Anti-convulsant effects of cultures bear bile powder in febrile seizure via regulation of neurotransmission and inhibition of neuroinflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Natural bear bile powder (NBBP) has been used to treat seizures for thousands of years, but its application is greatly restricted due to ethical reasons. Cultured bear bile powder (CBBP), which is produced by biotransformation, may be an appropriate substitute for NBBP. However, the anti-convulsant effects of CBBP and its mechanisms remain unclear. AIM OF THE STUDY: This study aimed to investigate the anti-convulsant effects and possible mechanisms of CBBP in a febrile seizure (FS) rat model. MATERIALS AND METHODS: FS was induced by placing the rats in a warm water bath (45.5 °C). The incidence rate and latency of FS, and hematoxylin-eosin staining (HE) were conducted for neurological damage. The levels of 4 bile acids and 8 main neurotransmitters in vivo were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The expression of bile acid related transports, neurotransmitter receptors, inflammatory factors, neurotrophic factors and glial fibrillary acidic protein (GFAP) in hippocampal tissues were detected by real-time PCR, western blotting, and immunohistochemistry. RESULTS: Pre-treatments with CBBP and similarly, NBBP, significantly reduced the incidence rate and prolonged the latency of FS. Additionally, CBBP alleviated the histological injury induced by FS in the rat hippocampus tissue. LC-MS/MS analyses revealed that CBBP markedly increased the levels of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA) in FS rats. Furthermore, the content of gamma-aminobutyric acid (GABA) was up-regulated in rats pre-treated with CBBP whereas GFAP was down-regulated. CBBP also significantly suppressed the expression of interleukin -1ß (IL-1ß), tumor necrosis factor α (TNF-α), nuclear factor kappa B (NF-κB), and brain-derived neurotrophic factor (BDNF) and its TrkB receptors, and improved the expression of GABA type A receptors (GABAAR) and farnesoid X receptors (FXR). CONCLUSIONS: The present study demonstrated that CBBP had anti-convulsant effects in a FS rat model. CBBP may protect rats against FS, probably by up-regulating FXR, which was activated by increasing brain bile acids, up-regulating GABAergic transmission by inhibiting BDNF-TrkB signaling, and suppressing neuroinflammation by inhibiting the NF-κB pathway.

A validated surrogate analyte UPLC-MS/MS assay for quantitation of TUDCA, TCDCA, UDCA and CDCA in rat plasma: Application in a pharmacokinetic study of cultured bear bile powder.

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC-MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975-0.9994), precision (RSD intra-day, 0.72-9.35%; inter-day, 3.82-9.02%), accuracy (RE, -12.42-5.67%) and matrix effect (95.53-99.80%), along with analyte recovery (95.90-98.82%) and stability (89.48-101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06- 11.51%; inter-day, 2.23- 11.38%), accuracy (RE, -7.40-10.76%) and stability (87.37-111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations.

Simultaneous determination of multiple compounds of Da-Huang-Xiao-Shi decoction in rat plasma by LC-MS/MS and its application in a pharmacokinetic study.

Da-Huang-Xiao-Shi decoction (DHXSD), a traditional Chinese medicinal formula, has been used mainly to treat jaundice for more than 1700 years in China. In this study, we developed a rapid, sensitive, and accurate LC-MS/MS method to simultaneously determine multiple, potentially bioactive compounds of DHXSD, including five alkaloids (berberine, phellodendrine, palmatine, jatrorrhizine, and magnoflorine), five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol, and physcion), two iridoid glycosides (geniposide and genipin 1-gentiobioside), and one iridoid aglycone (genipin) in rat plasma. Plasma samples collected from rats were treated immediately with 5% acetic acid to avoid the degradation of genipin. After protein precipitation with acetonitrile containing 5% acetic acid, the compounds were reconstituted in acetonitrile-water (50:50, v/v) solution containing 6.5% formic acid and separated on the ACQUITY™ UPLC BEH C18 column (2.1 × 100 mm; 1.7 µm) using a mobile phase composed of 2 mM ammonium formate in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Quantitation was performed on a Triple Quand 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) was used to quantify compounds in positive and negative ion modes. The method validation results showed that the specificity, linearity, precision and accuracy, recovery, matrix effect, and stability of the 13 compounds met the requirements for their quantitation in biological samples. This newly established method was successfully used in a pharmacokinetic study on rats orally treated with DHXSD. Besides, glucuronide and sulfate metabolites were also determined in rat plasma after hydrolysis. This is the first method developed for the simultaneous quantification of multiple compounds of DHXSD in vivo. Our study provides relevant information on the pharmacokinetics of DHXSD and the relationship between the compounds of DHXSD and their therapeutic effects.