The Research Of The Mechanism Of Hepatoprotective Effect And Pharmacokinetics Of Total Flavonoids From Scorzonera Austriaca Willd

Scorzonera austriaca Wild,also called black salsify,soil ginseng,and lycopene,as a traditional herbal medicine which belongs to Compositae,is naturally distributed in the northwest and northeast regions in China,and widely used for curing fever,carbuncle,detumescence and mastitis.So far,many bioactive compounds have been isolated and identified from S.austriaca including flavonoids,sesquiterpenes,triterpenes and coumarin.Among these compounds,flavonoids from S.austriaca（FSA）possesses various pharmacological activities,such as anti-bacterial,antioxidant,anti-inflammatory and anti-tumor.Our previous study had preliminarily determined the hepatoprotective effect of S.austriaca.However,the content of total flavonoids in the crude FSA is low,and the underlying mechanisms of hepatoprotective effect and distribution in vivo remain unclear,which need a further illumination.In addition,the relevant information of pharmacokinetics and tissue distribution of FSA has not been repotred.Therefore,the paper aims to determine the main composition and obtain the flavonoids compounds of FSA through the extraction and purification assays,and our paper mianly investigates the hepatoprotective effects of FSA and pharmacokinetics and tissue distribution in rats.The main researches are showing as follows:1.Extraction and purification of FSA and preparation of flavonoid compounds Objective:The purpose is to extract and purify the refined total FSA and prepare flavonoid compounds.Methods:The herbs of S.austriaca were extracted with ethanol solution at room temperature and then the extracts were purified through D101and D4020 column chromatographies to yield refined flavonoids.Normal and reversed phase silica gel column chromatographies and semi-preparative HPLC were used for preparing flavonoid compounds.Results:D4020 macroporous resin showed a good absorption and resolution to total FSA.The total flavonoids were obtained with high purity and reached at 92.5%.Six compounds were isolated from the total flavonoids of S.austriaca in this chapter and their structures were identified as follows:1:Isoorientin2:Orientin3:2′′-O-β-D-xylopyranosyl isoorientin4:2′′-O-β-D-xylopyranosyl isovitexin5:6-C-L-α-arabipyranosyl vitexin6:Vitexin2.The mechanism of hepatorotective effects of FSA on CCl₄-induced acute liver injury was explored in vivo and in vitroObjective:ICR mice were injected with 0.15%CCl₄ olive oil solution by intraperitoneal to induce the acute liver injury model,meanwhile,the mice were also administrated with solution of total FSA at the dosage of 60（low dose group）,100（medium dose group）,and 140 mg/kg（high dose group）,respectively.After the mice were sacrificed,the changes of enzymes,biochemical indexes and other specific proteins expression in mice were determined,and the protective effect of the total flavonoids on the liver injury induced by CCl₄ was investigated.Furthermore,the in vivo results were also confirmed by the L02 cell in vitro assay.Methods:A total of mice were randomLy divided into six groups（n=10 per group）.The mice from the normal control（NC）group and the model control（MC）group were administrated with 0.5%sodium carboxymethyl cellulose aqueous solution（SCCAS）for a week.The mice in FSA-treated group were orally administered with total flavonoids.After the oral administration（2 h later）,all the groups except the normal group were treated with 0.15%CCl₄（mixture with olive oil,V/V）by intraperitoneal injection at the dosage of 10 mL/kg,and the normal control group was treated with olive oil at the dosage of 10 mL/kg.Sixteen hours after the injection of CCl₄,blood samples were collected by eyeball extirpating and then the mice were sacrificed to collect the liver.Serum was obtained by centrifugation at 5000 rpm for 10 min,4℃,and used for detecting the levels of AST,ALT and LDH.The livers were removed quickly and dissected,and used for detecting the biochemical enzymes including SOD,MDA and GSH,histopathological studies（fixed in 10%formalin solution）and western-blot assays of in vivo and in vitro assays.Results:（1）Detection of biochemical indexes of serum in mice:Compared with normal group,the activities of ALT,AST and LDH were significantly higher in the model group,indicating the model was established successfully.And the activities of ALT,AST and LDH in FSA-treated groups obviously decreased（p<0.05）in a dose dependent manner;（2）Analysis of enzymes in liver tissue of mice:Compared with NC group,the content of SOD and GSH in the model group decreased significantly,and the content of MDA increased（p<0.05）,indicating that CCl₄ induced lipid peroxidation and oxidative stress in the MC group,which also represented that model was successful and effective.And activities of SOD and GSH in serum in the FSA-treated group increased remarkably,MDA significantly decreased and showed a dose dependent manner;（2）Changes in the liver tissue structures of the mice:The liver tissue organization of mice in control group was normal compared with MC group.The hepatic cords structure disappeared,and hepatocyte vacuolation degeneration,acidophilic degeneration,nucleus pycnosis,cataclastic and even necrosis appeared in the MC group,which indicated that the liver was seriously injured.However,compared with MC group,the liver tissue of FSA-treated groups ameliorated significantly.Hepatic lobule structure is basically complete and only part of the liver cells appeared vacuoles degeneration in the FSA-treated groups;（4）Liver-specific protein of mice and L02 cells:Compared with NC group,the expression of inflammatory protein including TLR4,TNF-α,P-P65,IL-6,IL-6 and autophagy protein LC3 were increased and P62 protein expression was decreased in the MC group.Whereas,FSA-treated groups were down-regulated on the expression levels of those of inflammatory protein in vitro and in vivo.Moreover,after incubation with autophagy inhibitor,the cell viability was decreased and the protective effect was reduced,which indicated that FSA-treated groups could alleviate the liver injury induced by CCl₄ through activating the autophagy pathways,thereby leading a well amelioration for liver tissue.Conclusion:FSA might serve as a potential material for drug development against chemical hepatic injury.3.Study on the pharmacokinetic and histological distribution of total flavonoids of S.austriacaObjective:LC-MS/MS,a rapid and sensitive method,was established to quantify six flavonoids in the plasma and tissues of rats and was validated for the study through pharmacokinetics and tissue distribution by oral administration and intravenous injection in vivo.Methods:Pharmacokinetics study of oral administration:A total of 15 Wistar rats were randomLy divided into 3 groups（n=5 per group）and the rats were oral-administrated with total flavonoids at doses of 42（low dose group）,70（medium dose group）,and98 mg/kg（high dose group）,respectively.0.25 mL of blood samples were collected from retinal vein into the heparin centrifuge tube before and 5 min,10 min,15 min,30 min,45 min,1 h,1.5 h,2 h,3 h,5 h,7 h,9 h,12 h and 24 h after administration,respectively,and then centrifugated at 1520×g（centrifugal force）for 10 min.The serum was taken and stored at-20℃to analyse.50μL of serum sample was disposed by precipitation protein method,and then the supernatant was transfered into the 1.5mL tube and blowed to dry by nitrogen at 45℃.All the residues were dissolved in the80μL mobile phase（acetonitrile-0.1%formic acid,20:80,v/v）,and analyzed by LC-MS/MS with 10μL injection.Pharmacokinetics study of intravenous injection:A total of 15 Wistar rats were randomLy divided into 3 groups（n=5 per group）and the rats were injected with total flavonoids through oral administration at dosages of 4（low dose group）,8（medium dose group）,and 16 mg/kg（high dose group）,respectively.And then the specific methods were according to the“Pharmacokinetics study of oral administration assay”.Tissue distribution study of oral administration:A total of 30 Wistar rats were randomLy divided into 6 groups（n=5 per group）and allowed free access to water,whereas the rats were fasted for 12 h before the experiment.After oral-administrated for 4 h,the rats were free to access the food.The rats were injected with total flavonoids at dosages of 70 mg/kg.0.25 mL of blood samples were collected from retinal vein into the heparin centrifuge tube after 5 min,30 min,1 h,3h,5 h and 7 h,the rats were sacrificed for anatomising to collect the heart,liver,spleen,lung,kidney,stomach and brain tissue into the normal saline and weight them.All the tissues were homogenized with saline（g:mL=l:10）,and the homogenized tissues were stored at-20oC.Using the same method as the above,all the homogenized tissues were analysed by LC-MS/MS.Tissue distribution study of intravenous injection:A total of 30 Wistar rats were randomLy divided into 6 groups（n=5 per group）and allowed free access to water,whereas the rats were fasted for 12 h before the experiment.After the injection for 4 h,the rats were free to access the food.The rats were injected with total flavonoids through tail vein at doses of 8 mg/kg.And then the specific methods were according to the“Tissue distribution study of oral administration”assay.Data analysis:The pharmacokinetic parameters were obtained by DAS2.1software which including area under the drug-curve(AUC_0-t)and(AUC_0-∞),elimination half-life（t_?）,mean retention time（MRT）,maximum blood concentration Cmax,etc.All the concentration and time of plasma of rats were analyzed and expressed as mean±SD.And the texture distribution histogram was denoted using software and expressed as mean±SD.Result:The pharmacokinetics and tissue distribution of six compounds of total flavonoids were denoted using LC-MS/MS method established by us.Although the low dose groups of compound 2 and compound 6 were not detected,which mostly probably owes to the low bioavailability,the results of pharmacokinetics is coincident with in vivo assays.The C_maxax and AUC_0-t-t of rest compounds showed reliable linear correlation.Tissue distribution studies also indicated that the compounds were rapidly and widely distributed in the most tissues which including kidney,lung,stomach,liver and other blood-filled tissues,and no accumulation in them.Furthermore,the brain with the lowest concentration of compounds indicated that flavonoid glycosides were difficult to penetrate the blood-brain barrier of rats.Meanwhile,it was noteworthy that the compound 4 had a higher content in the liver no matter oral administration or intravenous injection.Therefore,combined with the experimental results in vivo,these results also provided the experimental basis and theoretical basis of monomer compounds,and used for the further investigated.Conclusion:LC-MS/MS as a method established in this experiment,could be applied for researching the pharmacokinetics and tissue distribution of total flavonoids of S.austriaca.