Saururus Chinensis Hepatoprotective Active Constituents Screening,Structural Modification And Activity Study Of Main Constituent Sauchinone

Saururus chinensis is the dried aerial parts of Saururus chinensis(Lour.)Baill.,of the family saururaceae.It has been used as a diuretic and a detoxifying agent for the treatment of hepatitis,mastitis,gonorrhea and several inflammatory diseases in China.Pharmacological studies have demonstrated that S.chinensis possess a wide range of pharmacological and biochemical activities,such as hepatoprotection,anti-inflammatory and antioxidant,etc.Quite amounts of bioactivity lignans and flavonoids have been isolated from S.chinensis.Among them sauchinone is a particular structure lignan,the index component in S.chinensis which content can be as high as 0.4%in S.chinensis.Modern research shows that it has hepatoprotection and antioxidant,etc.All these means it has a very good value for development.But research reports about sauchinone are mainly in quality standards,pharmacological activity,few in solubility,pharmacokinetics and structure modification.Therefore,investigating solubility,absorption and metabolism of sauchinone,and improving its solubility by structural modification in order to obtain compounds with better hepatoprotective effect is very meaningful.In-this paper,hepatoprotective material foundation was initially revealed by screening monomers in S.chinensis in vitro.Hepatoprotective activity of sauchinone was found by investigated in vivo.Solubility,absorption and metabolism research findings explained that it was poor solubility that inhibited the activity of sauchinone.Therefore,it is meaningful to modifize the structure of sauchinone,and then to investigating solubility,in vitro and in vivo activity of sauchinone’s derivates in order to obtain compounds with better solubility,biological activity.We hope the selected compounds will be good hepatoprotective drug candidates for clinical research.This paper includes six parts,listing as follows.Part one:Overview of relevant literatures in order to offer ideas and methods for our subject.The research profile of liver disease drugs and the main of monomers from S.chinensis,the commonly used analysis method of pharmacokinetics,and the research profile of hepatoprotective drugs were reviewed.Part two:H2O2 damaged normal human hepatocytes LO2 model combined with MTT assay was used to screen the hepatoprotective constituents.The results show that the isoquercitrin and quercitrin have a significant protective effect on H2O2 injured LO2 cells.But licarin A and dihydroguaiaretic acid show significant injury to LO2 cells.They may be the toxic constituents in S.chinensis.Sauchinone have no significant protective effect on LO2 cells due to its poor solubility.Part three:the solubility,in vivo activity and pharmacokinetics dynamics of sauchinone were studied.The equilibrium solubility in different solvents of sauchinone was determined by HPLC.The apparent lipid-water partition coefficient in n-octanol-water/buffer solution systems was determined by shaking flask method.The results show that the equilibrium solubility of sauchinone in water is 0.16 mg/L at 25℃.The solubility was lower both in weak and strong polarity solvents,while higher in moderate polarity solvents.The 1gP(P:apparent partition coefficients)in n-octanol-water system was 3.34 at 25℃,Small equilibrium solubility in water and larger apparent lipid-water partition coefficient of sauchinone may hinder its in vivo distribution and absorption.In vivo hepatoprotective activity of sauchinone in CCl4 liver injury mice was investigated through three group mice of high,medium and low dosage administered orally suachinone 7 d continuously with DDB as a positive control.The results show that sauchinone could significantly decrease ALT,AST levels and partially alleviate the pathology of the liver damage in high dose group.In pharmacokinetics dynamic study six Sprague-Dawley rats were i.v.administrated 10 mg/kg of sauchinone,respectively.Then sauchinone plasma concentration was determined by HPLC-MS using di-O-methyltetrahydrofuriguaiacin B as an internal standard.The pharmacokinetic parameters of Cmax,AUC(0-t),T1/2,CL,Vd were(1.46±0.18)mg/L,(0.66±0.15)mg·h/L,(2.81±1.08)h,(14.3±2.03)L/h and(57.2±19.7)L,respectively.The results indicate sauchinone eliminate very soon in rats,which may affect the hepatoprotective activity of sauchinone.Part four:Sauchinone extraction process,separation and purification methods were studied.The optimum extraction conditions were obtained by orthogonal test.The best condition of extraction was that 14-fold 95%alcohol was added for 2 h reflux extraction after soaking for 2 h with 10 mesh powders,and then 10-fold 95%alcohol was added again for 1 h reflux extraction.Under these conditions,sauchinone average extraction rate was 0.21%and extract transfer rate was 96%.The ethyl acetate part of ethanol extraction was Preliminary separated by silica gel column.Then Sauchonine and licarin A were obtained by multiple recrystallizated according to their solubility differences in ethanol.Part five:Three derivates of sauchinone were synthesized.Sauchinone was reduced to derivate A by NaBH4 in tetrahydrofuran-methanol alkaline systems.A was turned to derivate B by hydroxyl group acetylation in dichloromethane with DMAP as a catalyst.A was turned to derivate C under a low temperature(-20℃～-10℃)with 4A molecular sieves activated in dichloromethane-anhydrous diethyl-TMSOTf systems.Part six:The solubility,in vitro and in vivo activity and pharmacokinetics dynamics of sauchinone derivates were studied.The solubility of the derivates was studied with the same method as part three.The equilibrium solubility of derivative A,B,C in the water were 12.3,0.14,0.66 mg/L,respectively at 25℃.The solubility of the derivatives was lower both in weak and strong polarity solvents,while higher in moderate polarity solvents such as sauchinone.The lgP in n-octanol-water system were 2.62,3.28 and 2.78,respectively at 25℃.The results show that the equilibrium solubility of A in water increased more than sauchinone and apparent lipid-water partition coefficient decreased more than suachinone,which may improve the distribution of A in the body and promote its absorption in the body.The in vitro hepatoprotective activity of the derivates was studied with the same method as part two.The results show that the derivates do not reflect the obvious role on LO2 cells due to the poor solubility.The in vivo hepatoprotective activity of the derivates and pharmacokinetics dynamics of A were studied with the same method as part three.The results show that,compared with sauchinone,A could significantly decrease ALT,AST levels and alleviate the pathology of the liver damage in CCl4 liver injury mice.B has fairly hepatoprotective activity as sauchinone.C can cause more serious liver damage in mice.Among these derivates,A has a better protective role on acute experimental liver injury.Six rats were i.v.administrated 10 mg/kg of A,respectively in pharmacokinetics dynamics study.The pharmacokinetic parameters of Cmax,AUC(0-t),T1/2,CL,Vd were(6.05±0.73)mg/L,(3.92±0.18)mg·h/L,(2.43±0.38)h,(2.49±0.12)L/h and(8.69±1.12)L,respectively.The results indicate the maximum plasma concentration of A was four times than that of suachinone.The plasma clearance was much lower than sauchinone.All these may help to improve the efficacy of A.In summary,it is found sauchinone has hepatoprotective activity in vivo.But its poor solubility,quick elimination in vivo inhibits sauchinone having a better hepatoprotective effect.Therefore,through structural modification to improve solubility and activity is very meaningful.Derivate A has higher solubility,lower apparent lipid-water partition coefficient,lower plasma clearance and better hepatoprotective effect in vivo than sauchinone.It is a better compound than sauchinone in hepatoprotection.