Development of a MD-LC-MS/MS Method to Analyze 3 Bioactive Compounds in Huoxuezhitong Rubber Patch and Application to a Pharmacokinetic Study in Rats.

Huoxuezhitong rubber patch, a well-known traditional Chinese medicine (TCM) prescription, is utilized to treat pain and inflammation. In this study, a microdialysis-ultra-high-performance liquid chromatography-tandem mass spectrometry (MD-LC-MS/MS) method was designed for the simultaneous determination of active constituents in the rubber patch, such as paeonol (Pae), eugenol (Eug), and piperine (Pip). A microdialysis probe was implanted in the subcutaneous tissue of a rat, which is intended to detect the subcutaneous concentrations of target components. Saline containing 30% ethanol acted as perfusion fluid. Analytes in the microdialysate were completely separated over an ACQITY UPLC RBEH C18 column (2.1mm×100mm, 1.7µm). The mobile phase was composed of 0.01% ammonia aqueous and acetonitrile-0.01% ammonia with gradient elution. The single-run analysis time was 10.0 minutes. The linear regression displayed good linear relationships in the ranges of 0.25-100 ng/mL for paeonol and eugenol and 0.001-5 ng/mL for piperine. The interday and intraday precision of the quality control samples exhibited relative standard deviations (RSD) <13.56%. The accuracy values ranged from -14.92% to 14.00%. The present method was successfully applied in pharmacokinetics studies following dermal administration of Huoxuezhitong rubber patch in rats. Pip's Tmax (488.00±150.73) min was greater than that of Pae (186.67±48.44) min and Eug (240.00±138.56) min, and the rank order of t1/2 was Pae > Pip > Eug. The rank order of AUC0-720 and Cmax was both Eug > Pae > Pip. MRT0-∞ of Pip was higher than that of Pae and Eug. Eugenol showed a faster elimination and a shorter half-life. Paeonol showed a stronger drug reservoir function after removing the drug source.

[Study on in vitro release and percutaneous absorption of Huoxue Zhitong gel].

To evaluate in vitro release and transdermal behaviors of Huoxue Zhitong gel, modified Franz diffusion cell methods was applied to investigate in vitro transdermal absorption of Huoxue Zhitong gel and the content of paeonolan in receptor fluid composed of PEG400%-95% ethanol-water (l:3:6)were determined by HPLC. The results were processed and different equations were fitted. The release law were in accordance with Weibull equation and the fitting equation was In[-1/(1 - Q)] = -0.790 51nt - 1.7012 (r = 0.9809). In 8 hours, cumulative release of paeonol was 85. 18% and the release rate was 2.827 µg . cm-2 h-1. Transdermal actions were consistent with zero-level model fit and the fitting equation was Q(t) = 1.7579t + 0. 7213 (r = 0.9991). In 8 hours, cumulative transdermal rate and transmission rate of paeonol was 54. 85%, 1. 820 µg . cm-2 h-1. So the Huoxue Zhitong gel had a good release and transdermal properties.

Protective effect of paeoniflorin on immunological liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide: modulation of tumour necrosis factor-alpha and interleukin-6 MRNA.

1. Paeoniflorin is one of the main effective components of the total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora which has been used for gynaecological problems and for cramp, pain and giddiness for over 1,500 years in Chinese medicine. Anti-inflammatory, antioxidative, antihepatic injury and immunoregulatory activities of TGP have been extensively proved in our laboratory for many years. Our present study investigates the effects and mechanisms of paeoniflorin on immunological liver injury in mice. 2. A model of immunological liver injury was induced by tail vein injection of bacillus Calmette-Guérin (BCG) and lipopolysaccharide (LPS) in mice. Activities of serum alanine aminotransferase (ALT) were measured by biochemical methods. Hepatic tissue sections were stained with haematoxylin and eosin and examined under a light microscope. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA (messenger ribonucleic acid) expression in mouse liver were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. 3. Immunological liver injury induced by BCG plus LPS was successfully duplicated. Serum ALT activities were significantly decreased by paeoniflorin. (25, 50, 100 mg/kg). Histological examination demonstrated that paeoniflorin could attenuate the area and extent of necrosis and reduce the immigration of inflammatory cells. The increase in TNF-alpha, LBP and CD14 mRNA expression in mouse liver after BCG and LPS injection was significantly decreased by paeoniflorin (100 mg/kg) and was changed by paeoniflorin (25, 50 mg/kg) at different time-point. The augmentation of IL-6 mRNA in mouse liver was markedly increased by paeoniflorin at 1 h and 3 h after LPS injection. 4. Paeoniflorin could significantly protect against immunological liver injury in mice. TNF-alpha, IL-6, LBP and CD14 mRNA expression in mouse liver may be involved in BCG plus LPS induced liver injury. The protective mechanism of paeoniflorin might be partially related to modulation of TNF-alpha, IL-6, LBP and CD14 mRNA expressions in mouse liver.

Upregulation of TNF-alpha and IL-6 mRNA in mouse liver induced by bacille Calmette-Guerin plus lipopolysaccharide.

AIM: To investigate the mechanism of immunological liver injury induced by bacille Calmette-Guerin (BCG) plus lipopolysaccharide (LPS). METHODS: Mice were injected via the tail vein with 125 mg/kg BCG, and 12 d later, the mice were injected intravenously with different doses of LPS (125, 250, or 375 microg/kg). Serum alanine aminotransferase (ALT) activity and liver pathological changes were examined. The expression of tumor necrosis factor (TNF)- alpha, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA, and NF-kappaB and IkappaB-alpha protein in mouse liver at different time points after BCG and LPS injection were measured using RT-PCR, immunohistochemistry and Western blotting analysis, respectively. RESULTS: The activity of serum ALT in mice treated with BCG and LPS was significantly increased. Different degrees of liver injury, such as inflammatory cell infiltration, spotty necrosis, piecemeal necrosis, even bridging necrosis, could be seen in liver sections from mice after BCG and LPS administration. Furthermore, the levels of TNF-alpha and IL-6 mRNA in mouse liver were significantly elevated after administration of BCG plus LPS (P<0.05). The levels of LBP and CD14 mRNA in mouse liver were markedly upregulated after treatment with BCG and LPS, and treatment with BCG alone led to an increase in CD14 mRNA in mouse liver. Finally, immunoreactivity for NF-kappaB p65 was predominantly detected in hepatocyte nuclei from mice treated with BCG plus LPS, compared with the normal group. Protein levels of IkappaB-alpha were strikingly decreased by LPS or BCG plus LPS treatment, compared with the normal group or BCG group. CONCLUSION: TNF-alpha and IL-6 mRNA were partially involved in early immunological liver injury induced by challenge with small doses of LPS after BCG priming. Upregulation of TNF-alpha and IL-6 mRNA might be related to increases in LBP and CD14 mRNA expression and activation of NF-kappaB. Furthermore, BCG priming in immunological liver injury may occur via upregulation of CD14 mRNA expression in mononuclear cell infiltration into the liver.