Research On The Acute Toxicity,Anti CCl₄-rinduced Liver Injury And Pharmacokinetics Of Nano-glycyrrhizic Acid Powder For Injection

In this paper,we studied the acute toxicity, pharmacodynamics and plasma pharmacokinetic of nano-glycyrrhizic acid injection formulations prepared by the supercritical anti-solvent (SAS) process.First,the single dose acute toxicity of nano-glycyrrhizic acid injection by intravenous injection in mice was studied by improved Cole’s method.The circumstances of the mortality rate and abnormal behavior of the mice was observed14consecutive days, followed by the gross anatomy observation of mice.We calculated the mouse median lethal dose and95%of the average confidence limit. The results show that single dose intravenous injection of nano-glycyrrhizic acid injection in mice has LD5o of340.28mg/kg and LD50of the95%average confidence limit302.73～382.49mg/kg. Observations on mice behavior showed that the nano-glycyrrhizic acid injection may have some degree of damage on the mouse central nervous system (CNS), neuromuscular, autonomic nervous and sensory. What’s more, it may possibly affect the respiratory system of mice indirectly through the influence of the autonomic nervous system, leading to the occurrence of respiratory failure. The gross anatomy of the dead mice reveals no significant organic damage.Second,the comparative efficacy of nano-glycyrrhizic acid injection were discussed. Chronic liver injury model in rats by gavage of carbon tetrachloride construct, followed by administration of nano-glycyrrhizic acid injection treatment, compared with the Glycyrrhetate ammonium.Then we determined the rat liver index, serum alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activity,measured liver tissue homogenates of hydroxyproline (Hyp) and glutathione peroxidase (GSH-Px) in content, superoxide dismutase (CuZn-SOD) activity, and light microscopic examination of HE staining of rat liver slices was observed. The results were as follow:compared to the glycyrrhizic acid monoammonium group, serum ALP, ALT and AST activities of nano-glycyrrhizic acid injection reduced by29.68%,5.51%and3.97%; liver index decreased by3.42%, and liver tissue Hyp15.67%reduction of SOD increased15.70%and GSH increased6.56%. Two experimental groups were compared with model group, the biochemical indicators the difference was significant (P<0.05,P<0.01). Light microscopic examination of HE stained liver tissue shows significantly reduction of degeneration of the nano-glycyrrhizic acid injection group, and can ease the process of liver tissue lesions.Third,the plasma pharmacokinetics of nano-glycyrrhizic acid injection was investigated. Through method validation, we established an accurate, reliable LC-MS/MS determination method for glycyrrhizic acid in rat plasma, and studied the drug concentration-time curve of nano-glycyrrhizic acid injection,calculated the pharmacokinetic parameters using the3p97software, and make judgment on the compartment model.The result show that we have established a reliable LC-MS/MS detection method of glycyrrhizic acid,in rat plasma,the chromatographic conditions:column, Agilent Eclipse XDB-C18column (150mm*4.6mm id,5um); mobile phase was0.1%formic acid:methanol (v:v=1:9), the column temperature was room temperature, flow rate1mL/min (Mass Spectrometry before shunt), the injection volume was10μL. MS conditions:ESI ion source, the MRM negative ion scan mode, the ion source spray voltage4500V; ion source atomization temperature of300°C; atomization gas12psi; air curtain gas10psi. Glycyrrhizinate m/z821.8â†'351.3, DP-150v,CE-60v, CXP-5v; chlorogenic acid m/z353.0â†'191.0,DP-130v, CE-27v CXP of-5v.In the above detection method, heparin anticoagulant rat blank plasma has little interference with glycyrrhizic acid and no interference with internal standard chlorogenic acid while the specificity is both good;the average recoveries of glycyrrhizic acid and internal standard chlorogenic acid were85.02%and81.84%, matrix effect factor were98.95%and94.32%; between-run precision of standard curve and quality control samples has a relative standard error of less than15%, accuracy within±15%; within-run precision of quality control samples has a relative standard error of less than15%and accuracy of±15%;the LLOQ samples has RSD of12.83%and relative error of-12.05%; extracted quality control samples placed in the2～8℃for48hours, laboratory bench at room temperature for8hours and one to three freeze-thaw situations are stable. The data of method validation show that the determination of glycyrrhizic acid in rat plasma in sodium heparin anticoagulant has reliable accuracy, high-precision and is determination specified and strong stability in the range of5-10000ng/mL.The method is accurate and reliable.The nano-glycyrrhizic acid injection metabolic behavior conforms the two compartment model, and relevant pharmacokinetic parameters are as follows:the distribution relative to the initial concentration A is1364.069±463ng/mL, eliminating phase concentration of B is95.199±15.3ng/mL The distribution phase rate constant a is2.699±0.576h-1, the eliminate the phase rate constant (3is0.221±0.025h-1, the distribution phase half-life t1/2α is0.257h, elimination phase half-life t1/2β is3.137h, the elimination rate constant K10is1.559h-1, the peripheral compartmentâ†'the central compartment rate constant of transport K21is0.383h-1, the central compartmentâ†'the peripheral compartment rate constant of transport K12is0.978h-1, drug-time area under the curve AUC is936.239ng/mL*h, the apparent volume of distribution Vd is3.426L/kg, clearance rate CL is5.341L/h.