ABSTRACT
BACKGROUND: Sanguisorba officinalis, a popular Chinese herb, called DiYu, has been shown to inhibit the growth of many human cancer cell lines, including colorectal cancer cells. The aims of this study were to discover the active compound and molecular mechanism of S. officinalis against Wnt/ß-catenin signaling pathway and develop Wnt inhibitors from natural products as anti-colorectal cancer agents. METHODS: 1,4,6-Tri-O-galloyl-ß-d-glucopyranose (TGG) was obtained by the preparative HPLC. The effect of DiYu on proliferation of NIH3T3 and HT29 was detected by MTT assay. Luciferase reporter assay was applied to investigate the activity of Wnt/ß-catenin signaling in NIH3T3. The expression levels of mRNA and protein were detected by RT-PCR and western blot. Immunofluorescence assay was used to measure the level of ß-catenin in cytoplasm and nucleus. Transcriptomic profiling study was performed to investigate the molecular mechanism of DiYu on the Wnt/ß-catenin signaling pathway. RESULTS: TGG significantly inhibited the Wnt/ß-catenin signaling pathway, down-regulated the expression of ß-catenin and Wnt target genes (Dkk1, c-Myc, FGF20, NKD1, Survivin), up-regulated the levels of cleaved caspase3, cleaved PARP and ratio of Bax/Bcl-2, which may explain the apoptosis of HT29. CONCLUSIONS: Our study enhanced the discovery of the materials and elucidation of mechanisms that account for the anti-Wnt activity of natural inhibitor (DiYu) and identified the potential of TGG to be developed as anti-colorectal cancer drugs.
ABSTRACT
A ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method was successfully developed and validated for the identification and determination of eight alkaloids: tetrahydropalmatine (A); palmatine (B); magnoflorine (C); columbamine (D); berberine (E); worenine (F); berberrubine (G) and coptisine (H) in rat plasma, which are the active components in Coptis deltoidea C. Y. cheng et Hsiao (CCY) and Coptis chinensis Franch (CF). The chromatographic separation of analytes was successfully achieved on an Agilent SB-C18 column (1.8 µm, 150 mm × 2.1 mm) using a programme with a mobile phase consisting of acetonitrile and water containing 0.3% acetic acid at a flow rate of 0.25 mL/min. The analytes were detected with a triple quadrupole tandem MS in multiple reaction monitoring (MRM) mode and an electrospray ionization (ESI) source in positive mode. The validated method showed good linearity over a wide concentration range (r² > 0.991), and lower limits of quantification (LLOQ) less than 1.1 ng/mL for all analytes, and matrix effects ranged from 85.2% to 106.8%. The mean extraction recoveries were no less than 86.4%, and the precision and accuracy were within the acceptable limits. All analytes were proven to be stable during sample storage and analysis procedures. The method validation results demonstrated that the proposed method was sensitive, specific, and reliable, which could lay a foundation for the pharmacokinetic study of eight analytes after oral administration of CCY and CF in subsequent studies.
