Hepatoprotective Effects Of Gentianella Turkestanerum Extracts And Its Mechanism Research

Objective:1.To investigate the contents of secoiridoid compounds?i.e.sweroside,swertiamarin and gentiopicrin?from Gentianella turkestanerum extracts.2.To observe its acute toxicity,and provide the basis for clinical medication safety.3.The liver protection and its mechanism of each polarities were studied by in vivo and in vitro pharmacodynamics experiments.The polarities with strongest hepatoprotective activity were screened.And to explore the polar parts of the iridoid glycosides the link between the content and efficacy.Method:1.The 95%ethanol extract of Gentianella turkestanorum followed by extraction with petroleum ether,ethyl acetate and n-butyl alcohol,to obtain different polarity.2.By using methanol-0.04%phosphoric acid solution as mobile phase?at a flow rate of 1.0 mL/min?for simultaneous determination of swertiamarin,gentiopicroside and sweroside content by HPLC.3.The maximum administration dosage method,within 24 hours after intragastric administration to give the maximum concentration and volume of GE,water?GW?,butanol?GBA?,ethyl acetate?GEA?extract mixed suspension of Gentianella turkestanorum,within 14 days of observed toxicity reaction on the mice.4.CCl₄ was used to induce acute liver injury in mice.The activities of aspartate aminotransferase?AST?,alanine aminotransferase?ALT?,alkaline phosphatase?ALP?and total protein?TP?and total bilirubin?TB?were measured The levels of superoxide dismutase?SOD?,malondialdehyde?MDA?,glutathione transferase?GSH?and catalase?CAT?in liver homogenate were measured;Tumor necrosis factor-??TNF-??,interleukin-1??IL-1??and interleukin-6?IL-6?in serum of mice were measured by Elisa kit;HE staining was performed to investigate the pathological changes of liver.5.The establishment of L02 cells in vitro liver injury model with different concentrations of CCl₄ and H₂O₂ in L02 cells after 24 h cells was detected by MTT method,to determine the optimal concentration of CCl₄ and H₂O₂ model;cells were divided into seven groups:control group,0.1%DMSO group,CCl₄/H₂O₂ group?the final concentration were 20 mmol/L,100?mol/L?,silymarin?final concentration of 5?g/mL?+CCl₄/H₂O₂ group,GBA?5,10,20?g/mL?+CCl₄/H₂O₂ 3 treatment group,each group had 8 holes;Cell viability was determined by MTT assay;The activity of lactate dehydrogenase?LDH?,the activity of ALT and AST in the culture supernatant were measured by the kit.The content of MDA in the cells was measured;Annexin V/PE method to detect cell apoptosis rate;The transcriptional level of Endoplasmic reticulum stress?ERS?related genes?CHOP,PERK,IRE1 and ATF6 mRNA?was determined by RT-PCR;The expression of CHOP,Caspase12 and NF-?B protein was detected by Western blot.Results:1.Extract cream rate of GE,GPE,GEA,GBA and GW of Gentianella turkestanorum was respectively 24.4%,8.20%,4.92%,11.47%and 19.67%.The good linear relationship were found in the range of 4.04¹29.54 mg/L?r=0.9994?,3.96¹26.73 mg/L?r=0.9993?,0.93²9.70 mg/L?r=0.9995?for swertiamarin,gentiopicroside and sweroside concentrations,respectively.The content of three compounds was the highest in the GBA,less in GEA and GW,these three compounds were not detected in the GPE.Contents of three compounds showed significant difference in these three parts of Gentianella turkestanorum.2.The maximum dose for oral administration GE,GW,GBA and GEA extracts of Gentianella turkestanorum were86.88g crude drug/Kg,203.35 g crude drug/Kg,348.73 g crude drug/Kg and 325.20 g crude drug/Kg.3.G.turkestanerum extracts induced significant decrease in the serum ALT,AST,ALP and TB compared with those in the mice with acute liver injury?P<0.01?.Obvious increase was noticed in serum TP?P<0.01?.Moreover,such effects presented in a dose-dependent manner.Compared with the control group,the MDA was significantly elevated in the model group?P<0.01?,while significant decrease was observed in the levels of SOD,GSH and CAT in model group compared with the control group?P<0.01?.The levels of IL-1?,IL-6 and TNF-?in serum of mice with acute liver injury induced by CCl₄ can also be reduced in different polarities.Whereas,such phenomenon was completely reversed by G.turkestanerum extracts in a dose-dependent manner.The results of liver biopsy also confirmed that the different polar parts of G.turkestanerum treatment group to a certain extent,improve liver cell damage.According to the serum liver function index,liver homogenate oxidative damage related indicators,inflammatory factors and liver biopsy results comprehensive analysis G.turkestanerum different polar parts can improve to some extent CCl₄-induced acute liver injury in mice,of which the strongest activity of GBA..4 With the increase of CCl₄ and H₂O₂ concentration,the survival rate of L02 cells decreased gradually.To use 20 mmol/L,100?mol/L CCl₄ and H₂O₂ for 24hours as a damage model of L02 cells;GBA pretreatment significantly increased the survival rate of L02 hepatocytes by CCl₄ and H₂O₂.Compared with the normal control group,GBA had a significant effect on the secretion of ALT and AST in culture supernatant of L02 hepatocytes by CCl₄ and H₂O₂?P<0.05?.GBA significantly reduced the content of MDA in L02 cells induced by CCl₄ and H₂O₂?P<0.05?,and decreased the leakage of LDH?P<0.05?.The apoptotic rate of the model group was significantly increased after CCl₄ and H₂O₂ injury?P<0.05?,Compared with the model group,the apoptosis rate of each GBA group was significantly inhibited?P<0.05?,and the dose-dependent.The expression of CHOP,PERK,IRE1 and ATF6 mRNA in ERS-related genes were significantly up-regulated by CCl₄ and H₂O₂?P<0.05?.and the expression of CHOP,PERK,IRE1 and ATF6 mRNA were significantly down-regulated by GBA pretreatment?P<0.05?,GBA also reduced the expression level of CHOP and Caspase12in total protein?P<0.05?.The expression of NF-?B p65 in the nucleoprotein was decreased and the expression of NF-?B p65 in cytoplasm was increased?P<0.05?.Conclusion:The ethanol extract of the paste was 24.4%,the rate of water extract GW?GBA?GPE?GEA.The method has the advantages of strong specificity,high accuracy and good reproducibility.It can be used to compare the distribution of active components in different parts of different parts.According to the acute toxicity grading standard of traditional Chinese medicine,under the condition of this study,the polar parts of G.turkestanorum were non-toxic and safe.Through in vivo and in vitro pharmacodynamics experiments have confirmed that G.turkestanorum can inhibit lipid peroxidation,improve antioxidant activity,inhibit the inflammatory response to protect the liver.There was a certain correlation between the hepatoprotective activity and the content of iridoid glycosides,Iridoid glycosides may be the main material basis of play the protective effect of G.turkestanorum.In this study,the mechanism of hepatoprotective effect of G.turkestanorum was further studied from the cellular and molecular level,It was found that the activity of ERS-related genes could down-regulate the expression of ERS-related genes and the expression of protein.And its mechanism of action is also related to the nuclear translocation of NF-?B p65,G.turkestanorum can inhibit the activation of NF-?B signaling pathway to reduce the inflammatory response,protect the liver cells.This study provides a theoretical basis for the subsequent rational development and application of G.turkestanorum.