Preliminary Study On Pharmacokinetic Parameters And Serum Medicinal Chemistry Of Different Proportions Of Qinghuang Powder

Objective:Qinghuang Powder is a compound composed of Indigo Naturalis and Realgar.It has the effect of detoxifying,cooling blood and dispersing phlegm.In recent years,the research on the chemical constituents and pharmacological effects of Qinghuang Powder has became deeper,but there are few reports on the study of the changes of Qinghuang Powder in vivo.In this paper,the content of tryptanthrin,indigo and indirubin,which were the active constituents in Qinghuang Powder,were determined for the first time to evaluate the quality of the Indigo Naturalis and provide a theoretical reference for the pharmacokinetic dose.The pharmacokinetic parameters and serum pharmacochemistry of Qinghuang Powder with different proportions were preliminarily studied to provide reference for the basic study of the pharmacodynamics of Qinghuang Powder.Method:1.In view of the pharmacodynamic activities of tryptanthrin,indigo and indirubin in the Indigo Naturalis,the three components were used as evaluation indicators to evaluate the quality of the three batches of Indigo Naturalis raw material,and to provide a dose reference for the pharmacokinetic study of Indigo Naturalis.The HPLC method was established to simultaneously determine the content of tryptanthrin,indigo and indirubin in Indigo naturalis,and the methodological verification was carried out,which was applied to different batches of products.2.Eighteen SD rats were randomly divided into three groups:A,B,and C.The different doses of Qinghuang Powder were administered,and the doses of Indigo Naturalis were5 g/kg,and the doses of Realgar were 26.25 mg/kg and 52.5 mg/kg,105mg/kg,respectively.Blood samples were taken before and after administration.UPLC-MS/MS method was established with carbamazepine as internal standard,multiple reaction monitoring model（MRM）and positive ion detection for determine the concentrations of tryptanthrin,indigo and indirubin in rat plasma The data were processed using DAS2.0software to calculate the main pharmacokinetic parameters of tryptanthrin,indigo and indirubin in rats.3.UPLC-Q-TOF-MS/MS technique was used to study serum pharmacochemistry of rats after oral administration of Qinghuang Powder with three different ratios of A,B and C,respectively,and the differences of migration components between the three groups were analyzed.The serum containing the group A,B and C of Qinghuan Powder group was applied to HL-60 cells,and the effect of drug-containing serum on the proliferation of HL-60 cells was detected by CCK-8 method.Result:1.The linear ranges of tryptanthrin,indigo and indirubin were 0.02-0.16μg/m L,8-64μg/m L and 0.5-4μg/m L,respectively.The average recovery of the method was more than 97%.The contents of the three components of tryptanthrin,indigo and indirubin in three batches of Indigo Naturalis are 0.065 to 0.10mg/g,17 to 31mg/g,and 2.4 to3.9mg/g,respectively.2.The t_max of tryptanthrin in rats of group A,B and C were（0.38±0.137）,（0.46±0.10）and（0.51±0.29）h,respectively,C_max were（1.52±0.32）,（1.97±2.27）,and（2.45±2.49）ng/m L.t_1/2 are（3.47±1.01）,（3.57±1.17）,（3.72±1.48）h,AUC_0-tis（5.21±1.63）,（6.67±8.06）,（6.06±5.37）ng·h/m L.The t_max of indirubin in rats of group A,B and C were（4.00±0）,（3.67±0.82）,（3.40±1.95）h,respectively,and C_max were（80.18±5.74）,（53.04±29.99）and（34.59±19.98）ng/m L.t_1/2are（1.79±0.84）,（1.98±1.05）,（2.11±0.81）h,respectively,AUC_0-t is（318.0±104.321）,（175.3±84.91）,（147.28±85.35）ng·h/m L.The t_max of indigo in rats of group A,B and C were（3.33±1.03）,（4.33±1.97）and（3.50±1.00）h,and C_maxwere（51.21±8.99）,（80.74±11.35）and（132.05±22.78）ng/m L,t_1/2were（2.71±1.03）,（2.08±1.14）,（2.43±0.55）h,AUC_0-twere（289.35±48.28）,（393.79±97.14）,（604.61±131.04）ng·h/m L.The experimental results showed that there were no significant change in C_max,AUC and MRT of tryptanthrin as the dose of Realgar increased.The C_maxand AUC of indirubin were decreased with the increase of the dose of Realgar.At the same time,the C_max and AUC of indigo were increased with the increased dose of Realgar.3.Comparing the serum mass spectra of the rats in group A,B and C of Qinghuang Powder,and 5 prototype components and 14 possible metabolites were found in the serum of group A of Qinghuang Powder.It was found that there were 5 prototype components and 17 possible metabolites in the serum of group A of Qinghuang Powder,and 5 prototype components and 20 possible metabolites were found in the serum of group C of Qinghuang Powder.Conclusion:The established HPLC method is simple,accurate and reproducible.It can be used to determine the content of active constituents of Indigo Naturalis.UPLC-MS/MS method is used to study the effect of Realgar on pharmacokinetic parameters of index components of Indigo Naturalis.The results showed that the main pharmacokinetic parameters of Indigo Naturalis were changed,suggesting that Realgar may affect the pharmacokinetic process of Indigo Naturalis in vivo.UPLC-Q-TOF-MS/MS method was used to compare the serum migration components of Qinghuang Powder in different proportions.The results showed that the increase of realgar could increase the metabolites of Indigo Naturalis.The CCK-8 method was used to detect the proliferative activity of different serum containing Qinghuang Powder on HL-60 cells.The group of Qinghuang Powder with larger Realgar proportion had the strongest inhibition on HL-60 cells.