Comprehensive characterization and identification of chemical constituents in Yangwei decoction using ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Yangwei decoction, a classical traditional Chinese medicine prescription, has been widely used to treat exogenous cold and internal injury with damp stagnation for many centuries. However, its systematic chemical profiling remains ambiguous, which has hampered the interpretation of pharmacology and the mechanism of its formula. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry method was successfully established for the first time to separate and identify the complicated components of Yangwei decoction. The accurate mass data of the protonated molecules, deprotonated molecules, and fragment ions were detected in positive and negative ion modes. A total of 226 compounds in Yangwei decoction were tentatively identified and unambiguously characterized by comparing their retention times and mass spectrometry data with those of reference standards and literature, including 24 lignans, 18 alkaloids, 9 phenylpropanoid glycosides, 76 flavonoids, 59 triterpenoids, 17 organic acids, 7 gingerols, 8 lactones, and 8 other compounds. The present study provides a novel method of constituents characterization for well-known Chinese medicine prescriptions. The study aims to lay a robust foundation for future research, providing the holistic quality control and pharmacology of Yangwei decoction.

Jianpi Yangwei decoction promotes apoptosis and suppresses proliferation of 5-fluorouracil resistant gastric cancer cells in vitro and in vivo.

BACKGROUND: The exploration of new therapeutic agents targeting 5-Fu resistance may open a new opportunity to gastric cancer treatment. The objective is to establish a 5-Fu resistant gastric cancer cell line and observe the effect of Jianpi Yangwei decoction (JPYW) on its apoptosis and drug-resistance related proteins. METHODS: MTT assay was used to measure the effect of JPYW on the BGC823 cells proliferation, and the apoptosis was observed by flow cytometry and Hoechst fluorescence staining. The BGC823 xenograft tumor nude mice models were established, the apoptosis was detected by Tunel method. BGC-823/5-Fu was established by repeated low-dose 5-Fu shocks, the drug resistance index and proliferation were detected by the MTT assay; MDR1 mRNA was detected by real-time RT-PCR; Western blot was used to detect the ratio of p-AKT to AKT; The BGC823/5-Fu xenograft tumor nude mice models were established and apoptosis was measured. The expressions of MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 were detected by immunohistochemistry and the AKT mRNA expression was detected by real-time RT-PCR. RESULTS: JPYW induced apoptosis in BGC823 cells; Drug-resistant cell line BGC-823/5-Fu was sucessfully established; JPYW induced apoptosis of BGC823/5-Fu cells, down-regulated the expression of MRP1, MDR1 and ABCG2 in vitro and in vivo, and further decreased MDR1 expression when combined with pathway inhibitor LY294002 (P < 0.05); JPYW down-regulated the ratio of p-AKT to AKT in vitro in a dose-dependent manner, the same as after the combination with LY294002 (P < 0.05). CONCLUSION: JPYW can induce apoptosis of BGC823 and BGC823/5-Fu cells, and down-regulate the expression of MDR1, MRP1, ABCG2 in vitro and in vivo. Its in vitro effect is related to the PI3K/AKT signaling pathway.

Jianpi-yangwei decoction inhibits DNA damage repair in the drug resistance of gastric cancer by reducing FEN1 expression.

BACKGROUND: Flap Endonuclease 1(FEN1) has been considered as a new tumor marker in recent years and Jianpi Yangwei Decoction (JPYW) is a basic Traditional Chinese Medicine (TCM) for the treatment of gastric cancer. This study aimed to explore the role of FEN1-mediated DNA damage repair in the drug resistance of gastric cancer and the effect of JPYW on it by employing BGC823/5-Fu drug-resistant cell model. METHODS: The DNA repair efficiency of BGC823 and BGC823/5-Fu was compared intracellularly and extracellularly using an extrachromosomal assay system and the reconstituted base excision repair assay. By comparing gene and protein expression and identifying cell survival rates after knockdown or high expression of FEN1, the correlation between FEN1 high expression and 5-Fluorouracil (5-Fu) drug resistance was revealed. The effect of JPYW on DNA damage repair and FEN1 expression was observed by the degree of γ-H2AX phosphorylation in the cells, DNA repair efficiency and enzyme activity, et al. RESULTS: BGC823/5-Fu had a higher DNA repair efficiency than BGC823(P < 0.001), which proved to be both intracellular and extracellular. FEN1 was highly expressed in BGC823/5-Fu regardless of gene level(P < 0.001) or protein level. Furthermore, manipulating FEN1 altered the sensitivity of cancer cells to chemotherapeutic drug 5-Fu. Different concentrations of JPYW were used to investigate the inhibitory effect on the expression of FEN1 and DNA damage repair. JPYW inhibited DNA damage repair both intracellularly and extracellularly: the phosphorylation of γ-H2AX increased, with more DNA damage in the cells; the synthetic 8-oxo dG damage repair was reduced; and the ability of cell lysates to repair DNA damage decreased. The decrease of FEN1 expression in BGC823/5-Fu had a concentration dependent relationship with JYPW. In addition, JPYW inhibited the activity of FEN1 at the enzymatic level, as the amount of cut-off synthetic 32p labeled DNA substrates were decreased. CONCLUSION: FEN1 was highly expressed in drug-resistance gastric cancer cells BGC823/5-Fu, which leading to BGC823 resistant to (5-Fu) by acting on DNA damage repair. JPYW inhibited DNA damage repair and reversed 5-Fu drug resistance by reducing FEN1 expression and inhibiting FEN1 functional activity.