Preliminary Study On Screening Methods Of Copper Excretion By Traditional Chinese Medicine

Copper is one of the most abundant critical trace elements in the human body,only less than iron and zinc.It plays a fundamental role in a variety of important physiological processes.For example,copper is the active ingredient of many enzymes in the body and it can influence the process of metabolism of the body;Copper participates in the process of hematopoiesis indirectly by affecting the metabolism of iron;Copper plays an important role in bone formation and tissue formation,as well as vascular health.In the body,the content of copper must be in a state of equilibrium.Once the balance is broken,so that the copper ion content is too much or too little,it will induce a series of pathological changes.When the body is in copper deficiency state,it will cause tissue hypoxia,endocrine disorders,developmental delay,low immunity and so on.On the contrary,when the body is in copper excess,it will also cause serious diseases,such as Alzheimer’s,Wilson’s,Parkinson’s,Prion and Menkes disease.Wilson’s disease(WD)is also known as Hepatolenticular degeneration(HLD),it is an autosomal recessive inherited disorder of copper metabolism caused by mutation of the ATP7B gene,which leads to copper accumulation,especially in the liver,brain,and cornea.The main clinical features of WD are hepatic,neurological,or psychiatric.If it is not treated in time,it will lead to disability and even death.Preliminary evidence from clinical studies suggests that traditional Chinese medicine(TCM)showed significant benefits for some patients with HLD.There are some prescription of TCM for the efficacy of HLD.However,the mechanism and material basis of the treatment are still unknown.It was speculated that some chelating agent might exist in the TCM decoctions that binds Cu2+ directly in blood and tissues and facilitates its excretion.To ascertain this,we detected the amount of free copper ions in the decoction first,and then measured the recovery of added copper ions.We proposed that recovery of added copper to TCM decoctions could indicate the chelating ability of constituents in the decoctions and the recovery is negatively correlated with the chelating ability.Thus,a screening method of TCM for copper excretion was established,and therefore the mechanism of TCM treatment of HLD was explored.This paper is based on the Cu2+ can catalyze the spiro structure of the o-vanillin fluorescein hydrazone(Chemosensor 1)by changing color from colorless to orange-red in aqueous solution at pH 7.5(10mM Tris-HCl buffer)to determine the content of free copper ion.The results show that the concentration of Cu2+ in the range of 2-24μM was consistent with Beer-Lambert law,R2 = 0.9990,the detection limit is 11.4nM,and the chemosensor 1 exhibits high selectivity and specificity toward Cu2+ over competing relevant metal ions in aqueous solution.Furthermore,Cu2+ with Chemosensor 1 through a 1:1 binding mode,and this change is reversible by the addition of EDTA.Moreover,We further used this method to screen the chelating ability of fifty-one kinds of TCM compounds for treating WD.It is found that the decoctions of Chinese Gall,Cortex Granati,Flos Rosae Rugosae,Flos Rosae Sinensis,Syzygium Aromaticum and Radix Scutellariae showed strong Cu2+ chelating ability.Therefore,these herbs should be key drugs for the treatment of HLD.We explored the specific complex components of this six kinds of TCM and found that tannin,two adjacent hydroxy benzoic acid and baicalin may be the key ingredients of six TCM,which can complex with Cu2+.