The Content Determination Of Raw And Fried Cassia Seeds And The Comparative Study Of Their Liver-protecting Effects

Purpose:1.To establish an HPLC method for determination ofsix aglycones?aurantio-obtusin,chryso-obtusin,obtusin,emodin,chrysophanol,physcion?in the processed products of Cassiae Semen.2.To establish an HPLC method for determination of glucose,galactose,xylose,mannose,arabinose,glucuronic acid and fucose in Cassiae Semen,and to make a comparison between the Raw and Fried CassiaeSemen.3.To investigate the protective effects of Raw and Fried Cassiae Semen on immunological liver injury induced by concanavalin A?Con A?.4.To study the protective effect of Raw and Fried Cassiae Semen on acute liver injury induced by Acetaminophen?APAP?in mice.5.To explore the bioactive constituents of Cassiae Semen for providing evidence for serum pharmacochemistry research.Material and method:1.The samples wereseparated on an Agilent ZORBAX SB-C18?4.6 mm×250 mm,5?m?column at a flow rate of1.0 mL/min and the column temperature was at room temperature.The mobile phase?was acetonitrile-0.1%phosphoric acid aqueous for determination of aglycones with gradient elution,and the detection wavelength was 284nm.2.Chromatographic conditions concluded Kromasil C₁₈?150 mm×4.6 mm,5?m?column at the flow rate of 1.0 mL/min.The detection wavelength was set at 245 nm.The mobile phase?was acetonitrile-0.1%phosphoric acid in gradient elution mode for determination of glucose,galactose,xylose;The mobile phase?was acetonitrilewater in gradient elution mo-de for determination of mannose,arabinose,glucuronic acid and fucose.3.Immunological liver injury in mice was induced by Con A?20 mg/kg?by injection via tail vein.Ninety mice were randomly divided into 9 groups:normal control group,model group,biphenyl group,Raw Cassiae Semen high does group,Raw Cassiae Semen middle dose group and Raw Cassiae Semen low dose group,Fried Cassiae Semen high does group,Fried Cassiae Semen middle dose group and Fried Cassiae Semen low dose group.Each group was treated with oral administration once a day for successively 7 days.Normal control group and model group were given the same amount of normal saline.In the 1 hour after the last administration,in the treatment groups,mice were injected with Con A at the dose of 20 mg/kg.The control group was given equal volume of saline.The contents of alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,malondialdehyde?MDA?and superoxide dismutase?SOD?in mice serum were measured.Pathological changes of liver and kidney were observed by HE staining.4.Drug-induced liver injury in mice was induced by APAP?400mg/kg?by intraperitoneal injection.Ninety mice were randomly divided into 9 groups:normal control group,model group,biphenyl group,Raw Cassiae Semen high does group,Raw Cassiae Semen middle dose group and Raw Cassiae Semen low dose group,Fried Cassiae Semen high does group,Fried Cassiae Semen middle dose group and Fried Cassiae Semen low dose group.Each group was treated with oral administration once a day for successively 7 days.Normal control group and model group were given the same amount of normal saline.In the 1 hour after the last administration,In the treatment groups,mice were injected with APAP at the dose of 400 mg/kg.The control group was given equal volume of saline.The contents of alanine aminotransferase?ALT?and aspartate aminotransferase?AST?in mice serum were measured.The content ofGlutathione peroxidase?GSH-Px?and superoxide dismutase?SOD?in liver homogenate were detected.5.Take rats as research object,prepare medicated serum by the way of serum pharmacology of traditional Chinese medicine and establish the chromatogram of serum pharmaeoehemical ingredients by HPLC.The blood migrating constituents of Cassiae Semen were determined by comparing the HPLC fingerprints of the crude and processed Semen Cassiae extract,containing serum and blank serum.Results:1.aurantio-obtusin,chryso-obtusin,obtusin,emodin,chrysophanol,phy-scion had good linearity in the ranges of 0.01884-0.1884?0.05760-0.5760?0.01206-0.1206?0.02482-0.2482?0.02112-0.2112?g,respectively.The average recoveries of the nine constituents were 97.1%-99.1%and RSDs were 0.9%-2.6%.The contents of six constituents in Cassiae Semen had significant differences.2.Glucose,galactose,xylose,mannose,arabinose,glucuronic acid and fucose had good linearity in the ranges of 0.0288-1.15?r=0.9998??0.0220-0.882?r=0.9997??0.0258-1.03?r=0.9997??0.0514-2.05?r=0.9992??0.0106-0.426?r=0.9996??0.0314-1.26?r=0.9995??0.0478-1.19?r=0.9994?,respectively.The average recoveries of the seven constituents were 98.1%-99.3%and RSDs were 0.9%-1.7%.3.Raw and Fried Cassiae Semen remarkably decreased the contents ALT,AST levels in serum,and MDA in liver homogenately,increased the levels of SOD in liver homogenately?P<0.05?.4.The administration of APAP to mice caused acute liver injury,as evidenced by significantly increased ALT and AST levels.Compared with the APAP-treated mice,Raw and Fried Cassiae Semen showed a remarkably decreasing trend of ALT,AST,as well as the reduced liver injury.Besides,the GSH-Px and SOD were significantly decreased in APAP-treated group,these indexes of the Raw and Fried Cassiae Semen-treated group were increased?P<0.05?.5.Twenty-one compounds absorbed in blood were detected,including four endogenous substances in the blank serum of rats,eight prototype constituents of Cassiae Semen and nine metabolites.Conclusion:1.The method provides reference for comprehen-sive quality control of Cassiae Semen,andthe contents of six aglycones in Fried Cassiae Semen decreased.2.The contents of seven monosaccharides in Fried Cassiae Semen decreased.3.Raw and Fried Cassiae Semen can obviously protect immunological injury liver in mice.4.Raw and Fried Cassiae Semen can obviously protect acute liver injury induced by Acetaminophen?APAP?in mice.5.These seventeen constituents in blood could be bioactive components of Cassiae Semen,further studies on which could be useful to clarify the bioactive constituents and mechanisms of action of Cassiae Semen.