma stem cell lines that were established by Kunming institute of zoology from two human glioblastoma multiform samples. The glioma stem cell was cultured in serum-free medium DMEM F12 supplied with 1xB27 and 50 ng/mL EGF, BFGF and 1% penicillin/streptomycin. The glioma stem cells were seeded in the laminin pre-coating dishes and cultured in 37°C, 5% CO 2 incubator. Cell viability assay was performed by the MTS method as previously described. GSCs were digested and counted, seeded in laminin pre-coating 96-well-plate with 20000 cells/well. The compounds were added with a serial gradient concentration (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 lg/mL) and cultured in cell incubator for 72 h. MTS reagent was diluted 1:5 with fresh medium and mixed well. The old medium was removed and subsequently the fresh medium was added with 100 lL/well. The cells were incubated for 1.5 h. Absorbance was measured by Hybrid Reader (BioTek synergy H1) at 490 nm. The cell viability was evaluated by percentage compared with DMSO control group. The half-maximal inhibitory concentration (IC 50 ) was measured and calculated by Graph Pad Prism 5 software.

Supporting Information
1D, 2D NMR spectra, ESIMS/MS, HRESIMS, UV and IR of compounds 1-4 and influence of deuterated solvent on the 1 H NMR spectra of compounds 3 and 6 are available).