Study On Extraction Of Pharmacodynamic Components Of Bupleurum And Its Antioxidant And Liver Protective Effect

Background:Chinese traditional medicine Bupleurum is the dry root of Bupleurum Chinennse Dc.Or Bupleurum scorzonerifolium Willd.It has the functions of evacuating fever,relieving depression of liver and lifting Yang qi.In recent years,the compound preparations of Chaihu Shugan San,Xiaochaihu Tang and Chaihu Lizhong Tang,which take Bupleurum as the main medicine,have been widely used in the prevention and treatment of liver diseases with remarkable curative effects.In terms of liver diseases,non-alcoholic fatty liver disease（NAFLD）is one of the most common chronic liver diseases in the world,and its pathogenesis is complex.Based on the imbalance between Yin and Yang of the liver,kidney and spleen,NAFLD is divided into Stagnation of Qi due to depression of the liver,deficiency of liver and kidney Yin,liver Yang phlegm and fire and et al.Modern medicine believes that oxidative stress（OS）is one of the factors affecting the occurrence and development of NAFLD,and the imbalance between the oxidative and antioxidant systems of the body is similar to the imbalance between Yin and Yang in traditional Chinese medicine.With the continuation of OS,liver damage becomes more serious,leading to the development of liver cirrhosis and even liver cancer.At present,the therapeutic target of western medicine is single,and there is no specific drug for the treatment of NAFLD,while traditional Chinese medicine has the characteristics of multi-component,multi-pathway and multi-target.Bupleurum-related compound preparations are widely used in the clinical treatment of chronic liver disease and improvement of abnormal liver function.However,the material basis and mechanism of the liver-protecting effect of Bupleurum-related compound preparations are still unclear.In this study,different polarity parts and main active components of Bupleurum were extracted,and their free radical scavenging ability,iron reducing power and protective effect on oxidative damage of liver cells ere compared to evaluate the role of Bupleurum in protecting liver based on antioxidant.Exploring the effects of saikosaponin d（SSd）on Nuclear factor Erythroid2-related factor 2（Nrf2）and Heme oxygenase 1（HO-1）signaling molecules,to lay a theoretical foundation for the comprehensive utilization and preparation development of Bupleurum.Objective:Exploring the effect of Bupleurum on liver protection based on antioxidant and the effect of active ingredient（SSd）on Nrf2 and HO-1 signal molecules,to lay a theoretical foundation for rational utilization of Bupleurum resources and preparation research and development,quality control and clinical research.Methods:1.Study on extraction of different polar parts of Bupleurum and antioxidant activity in vitro.The water parts of Bupleurum were extracted by reflux of distilled water,and the different polar parts of Bupleurum were extracted by 70%（v/v）ethanol reflux extraction and extraction methods（ethanolic,petroleum ether,ethyl acetate,and n-butanol extracts）.The antioxidant capacities of different polarity parts of Bupleurum were determined by salicylic acid,DPPH,ABTS and Prussian blue methods.Cell oxidative damage model was established,and the cell survival rate,antioxidant enzyme activity and malondialdehyde of LO2 cells with differentpolarity parts was measured,to investigate the protective effects of polarity parts on oxidative damage of LO2 cells.2.Study on extraction of main active constituents of Bupleurum and antioxidant activity in vitro.Polysaccharides,flavonoids and volatile oil of Bupleurum were extracted by water extraction and alcohol precipitation,alkali dissolution and acid precipitation and steam distillation,and their contents were determined by phenol sulfuric acid method and ultraviolet spectrophotometry.The free radical scavenging ability of Bupleurum polysaccharides,flavonoids,volatile oil and saikosaponins was etermined by DPPH method and ABTS method,and the antioxidant ability of Bupleurum active components was compared in vitro.The protective effects of Bupleurum polysaccharide,flavone and saikosaponins on H₂O₂-induced oxidative damage of LO2 cells were investigated by measuring cell survival rate,antioxidant enzyme activity and malondialdehyde.3.The protective effect of SSd on H₂O₂-induced oxidative damage of LO2 cells and its potential mechanism were explored by measuring cell survival rate,liver cell damage index,changes in levels of malondialdehyde and reactive oxygen species,and expression levels of Nrf2 and HO-1 signaling molecules.Results:1.Study on extraction of different polar parts of Bupleurum and antioxidant activity in vitro.Five polar fractions of Bupleurum were extracted from 100 g of Bupleurum decoction pieces to obtain 11.36 g of water fraction,14.01 g of ethanol fraction,1.86 g of n-butanol fraction,1.74 g of ethyl acetate fraction and 1.48 g of petroleum ether fraction.The results of in vitro antioxidant activity of different polar parts of Bupleurum showed that all the five polarity parts of Bupleurum had good antioxidant activity.Among the five polarity parts of Bupleurum,the ethyl acetate fraction had the strongest antioxidant activity,the IC₅₀of hydroxyl radical,DPPH radical and ABTS radical were 0.177 mg/mL,0.171 mg/mL and 0.095 mg/mL,respectively,and the iron reducing ability A_0.5was 0.491 mg/mL.The order of comprehensive antioxidant activity of different polar fractions of Bupleurum in different in vitro was as follows:ethyl acetate fraction,water fraction,ethanol fraction,n-butanol fraction,petroleum ether fraction,The experimental results showed that after treated with 800μmol/L H₂O₂reagent for 24 h,the survival rate of LO2 cells decreased significantly to（51.09±2.21）%（P<0.001）.After pretreatment with different polarity arts of Bupleurum,the ethyl acetate fraction（24μg/mL）had the most significant protective effect,the cell survival rate increased to 70.63%,the activities of GSH-Px and SOD were significantly increased to 15.44 U/mg and 49.43 U/mg（P<0.05）,and the content of MDA was significantly decreased to 1.85μmol/g（P<0.05）,the protective effect of petroleum ether extract（24μg/mL）on oxidative damage of LO2 cells induced by H₂O₂was not obvious.2.Study on extraction of main active constituents of Bupleurum and antioxidant activity in vitro.The concentration of Bupleurum polysaccharide was 5.78 mg/mL,the proportion of flavonoids was 61.7%,the volatile oil was 1.8938 g/mL,saikosaponins were purchased products,and the purity was 70%.The antioxidant activities of the main active components of Bupleurum in vitro showed that the scavenging ability of Bupleurum polysaccharide,flavonoids and saikosaponins was significantly better than that of Bupleurum volatile oil.In the experiment of protecting LO2 cells from oxidative damage,Bupleurum polysaccharide（6.936μg/mL）,Bupleurum flavone（32μg/mL）and saikosaponins（60μg/mL）groups could increase the survival rate of LO2 cells to63.92%,60.85%and 67.47%,and the differences were statistically significant（P<0.05）,and the activities of GSH-Px and SOD were significantly increased to 15.81,17.10,17.06 U/mg and 46.79,47.11,52.05 U/mg（P<0.05）,and the content of MDA was significantly decreased to 2.34,2.28 and 2.11μmol/g（P<0.05）.3.Protective effect of SSd on H₂O₂-induced oxidative damage in LO2 cells.H₂O₂（800μmol/L）significantly decreased cell survival to（51.09±2.21）%（vs control,P<0.05）.SSd（1 and 2μmol/L）reversed H₂O₂-induced cell injury and increased cell survival to（61.92±1.65）%and（64.99±2.58）%（vs H₂O₂,P<0.05）.H₂O₂increased the ALT,AST,LDH,and MDA levels to 11.70 U/L,33.70 U/L,570.75 U/L,and 3.97μmol/g（vs control,P<0.05）,SSd（2μmol/L）decreased the value of these indicators to7.27 U/L,19.04 U/L,478.39 U/L,and 1.75μmol/g（vs H₂O₂,P<0.05）.H₂O₂significantly increased ROS production（vs control,P<0.05）,while SSd（2μmol/L）inhibited H₂O₂-induced ROS production（vs H₂O₂,P<0.05）.According to the immunofluorescence assay,SSd promoted the nuclear transmigration of Nrf2,was similar to tBHQ group.According to western blotting,SSd（2μmol/L）protected H₂O₂-induced LO2 cell injury and increase the expressions of Nrf2 and HO-1（vs H₂O₂,P<0.05）.Conclusion:1.The extraction and content determination methods of different polarity parts and main active components of Bupleurum were simple,stable,sensitive and reproducible.2.The antioxidative and hepatoprotective effects of Bupleurum may be related to its main pharmacodynamic active substance groups,such as saponins,polysaccharides and flavonoids.3.SSd can effectively protect LO2 cell injury induced by H₂O₂treatment,which may be related to the increased expression of Nrf2 and HO-1 signal molecules.