Simultaneous determination of forsythin and its major metabolites in human plasma via liquid chromatography-tandem mass spectrometry.

Forsythiae suspensa is widely used in China as a traditional Chinese medicine. Forsythin is extracted from Forsythiae Fructus and has undergone phase II clinical trials as an antipyretic drug in China. The main metabolites of forsythin in human plasma are aglycone sulfate (KD-2-SO3H) and aglycone glucuronide (KD-2-Glc). In the present study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous analysis of forsythin, KD-2-Glc, and KD-2-SO3H, in human plasma. After precipitating proteins with methanol, these three analytes were separated on a Gemini-C18 column along with teniposide as an internal standard. Mass spectrometry detection, under multiple reaction monitoring, was then carried out in negative mode using the Triple Quad™ 6500+ LC-MS/MS system coupled with an electrospray ionization (ESI) ion source. The transitions of m/z 371.1→356.1 for forsythin, m/z 547.2→356.0 for KD-2-Glc and m/z 451.2→356.2 for KD-2-SO3H were chosen to effectively maintain the balance between selectivity and sensitivity. The developed method was linear over the following concentrations in human plasma samples: 1.00-1000 ng/mL for forsythin, 2.50-2500 ng/mL for KD-2-Glc, and 5.00-5000 ng/mL for KD-2-SO3H. Assays were validated and satisfied the acceptance criteria recommended by the CFDA guidance. Furthermore, this LC-MS/MS method was successfully implemented in a Phase I, first-in-human, dose-escalation pharmacokinetic study among Chinese healthy participants after single oral administration of forsythin tablets.

The role of Ntcp, Oatp2, Bsep and Mrp2 in liver injury induced by Dioscorea bulbifera L. and Diosbulbin B in mice.

Dioscorea bulbifera L. (DB) is a traditional Chinese herb used in thyroid disease and cancer. However, the clinical use of DB remains a challenge due to its hepatotoxicity, which is caused, in part, by the presence of Diosbulbin B (DIOB), a toxin commonly found in DB extracts. As abnormal expression of hepatobiliary transporters plays an important role in drug-induced liver injury, we assessed the hepatotoxicity induced by DB and DIOB, and explored their impacts on hepatobiliary transporter expression levels. Following liquid chromatography-tandem mass analysis of the DIOB content of DB extract, male ICR mice were randomly orally administered DB or DIOB for 14days. Liver injury was assessed by histopathological and biochemical analysis of liver fuction. The levels of transporter protein and mRNA were determined by western blotting and real-time PCR. Liver function and histopathological analysis indicated that both DB and DIOB could induce liver injury in mice, and that DIOB might be the primary toxic compound in DB. Moreover, down-regulation of Mrp2 blocked the excretion of bilirubin, glutathione disulfide, and bile acids, leading to the accumulation of toxic substrates in the liver and a redox imbalance. We identified down-regulated expression of Mrp2 as potential factors linked to increased serum bilirubin levels and decreased levels of glutathione in the liver and increased liver injury severity. In summary, our study indicates that down-regulation of Mrp2 represents the primary mechanism of DB- and DIOB-induced hepatotoxicity, and provides insight into novel therapies that could be used to prevent DB- and DIOB-mediated liver injury.

[Clinical and experimental study of xiaoshui decoction in the treatment of primary liver cancer caused ascites].

OBJECTIVE: To observe the clinical efficacy of Xiaoshui Decoction (XSD) in treating ascites in patients suffered from primary liver cancer of Pi-deficiency with damp harassment syndrome (PDDHS) as well as to study the effect through the experiment in mice. METHODS: Sixty-one patients confirmed to be primary liver cancer of PDDHS and accompanied with ascites were randomly divided into the treated group (n=33) and the control group (n=28). The treated group was treated by XSD combined with chemotherapy by locally applying of DDP via abdominal infusion, while the control group treated by DDP infusion alone. The treatment lasted for two months. The conditions of ascites, quality of life (QOL), survival period, and TCM syndrome after treatment were observed. In the experimental study, the mice models of ascites were grouped and treated to observe the conditions of ascites and their survival period. RESULTS: The short-term total effective rate of the treated group and the control group was 42.4% and 21.4%, the interval of aspirating ascites after treatment was 17.95 +/- 9.63 days and 10.87 +/- 7.76 days, and the 1-year survival rate 33.3% and 14.3%, respectively, significant difference was shown between the two groups in the three parameters (all P < 0.05 ). QOL was improved in both groups with insignificant difference (P > 0.05). Besides, the main symptoms were improved in patients of both groups, especially in the ameliorating of fatigue, abdominal distension, nausea and vomiting. Evaluation on safety of treatment showed that XSD had no obvious toxic and adverse reaction, and so it was safe in use. Experimental study showed that on the two mice models of ascites induced by inoculating two kinds of tumor cell, the effect of XSD was superior to that of the control group in aspects of reducing ascites and prolonging survival period, showing significant difference (P < 0.05). CONCLUSION: Satisfactory short-term efficacy in treating primary liver cancer with ascites of the Pi-deficiency with damp harassment syndrome could be obtained by XSD. Its effect in prolonging survival period was confirmed by experimental study. XSD can also improve the symptoms and QOL of patients, therefore, it is an effective and reliable remedy for treatment of primary liver cancer with ascites.