Study On Purification Of Active Components From Inula Britannica L. And Its Protective Mechanism On Liver Injury

Inula britannica L.is the dry head of the Inula japonica Thunb.or Inula britannica L.It has the functions of reducing stomach qi,relieving vomiting,resolving phlegm and relieving asthma,eliminating phlegm and hydrating to eliminate fullness,etc.It is an important medicine for treating lung and stomach qi adversity syndromes.Eurasian Inula is the most representative species in the research of Inula.Its chemical components are more abundant.Studies have shown that sesquiterpenes and flavonoids are the main chemical components of Inula britannica L.and it is the important material basis for its medicinal effects.Based on HPLC fingerprinting technology,this study explored the chemical composition differences of 33 commercial sources of Eurasian Inula,and used flavonoids and sesquiterpene lactones as the inspection indicators,using orthogonal test and Box-Behnken design response surface Method to optimize the extraction and purification process.In addition,through animal and cellular pharmacological experiments,the effects of total flavonoids from Inula Britannica L(TFIB)on 4 types of liver damage(chemical liver damage,immune liver damage,alcoholic liver damage and drug Hepatic Injury),and systematically explained its main indicator component——1-O-actylbritannilactone(ABL)on the protective effect and the mechanism of alcohol-induced L02 cell damage.Specifically include the following aspects:1.The analysis and purification of the main chemical components of Inula britannica L.1.1 HPLC fingerprint analysis of Inula britannica L.Using HPLC fingerprinting technology,the chemical composition differences of 33 commercial sources of Inula vulgaris were explored.After multi-point calibration and full peak spectrum matching,the inula HPLC fingerprint spectrum was formed,and 40 common peaks were calibrated.Compared with the reference solution,the Inula britannica L.contains chlorogenic acid,caffeic acid,rutin,quercetin,douglas fir,luteolin and 1-O-actylbritannilactone.In addition,using fingerprint similarity evaluation and cluster analysis,the results showed that the fingerprint similarities of 33 commercial sources of Inula vulgaris were mainly concentrated in the range of 0.89 to 0.99,and the similarity was over 0.90%,reaching 84.85%.It shows that the chemical components in the samples of Inula have good consistency.The chemical components of the medicinal materials are stable,but there are obvious differences in the content of the components.The reasons may be related to factors such as the harvest time,weather,soil physical and chemical properties,topography and geomorphology,and man-made factors during processing.The influence of factors has led to certain differences in inula medicinal materials from commercial sources.1.2 Optimizing the extraction process of the main chemical components in Inula britannica L.by orthogonal testThrough orthogonal experiments,different extraction solvents(90%,60%,30%,water),different extraction methods(ultrasonic-assisted extraction,hot reflux extraction,Soxhlet extraction)were investigated through orthogonal experiments,combined with extraction times,extraction time and other factors to establish The orthogonal experiment of "three factors and three levels" systematically studied the extraction process of TFIB and ABL,which are rich in Inula.After scientific data analysis,combined with the mass fraction of TFIB and ABL in the Inula medicinal materials,it was finally determined that 60% ethanol was used as the solvent,and the reflux method extraction method could simultaneously maximize the extraction rate of TFIB and sesquiterpene lactone.The extraction of TFIB The rate can reach more than 10.95%,while ABL reaches 2.75%;in addition,this chapter also shows through orthogonal experiments that the number of extractions(A)is the main factor affecting TFIB extraction,and the extraction time(B)and solvent volume(C)are TFIB extractions.The extraction process is:A3Bl Cl,to further optimize the purity of TFIB,and to use chemical methods such as silica gel column chromatography to prepare and separate ABL,which provides a material basis for subsequent research.1.3 Box-Behnken design response surface method to optimize the purification process of TFIBBased on the determination of the best extraction process for TFIB and ABL in Inula,this chapter systematically explores the effect of 6 different types of macroporous resin on the adsorption of TFIB through the method of combining the static adsorption and dynamic adsorption of macroporous resin,and clarifies AB-8 has the best effect on TFIB purification.Furthermore,using Box-Behnken design response surface method to explore the best purification process of TFIB,factors such as sample concentration,elution solvent,and eluate volume are used as inspection indicators.The order of influence is as follows: elution solvent<loading concentration <Volume of eluent,preferably 70% ethanol as the elution solvent,the loading concentration is 30 mg/m L,the eluent volume is 3BV,and the flow rate is 2m L/min.The research results show that the best extraction and purification method for TFIB is: add 60%ethanol to extract 3 times,each extraction is 1 h,the amount of alcohol added is 10 times that of medicinal materials,the extracts are combined and filtered.TFIB was purified with AB-8macroporous resin,the loading concentration was 30 mg/m L,and the eluent volume was 3 BV,the TFIB content could reach 45.58%,the eluent was 70% ethanol,and the elution rate was 2m L/min,the eluent recovers ethanol until there is no alcohol smell,concentrates to a thick paste with a relative density of 1.30?1.35(80?),dried under reduced pressure(control vacuum?0.08 MPa,temperature: 80?85?),crush Become a fine powder,sieved,and get ready.2 Study on the protective effects of the main chemical components of Inula britannica L.on different types of liver tissue damage2.1 Preliminary study on the protective effect of TFIB on different types of liver tissue damageBased on the determination of the optimal preparation process of TFIB,combined with the traditional pharmacodynamics research background of Inula,this research will focus on the in-depth exploration of the main pharmacodynamics of TFIB,and establish the induction of carbon tetrachloride(CCl4)Acute and chronic liver injury,D-galactosamine(D-Gal)combined with lipopolysaccharide(LPS)-induced acute liver injury,BCG and LPS combined-induced acute liver injury,alcohol-induced acute liver injury,and thio B Thioacetamide(TAA)induces liver fibrosis in mice and conducts experimental research.The use of serum biochemical index detection(ALT,AST,MDA,SOD,etc.),histopathological staining(H&E staining,Masson staining)and Western Blot detection,etc.,have preliminarily proved that TFIB is effective against chemical liver damage and immunity.Liver injury,alcoholic liver injury and drug-induced liver injury have obvious protective effects.2.2 The protective effect and molecular mechanism of ABL on alcohol-induced damage to L02 cellsPrevious studies have fully demonstrated through in vivo experiments that TFIB has a significant protective effect on alcohol-induced liver injury in mice,and has a significant pharmacodynamic effect on different types of liver injury such as chemical and drug properties.In order to further explore the liver-protecting effect of Inula britannica L,this study was based on the preparation of high-purity ABL and used in vitro experiments to explore the protective effect of ABL on alcohol-induced damage to L02 cells.It was stained by Hoechst 33258 and used fluorescent probes.(DCFH-DA)detection of intracellular reactive oxygen species(ROS),flow cytometry detection of cell apoptosis and Western Blot(Western Blot)and other pharmacological methods,through the two aspects of cell apoptosis and inflammatory response in-depth elaboration The pharmacological effect of ABL in the treatment of liver injury and revealed the unique role of the main components of Inula(TFIB,ABL)in protecting the liver.The specific pharmacodynamic mechanism may be related to the partial inhibition of activated caspase 8,caspase 9,Bax,etc.Protein expression in turn inhibits ERK,JNK and MAPK signaling pathways,and regulates Akt/NF-?B signaling pathways,and is related to inhibiting oxidative stress,reducing liver cell apoptosis and preventing inflammation.In summary,this study used HPLC fingerprinting technology to explore the overall chemical composition of Inula britannica L,and used orthogonal experiments and response surface methods to clarify the total flavonoids and sesquiterpene lactones of Inula britannica L The extraction and purification process,through a combination of in vivo and in vitro experiments,explained that TFIB and ABL have a significant improvement effect in the treatment of liver injury caused by various factors,and also revealed its main pharmacodynamic mechanism.The quality evaluation of anthers and the in-depth development of medicinal substances provide a sufficient theoretical basis.