Studies On The Toxicological Effects Of Gegenqinlian Decoction And Serum Pharmacochemistry Of Main Reconstituent

Objective:To observe the different change of erythrocyte osmotic fragility(EOF) of rats with G6PD deficiency and bacterium dysenteriae infection(G6PD-BD) by method of intragastric administration of coptis root's different compatibility of Gegen Qinlian Decoction,and to investigate the effect of compatibility on reducing the EOF of coptis root,Compared the efficacy of the each combined group,through changes in the state of rat feces.By observing the biopsy results of Gegenqinlian Decoction and Coptis for rat model of liver,study the safety of Gegenqinlian decoction.Using HPLC-MS" and mass spectrometry to analyse content changes of main ingredients of puerarin,berberine and Gegenqinlian decoction in Beagle,to explore the drug in vivo pharmacokinetics of the law and elucidate the principle of the effectiveness enhanced and the toxicity reduced.Methods:1.Establish animal model:First infect rat by dysenteriae,and establish experimental rat model of G6PD-BD defected on the second day2.Occult blood test:Give the animal model the split group decoction containing Gegenqinlian by intragastric administration twice a day for three days.Then to observe the state of rat feces every day.The fourth day take abdominal aortic blood and measure starting Hemolyticcon centration.3.Pathology experiment:Give normal and G6PD-BD rats with high,medium and low doses of berberine and Gegenqinlian decoction twice a day for three days.The fourth day take their liver and kidney postanesthesia,then make biopsy to Observe the results of pathological biopsys.4.Pharmacokinetic experiment:Using WinNonlin pharmacoinetic software to establish suitable HPLC- MSⁿ conditions for puerarin and berberine to study the major components of serum at different times.Take the Chinese Medical Herbs according to prescription Gegenqinlian decoction(Puerarin 24g,Baicalin 9g,Coptis 9g,licorice 6g),soak for 15min by adding water 2400ml.At first decoct Puerarin for 20min,and then decoct the remaining drugs twice.Filter them by 120 mesh while they are hot and combined.Administration to different Puerarin,Huanglian Gegenqinlian decoction and take the femoral vein blood at different time points.Then centrifuge the blood sample for 20min at the 3000r/min rotation speed.Shift the supematant,then precipite impurity with acetonitrile.Shocks on the vortex mixer for 5min,centrifuge the sample for 20min at the 3000r/min rotation speed. Obtained supernatant dry the solvent by nitrogen.Dissolved with methanol and metered the volume,0.22μm membrane filter,getthe sample solutions.Analyze by HPLC-MSⁿ and high performance liquid instruments.Use the column of Zorbax Eclipse XDB-C18(4.6×100mm, 1.8um),Take 0.02mol/L ammonium acetate as solution A and acetonitrile as solution B.Adopt the solutions as mobile phase gradient elution to detect the content of the main components of puerarin.At the same time,take 0.2%triethylamine as solution A and acetonitrile as solution B. Adopt the solutions as mobile phase gradient elution to detect the content of the main components of huanglian.Results:1.G6PD-BD rats with body cooler,melena,air shaft.2.Occult blood test shows that Gegenqinlian decoction of the whole prescription and Berberine, baicalin,puerarin group Osmotic fragility of red blood cells can return to normal levels.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian3.Give G6PD-BD and normal rats with high,medium and low doses of Single Coptis Decoction and Gegenqinlian Huanglian Decoction,compared to the results of liver biopsy of the kidney.After administration,compared renal hyperaemia of the model group:High dose group of Huanglian is higher than medium and low doses,and the latter two is equivalent.As the same for Gegenqinlian decoction.Normal group comparison of renal hyperemia after administration:The degree of congestive is reduced successively.Comparison of liver congestive of model group and normal group:High and medium dose groups are serious than the low dose group of Huamglian.However the first two are equivalent.Compared edema of the liver of Huanglian after administration for model group.The degree of edema of the liver is reduced successively.For Gegenqinlian decoction,High dose group is serious than medium and low doses,and the latter two is equivalent.Compared edema of the liver of Huanglian after administration for normal group,The degree of edema of the liver is reduced successively for high,medium and low doses.However,for For Gegenqinlian decoction,it is reduced successively for medium,low and high doses.4.Using WinNonlin phannacokinetic software to process the results of LC-MSⁿ(1) Compared the change of phmarrnacokinetic parameters of Gegenqinlian decoction and singal Puerarin.â‘ The change of main components of Radix Puerariae:Gegenqinlian decoction contrast to single Radix Puerariae.â‘ Puerarin:The AUC of the former is bigger than the latter, CL is higher,t_1/2 is slower,Tmax is slower and Cmax is higher than the latter.â‘¡Daidzein:The AUC of the fonner is bigger than the latter,CL is lower,t_1/2 is faster,Tmax is slower and peak Cmax is higher than the latter.â‘¢lsoflavoues Aglycone:The AUC of the former is bigger than the latter,CL is lower,t_1/2 is faster,Tmax is same and Cmax is higher than the latter.There is not standard reference substance(material A)in Ggenqinlian decoction and Puerarin,but there is blood metabolites(material B)â‘£Material A:The AUC of the former is bigger than the latter,CL is faster,t_1/2 is faster,Tmax is faster and Cmax is higher than the latter.⑤Material B:The AUC of the former is smaller than the latter,CL rate is slower,t_1/2 is faster, Tmax is faster and Cmax is higher than the latter.(2) Compared the change of phmarmacokinetic parameters of Gegenqinlian decoction and singal Coptis.â‘ Berberine Hydrochloride:The AUC of the foriner is smaller than the latter,CL is faster,t_1/2 is slower,Tmax is faster and Cmax is slower than the latter.â‘¡Palmatine:The AUC of the fonner is smaller than the latter,CL is higher,t_1/2 is faster,Tmax is slower and Cmax is lower than the latter.â‘¢Coptisine:TheAUC of the former is smaller than the latter,CL rate is higher,t_1/2 is slower,Tmax is faster and Cmax is lower than the latter.Conclusions: 1.The effect of EOF were lower when G6PD-BD rats ate CR plus KR and PRG,CR plus KR,CR plus BSR and PRG,CR plus BSR or CR plus PRG than ate CR.The GegenQinlian Decoction or three traditional Chinese medicine including CR,KR and BSR can relive the effect of CR on EOF.Based on the above descriptions that Gegenqinlian decoction can reduce EOFtoxicity after combination.The change of the stool state intially indicated that the effective of Gegenqinlian decoction,Huanglian Puerarin licorice,Huanglian Puerarin combined groups than a single Huanglian.2.Under normal conditions of the body and administration the Huanglian Gegenqinlian decoction produce the side effects of kidney and liver than under the pathological state,from the impact of the study about the kidneys and liver show that in the case of treatment of disease Huanglian gavage or Gegenqinlian decoction to the liver and kidney is safe.The results of iver and kidney slices suggest that the toxicity of single is higher than the whole Gegenqinlian decoction.The high dose of single Huanglian and Gegenqinlian decoction have certain side effects for the liver and the side effects for Kidney is lower.3.Measure the four components of Gegenqinlian decoction and single Radix Puerariae.The AUC of the AUC of the former is bigger than the latter and peak concentration is higher than the latter.It showed that Baicalin,Huanglian and licorice can increase the main ingredients of Puerarin into the blood and serum containing an effective concentration.Meanwhile,Highlighted the drug-jun status of Puerarin in Gegenqinlian decoction.The elimination half-life,the clearance rate and peak time of the four components of Gegenqinlian Decoction and single Puerarin is different.It showed that absorption and metabolism of Puerarin in the body is diverse.The AUCs of berberine,jatrorrhizine and tetrahydropahnatine of the whole Gegenqinlian decoction are smaller than the single herbs.Indicate that Huanglian after combination can reduce the entry of these three components into blood.CL become high Descript that the metabolism of Huanglian step up after compatibility.Cmax of the whole decoction is higher than single component of berberine,jatrorrhizine and tetrahydropalmatine.It shows the whole decoction can restrain the absorption of Huanglian to blood.Coptiscan produce a marked effect in intestinal tract. Tmax and t_1/2 of the three components are different in the single and whole decoction.It Prompts that study the role of traditional Chinese medicine having the complexity and diversity in vivo once again.In the total,the results of the occult blood of experimental rats and pathology described that the part toxicity of berberine and berberine can be reduced after combining.Through the experiment of Beagle Pharmacokinetics to explanate the reasons for reducing the toxicity of Huanglian.The results of pharmacokinetic of the main components of puerarin and -wide side showed the puerarin can increase the absorption of the major components,whatever support the combined law of puerarin as King drug.