The Study Of Anti-CIKI Ingredients From Compound Herba Lysimachiae Based On Comprehensive Two-dimensional HK-2 And HK-2/CIKI CMC Comparative Analysis System

Renal calculi?RC?is caused by abnormal accumulation of crystalline substances?common ingredient is calcium oxalate monohydrate?in the kidney and is a clinically common type of urinary system disease.Epidemiological investigation shows that the prevalence of RC in China is increasing year by year,and it is more common in adult men.Crystal-induced kidney injury?CIKI?is the early stage of RC,and without the timely intervention,the renal function of patients will gradually be damaged.Traditional Chinese Medicine?TCM?is a treasure of the Chinese nation and possesses profound historical and cultural heritage,which is worthy of further exploration.At present,TCM treatment is more and more valued by clinicians for its unique advantages of multiple components,multiple targets,and integration,so it is often used as an adjuvant treatment method for diseases.Compound herba lysimachiae is a Chinese medicine prescription which is commonly used for treating urinary system stones and urinary tract infections in clinical practice.It has the effects of clearing heat and dampness,diuresis and discharging stones,and reducing inflammation and pain.Cell membrane chromatography?CMC?is a biological affinity chromatographic method that combines extracted cell membrane fragments with silica gel to form a cell membrane stationary phase?CMSP?,then uses chromatographic analysis to study the interaction between drug components and cell membrane receptors.It is simple,stable,and efficient,and it has the dual characteristics of chromatographic separation and receptor affinity.The research content of this topic is mainly explained from the following three parts.1.The study of optimization of the compound herba lysimachiae extraction process and the chemical ingredients identificationCompound herba lysimachiae has attracted wide interest from researchers because of its good curative effect in treating urinary system stones.However,at present,there is no universal extraction process for compound herba lysimachiae,and the extraction methods in each laboratory are inconsistent,which leads to the poor stability of its results,and the ingredients of compound herba lysimachiae are complex and diverse,which have not been reported yet.Orthogonal test and response surface methodology have the characteristics of high scientificity,short operation time,and simple experimental steps,and response surface methodology can predict the optimal extraction parameters through regression model.Therefore,these two methods are widely used in the research of Chinese medicinal material extraction process.In order to optimize the extraction process of compound herba lysimachiae and clarify its chemical ingredients,in this chapter,a method for determining the content of ingredients in compound herba lysimachiae was established,and the methodology validation was performed by taking the three ingredients apigenin,luteolin,and soyasaponin I in compound herba lysimachiae as representatives,examining its linearity,precision,repeatability,recovery,and stability.Then,combined with the two statistical methods of orthogonal test and response surface methodology,the sum of the peak areas of fourteen indicative active ingredients in compound herba lysimachiae was used as the evaluation index to optimize the extraction process to maximize the active ingredient content.The results showed that the methodology validation performed with apigenin,luteolin,and soyasaponin I had excellent linearity,high precision,good repeatability,high accuracy,and good stability.Therefore,the method was suitable for the determination of the content of ingredients of compound herba lysimachiae.Combined with orthogonal test and response surface methodology,the optimal extraction condition for compound herba lysimachiae was10 min ultrasonic extraction time,55?extraction temperature,and 55%ethanol ratio.It was verified that the result was stable and reliable according to the above condition.In addition,the UHPLC-QTOF/MS was used to analyze the chemical ingredients in compound herba lysimachiae extract,and sixty-nine compounds were identified finally,most of which were terpenoids,phenolic acids,and flavonoids.2.Construction of comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system and its application for screening anti-crystal-induced kidney injury active ingredients from compound herba lysimachiaeCompound herba lysimachiae is often used to treat patients with RC,however,the pharmacodynamic substances and mechanism of its anti-RC effect are still unclear,which limit its popularization and application.CMC is a biological affinity chromatographic method that is often used to screen potential active ingredients from complex systems such as TCM.However,due to the combination of the TCM ingredients with the non-functional receptors on the cell membrane,the screened active ingredients often do not possess good pharmacological effects,and the target specificity of the membrane receptors is not high,which may waste lots of time in the subsequent efficacy verification.Therefore,it is necessary to construct a cell model of the specific disease that is used to realistically simulate the interaction between drugs and receptors.In order to screen out the potential active ingredients with anti-crystal-induced kidney injury efficacy from compound herba lysimachiae,in this chapter,crystal-induced kidney injury cell model was successfully constructed by stimulating HK-2 cells with 1000?M Na₂C₂O₄ solution for 24 h,and the Western blotting experiment confirmed that the stimulation of COM could down-regulate the expression of integrin?₁on the membrane of HK-2 cells,and a comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system was constructed based on the cell model,next,the selectivity of the comparative analysis system was examined by the positive drug GLPG0187 and the negative drug dexamethasone,afterward,twenty-five compounds from compound herba lysimachiae extract were initially identified by the comparative analysis system,among them,nine compounds were retained on the HK-2 CMC model,and twelve compounds were retained on the HK-2/CIKI CMC model,finally,the seven potential active ingredients?soyasaponin I,baicalein,apigenin,quercetin,luteolin,6-hydroxyl luteolin 7-O-glucoside,mangiferin?were verified using standards.3.Pharmacodynamic validation of potential active ingredients against crystal-induced kidney injury in compound herba lysimachiaeDue to the complexity and diversity of membrane receptors on the cell surface,there may exist some false positives for the active ingredients screened by CMC.Therefore,further pharmacodynamic studies on the ingredients are needed.In order to validate the anti-crystal-induced kidney injury pharmacodynamic effects of the potential active ingredients screened by the comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system in chapter three,in this chapter,six compounds including soyasaponin I,baicalein,apigenin,luteolin,6-hydroxyl luteolin 7-O-glucoside,and mangiferin which were significantly retained on the HK-2/CIKI CMC model were selected.First,their protective effects on cell viability of HK-2/CIKI cells were investigated,the results showed that baicalein,apigenin,luteolin,and mangiferin had certain protective effects on cell viability of HK-2/CIKI cells,soyasaponin I and 6-hydroxyl luteolin7-O-glucoside had significant protective effects on cell viability of HK-2/CIKI cells.In addition,the effect of 6-hydroxyl luteolin 7-O-glucoside on the cell apoptosis of HK-2/CIKI cells was also investigated using flow cytometry,the result showed that 6-hydroxyl luteolin7-O-glucoside could inhibit the cell apoptosis of HK-2/CIKI cells,and the higher the drug concentration,the stronger the ability of inhibiting cell apoptosis.Although the content of 6-hydroxyl luteolin 7-O-glucoside in compound herba lysimachiae is very low,the anti-crystal-induced kidney injury pharmacodynamic effect is much better than other ingredients,which deserves further study.The effect of mangiferin on crystal-induced kidney injury in mice was also investigated,the results showed that mangiferin could decrease the deposition of calcium oxalate crystals in the mice kidneys,alleviate the pathological damage of kidney tissue induced by calcium oxalate crystals,and inhibit the up-regulation of the expression of OPN,MCP1,and CD44 caused by calcium oxalate crystals.This topic optimized the extraction process of compound herba lysimachiae by orthogonal test and response surface methodology,used UHPLC-QTOF/MS to clarify the chemical constituents of compound herba lysimachiae,screened the anti-crystal-induced kidney injury active ingredients from compound herba lysimachiae by the constructed comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system,carried out the related experiments to validate the pharmacodynamic effects of some active ingredients.Through the study of this topic,the intrinsic value of compound herba lysimachiae was further explored,which laid a solid foundation for the subsequent development of new drugs.