| Objective: Hepatolenticular degeneration(HLD),an autosomal recessive genetic disorder,was characterized by impaired copper transport and excretion due to the mutation of the ATP7 B gene,which encodes a copper-binding ATPase.The resulting defects in the excretory pathway of the liver and impediments to the excretion of copper into bile lead to copper accumulate in the liver kidney,brain and other organs,causing damage to these organs to varying degrees.Gandou decoction(GDD),a classical formula of Traditional Chinese medicine(TCM),has been clinically applied to treat hepatocellular degeneration(HLD)in China for decades due to its remarkable efficacy.However,no detailed study of the quality control methods and the pharmacodynamic of GDD has been reported yet.In this study,on the one hand,we established the fingerprint and the determination of the 8 biochemical active composition of GDD by high performance liquid chromatography(HPLC),for controlling quality of GDD.On the other hand,the rat model of copper-laden hepatolenticular degeneration was established.The liver tissue was stained with hematoxylin-eosin(H&E)and the histological changes were observed to judge the pathological model.Meanwhile,the copper levels in the liver,blood,urine and feces were quantified to investigate the effect of the copper excretion of GDD.And the analysis of by following: alanine aminotransferase(ALT),aspartate aminotransferase(AST),and alkaline phosphatase(AKP)in serum;superoxide dismutase(SOD)and glutathione(GSH)as well as the total antioxidant capacity(T-AOC)in liver homogenate to investigate the effect of GDD on metabolic enzymes and lipid peroxidation injury in a rat model of copper-laden hepatocellular degeneration.Methods:Firstly,The HPLC conditions of GDD were optimized to establish GDD HPLC fingerprint,while the content of 8 representative active components in the compound GDD was determined and the related methodological study was investigated.The 8representative active components were quercetin,aloe-emodin,rhein,kaempferide,curcumin,emodin,chrysophanol and physcion,respectively.Secondly,the rat model of copper-laden was establish.To observe the histological changes and success of the model,the liver tissue was stained with hematoxylin-eosin(H&E).The copper levels in the liver,blood,urine and feces were quantified to investigate the effect of GDD on copper excretion.The serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)of rats in each group were detected by aspartate aminotransferase and aspartate aminotransferase assay kits(Reitman-Frankel method),and the serum alkaline phosphatase(AKP)of rats in each group were detected by the AKP microplate method,which were to investigate the liver protection of GDD.The activity of superoxide dismutase(SOD)in rat liver tissue was detected by the superoxide dismutase method,and the levels of glutathione(GSH)and total antioxidant capacity(T-AOC)in liver were measured by the oxidative stress method kits to explore the protective effect of GDD on a rat model of copper-laden hepatocellular degeneration.And these results were as follows.Results:(1)The related HPLC conditions of GDD were optimized and the results were as follows: An Agilent Eclipse Plus SB-C18 Column(4.6 mm×250 mm,5 μm)is employed for the separation of compounds at 30℃;the mobile phase A is water with0.1% FA and mobile phase B is CAN by a gradient elution;flow rate and sample injection volume were 1.0 m L/min and 3 μL,respectively;detection wavelength is260 nm.Meanwhile,10 batches of GDD samples(named S1-S10)were prepared to establish GDD HPLC fingerprint.30 common peaks were identified and the similarity were over 0.980.The results of methodological study were as follows: the concentration of the analytes demonstrated a satisfactory linearity(r2>0.999)with the peak area within the test ranges.The LOD and LOQ values of eight analytes ranged from 0.21 to 0.50 μg/ml and 0.70 to 1.67 μg/ml,respectively.The RSD values of precision,stability and recovery of the seven analytes were all less than 3%.The overall recoveries laid between 96.91% and 100.13% with RSD less than 2.09%.The method has good linearity,better precision,stability,recovery and reproducibility.It can be used for the quality control of GDD.(2)the rat model of copper-laden hepatolenticular degeneration was established,and three model groups were co-treated with the freeze-dried powder of GDD(0.65-2.6 g/kg)during the last 30 days.H&E staining revealed the damage of liver cells after GDD intervention was significantly less than that of model group.Compared to the model rats,GDD had remarkably reduced copper contents in the serum and liver,while treatment with GDD also resulted in a significant increase in urinary and fecal copper levels,showed that GDD could reduce the copper content in the body by promoting the excretion of copper in urine and feces.In addition,compared with the normal control group,the activity of alt,AST and AKP in the model group increased significantly(P < 0.05);the treatment of model rats with medium-dose and high-dose GDD had significantly reduced the activities of serum ALT,AST,AKP(P<0.01 or P<0.05).And the activity of SOD and levels of GSH and T-AOC were significantly increased after the treatment of model rats with medium-dose and high-dose GDD(P <0.01 or P <0.05).Conclusion: In our research,the GDD HPLC fingerprint was established successfully,which accurately showed holistic characteristics of GDD components.And this HPLC method is highly sensitive,simple,precise and stable,which can powerfully support for the quality control of GDD.GDD can significantly promote the excretion of copper in urine and feces,reduce the content of copper in blood and liver,and reduce the accumulation of copper in the body of rats with HLD.Meanwhile,GDD can effectively improve liver injury in the model through the metabolism of enzymes in liver and the role of antioxidant stress,which can provide a strong data basis on treatment of HLD patients. |
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