| Objective:The present thesis aimed to establish a multi-components content determination method of Lomatogonium rotatum（LR）,and to optimize the extraction criteria of LR with swertiamarin as a main indicator.In addition,the protective effect of LR was observed in a high-fat diet induced nonalcoholic fatty liver disease in mice and cell culture.Methods:1)Study on multicomponent content of Mongolian medicine Lomatogonium rotatum.The mixed stock solution of seven compounds was prepared by dissolving accurately weighted portions of the standards in methanol at a stock concentration of Swertimarin 0.7872 mg/ml,swertiside 0.1724 mg/ml,Hesperetin0.4404mg/ml,Hydroxymethylcoumarin 0.8248 mg/ml,quercetin 0.0804 mg/ml,Kaempferol 0.0472 mg/ml,1.7-dihydroxy-3.8-dimethoxyxanthone 0.099 mg/ml.In Sample Solution Preparation.a weight of 1.0 g LR was accurately taken place in 25 ml conical bottle ultrasonic extraction filtration stand by.A constant flow rate of 1.0m Lmin-1was employed throughout the analysis.All analysis was performed at 30℃.Then,5ul aliquot of the solution was injected into the HPLC DAD-HS system for analysis.2)In this study,the whole herb of Lomatogonium rotatum was used as raw material,swertiside was used as the active index component,and L9（34）orthogonal test was carried out in combination with HPLC method.In the Extraction process,first,the single factor experiment was carried out with the concentration of ethanol,the quantity of ethanol,the time of extractionand the number of times of extraction as four factors,On this basis,the main factors affecting the extraction rate were selected and optimized by orthogonal L9（34）experiment.Finally,the optimal extraction process of swertiside was obtained.3)To elucidate the effect of Lomatogonium rotatum on proliferation and differentiation of adipocyte 3T3-L1 and protective effect on nonalcoholic fatty liver disease in mice.（1）When 3T3-L1 was overgrown to 80%to 90%,the cells were divided into 5 experimental groups,which were respectively the administration group（high,medium and low concentration）blank group and control group.After that,CCk-8 method was used to detect the inhibition of 3T3-L1preadipocyte proliferation by Lomatogonium rotatum.（2）After adaptive feeding for one week,C57BL/6J mice were randomly divided into normal group and model group,and NAFLD mouse model was established through 12 weeks of high-fat diet induction,Then model group mice were randomly divided into model group,LR extract low medium high（0.36 g/kg,1.08 g/kg,2.16 g/kg）to drug group,pioglitazone positive control group（40 mg/kg）.The body weight,liver index and adipose tissue quality of the mice were measured after continuous intragastric administration for 15 weeks,AST（Aspartate Transaminase）,ALT（Alanine Transaminase）,（TC）Total Cholesterol,（TG）Triglyceride,（HDL-C）High Density Lipoprotein and（LDL-C）Low Density Lipoprotein were detected by serum biochemical analyzer.HE staining was used to observe liver pathological symptoms.The contents of inflammatory factors such as（IL-6）Interleukin-6,（IL-1β）Interleukin-1βand,（TNF-α）Tumor Necrosis Factor-αin liver were determined by enzyme-linked immunosorbent assay（ELISA）.Results:1)The coefficient of determination of the calibration curves in analysis systems showed good linearity（R2>0.99）.According to the methodological investigation,the precision and repeatability were good,and the sample was stable within 24 h.Moreover,named Swertimarin,swertiside,quercetin,Kaempferol,Hesperetin,1.7-dihydroxy-3.8-dimethoxyxanthone and Hydroxymethylcoumarin were recovery rate ranged from 92%to 100%.2)The content of bitter principle in Lomatogonium rotatum was determined by HPLC.From the results of single factor experiment,the peak area was the highest when the ethanol concentration was 60%,the peak area was the highest when the ethanol extraction times were 2 times,and the peak area was the highest when ethanol extraction time was 2.0 h,the peak area was the highest when the ratio of ethanol material to liquid was 1:10.According to the results,L9（）34</sub>orthogonal experiment was designed,from the analysis of variance,it was found that the order of influencing factors was extraction time>ethanol concentration>extraction dosage>extraction times,and extraction time and extraction concentration were the most significant factors.3)Compared with the normal group,different concentration of the extract of LR could effectively inhibit cell activity.Compared with model normal group,the indexes of body weight,liver index,fat mass in tissues and Serum AST,ALT,TG,TC,LDL-C indicators of mice in model group fed high fat diet were significantly up-regulated（P≪0.05）.The Body Weight,liver index,fat tissue quality,Serum ALT,AST,TG,LDL-C and so on were significantly decreased in the LR low,medium and high dose groups（P≪0.05）.The quality of Perirenal adipose tissue and periintestinal adipose tissue was significantly decreased in the LR low,middle and High Dose Groups（P≪0.05）.Compared with the normal group,the levels of IL-6,IL-1βand TNF-αincreased in the model group,but not significantly.The levels of Il-1βwere decreased in the middle and High Dose Groups（P≪0.05）.Conclusion:The Multi-component quantitative analysis method established in this experiment has high precision,good repeatability and stability,and reliable determination results,which can provide scientific basis for the quality control and standard improvement of Mongolian medicine LR.The optimal preparation process of swertiamarin was as follows:ethanol concentration of 60%,solid-liquid ratio of1:10,reflux extraction for 1 time,each extraction time 2 h.In vivo and in vitro studies illustrates that LR can reduce bodyweight and lipid,improve liver function,inhibit liver inflammation in NAFLD mice induced by high fat diet,and inhibit the proliferation and differentiation of 3T3-L1 adipocytes. |
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