Pharmacokinetics Of Schisandra Lignans And Preliminary Pharmacodynamic Study Of Schisandrin

Objective:（1）To establish and verify a quantitative method for the determination of lignans in biological samples;（2）To study the pharmacokinetic profiles of lignans in rat plasma after intragastric administration of Schisandra chinensis extract;（3）To study the pharmacokinetics of lignans in rat plasma after intragastric administration of Wuweizi syrup;（4）To study the absolute bioavailability and tissue distribution of schisandrol B in rats;（5）Preliminary study on the protective effect of schisandrin on isoproterenol-induced myocardial injury in rats.Methods:1.Pharmacokinetic study（1）A HPLC-MS/MS method was developed to simultaneously determine the content of seven lignan components（schisandrin,schisandrol B,schisandrin A,schisandrin B,schisandrin C,schisanhenol,and schisantherin A）in biological samples.The chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18（4.6 mm×150 mm,5μm）column with the mobile phase consisting of methanol（containing 0.1%formic acid）-water（containing 0.1%formic acid,5 mmol ammonium acetate）at a flow rate of 0.5 mL/min,the column temperature was 30℃and the injection volume was 10μL.An electrospray positive ion multiple reaction monitoring was used as a detector.（2）Six rats were intragastrically administrated with 0.95 g/kg Schisandra chinensis extract,and about 0.25 mL of blood samples were collected from the fundus vein at0.25,0.5,1,2,4,6,8,12,24,36 h after administration.The plasma drug concentration was measured by HPLC-MS/MS,and the pharmacokinetic parameters of seven lignans were calculated by Phoenix WinNonlin.（3）Six rats were intragastrically administrated with 12.5 ml/kg Wuweizi syrup,and about 0.25 mL of blood samples were collected from the fundus vein at 0.25,0.5,0.75,1,1.5,2,4,6,8,10,12 h after administration.The plasma drug concentration was measured by HPLC-MS/MS,and the pharmacokinetic parameters of schisandrin were calculated by Phoenix WinNonlin.（4）（1）Six rats were intragastrically administrated with 10 mg/kg schisandrol B,and about 0.25 mL of blood samples were collected from the fundus vein at 0.25,0.5,1,2,4,6,8,10,12 h after administration.The plasma drug concentration was measured by HPLC-MS/MS,and the pharmacokinetic parameters of schisandrol B were calculated by Phoenix WinNonlin.（2）Six rats were injected with 2 mg/kg of schisandrol B by tail vein,and about 0.25 mL of blood samples were collected from the fundus vein at 0.083,0.25,0.5,0.75,1,2,4,6,8,10,12 h after administration.The plasma drug concentration was measured by HPLC-MS/MS,and the pharmacokinetic parameters of schisandrol B were calculated by Phoenix WinNonlin.（3）Eighteen rats were intragastrically administered with 10 mg/kg schisandrol B and three of the rats were euthanized at 0.5,1,2,4,8 and 12 h after administration,respectively.The drug concentrations in heart,liver,spleen,lung,kidney,and brain were measured.2.Pharmacodynamic studyRats were randomly divided into four groups（n=10）:1-28 days,the control group and the model group were received normal saline（1 ml,i.g.）.Shengmaiyin group（6.25ml/kg）and schisandrin group（20 mg/kg）were received drugs by gavage once a day.15-28 days,the model group,shengmaiyin group and schisandrin group were injected subcutaneously with isoproterenol（2.5 mg/kg）to establish a rat model of myocardial injury.On the 29th day,the rats were anesthetized after weighing.Heart weight/body weight（HW/BW）,left ventricular weight/body weight（LVW/BW）and electrocardiogram（ECG）were recorded.The contents of malondialdehyde（MDA）,type B natriuretic peptide（BNP）,superoxide dismutase（SOD）,lactate dehydrogenase（LDH）,creatine kinase isoenzyme MB（CK-MB）and endothelium dependent nitric oxide synthase（eNOS）were detected according to the kit instructions.Results:1.Pharmacokinetic study（1）A sensitive and efficient HPLC-MS/MS method was developed to simultaneously determine the content of seven lignan components in rat plasma.The seven lignans had good linear relationship within the determination range（r²>0.9950）;The RSD of inter-day and intra-day were<12.08%,and the accuracy was 88.64%～111.61%.The extraction recovery was 76.36%to 92.72%,the matrix effect was 85.20%to 95.81%,and the stability was in the range of 89.93%～107.78%,which all meet the biological samples analysis requirements.（2）After intragastric administration of Schisandra chinensis extract at 0.95 g/kg in rats,all seven lignans were detected within 15 min,indicating that these components were quickly absorbed into the blood.The Cmax and AUC of schisandrin were（1078.54±179.30）ng/mL and（5775.05±1457.02）ng.h/mL,which were much higher than those of the other six lignans.（3）After intragastric administration of Wuweizi syrup,it was found that the plasma drug concentration of schisandrin was the highest among the seven lignans,while that of the other six lignans was lower than the lower limit of quantification.The Tmax,T1/2and Cmax of schisandrin were（0.25±0.00）h,（1.65±1.04）h and（8.95±1.91）ng/mL,respectively.The MRT_0-∞and AUC_0-∞were（1.52±0.55）h and（13.05±2.1）ng.h/mL,respectively.（4）After intragastric administration of 10 mg/kg schisandrol B in rats,the AUC0-∞was（396.33±32.13）ng.h/mL;after injection of 2 mg/kg schisandrol B by tail vein,the AUC_0-∞was（456.84±91.63）ng.h/mL,the absolute bioavailability of schisandrol B was about 17.35%.Tissue distribution showed that schisandrol B was distributed in the heart,liver,spleen,lung,kidney and brain,especially in the liver.2.Pharmacodynamic study（1）The HW/BW and LVW/BW of model group were significantly higher than those of normal rats（P<0.01）.Compared with the model group,the HW/BW and LVW/BW of schisandrin and shengmaiyin groups were significantly decreased（P<0.01）.（2）Compared with the control group,the ST-segment of ECG in the model group was significantly elevated（P<0.01）.Compared with the model group,schisandrin and shengmaiyin groups could effectively reduce the ST-segment elevation of ECG in the process of myocardial injury.（3）The activity of LDH and CK-MB,the content of MDA and BNP in the model group were higher than those in the control group（P<0.05）.Compared with the control group,the SOD and eNOS activities in the model group were significantly decreased（P<0.01）.Compared with the model group,the SOD and eNOS activities were increased after administration of schisandrin and shengmaiyin（P<0.05）.（4）Pathological sections showed that in the model group,coagulative necrosis of cells was observed in multiple regions,and interstitial was infiltrated with acute and chronic inflammatory cells,and fibrous tissue was formed in some regions.After the pretreatment of schisandrin and shengmaiyin,myocardial necrosis was significantly reduced,a small amount of acute and chronic inflammatory cells infiltrated into the stroma,and the proliferation area of fibrous tissue was reduced.Conclusion:（1）A HPLC-MS/MS method for the simultaneous determination of seven lignans in biological samples was established and verified,which can be used for further pharmacokinetic studies of schisandra lignans;（2）The seven lignans of Schisandra chinensis extract can be absorbed into the blood rapidly after intragastric administration,and schisandrin is superior to the other six lignans in absorption speed and degree;（3）After administration of Wuweizi syrup,only schisandrin was the highest concentration in plasma among the seven lignan components,suggesting that schisandrin may be the pharmacodynamic substance basis of Wuweizi syrup;（4）The absolute bioavailability of schisandrol B was about 17.35%in rats,and it was distributed in the heart,liver,spleen,lung,kidney and brain of rats,with the highest concentration in the liver;（5）Schisandrin has protective effect on isoproterenol-induced myocardial injury in rats.