| Kudingcha is not only a traditional widely used tea in China,but also a folk medicinal plant.Various kinds of triterpenoid saponins are one of the main active ingredients in Kudingcha.It possessess anti-oxidant ability,anti-aging,enhanced immunity,hypoglycemic,hypolipidemicandother pharmacodynamic activities.Currently,most of the studies have focused on the pharmacological activities of their total extracts or saponins.There are few research on the preparation technology of their active purely components and their pharmacodynamics of lowing blood lipids.This porject will systematically study the extraction and purification of saponins from Kudingcha and its hypolipidemic activity.First,the Box-Behnken response surface method was used for the first time to optimize the extraction conditions of total saponin alcohol extraction from Kudingcha and determined the best extraction process.The study used single factor experiments,with the content of kudinoside A as an indicator.The effects of ethanol concentration,extraction time and ratio of material to liquid on the yield of total saponins were investigated,respectively.The experiment provided the basis for setting the factor level.Then the response surface methodology was used to optimize the process conditions.As a result,the optimum extraction conditions were as follows:ethanol concentration 63%,solid-liquid ratio 1:16 and extraction time 90 min.The total saponin yield of Kudingcha was 5.59%.This experiment establishes quantitative analysis method of triterpenoid saponin in Kudingcha.High performance liquid chromatography coupled with evaporative light scattering detector was used to determine the content,A Agilent XDB-C18column（4.6mm×250mm,5μm）was used at a flow rate of 0.8mL?min-1.Mobile phase A was methanol and mobile phase B was water-formic acid（1000:1,v/v）,the column temperature is40℃,and the injection volume is 10μL.The detector drift tube temperature is 110℃,the carrier gas flow rate is 3L?min-1,the evaporative light scattering detector uses compressed air as atomizing gas,and the carrier gas pressure is 0.4MPa.The results showed that the concentration of five saponins in Kudingcha showed a good linear relationship in the range of50μg?mL-1to 800μg?mL-1.The method is stable,accurate,reproducible and the recovery rate is good,which accords with the requirement of quantitative analysis.Meanwhile,the content of three terpenoid saponin in Kuding tea from two different habitats was determined by the method.The results showed that the content of saponin in Hainan Kudingcha was higher.In order to optimize the separation and purification process parameters of saponins in Kudingcha,nine different types of macroporous resins were selected.The best resin was selected by static adsorption and desorption test to be HP20SS.The related factors were investigated through adsorption and desorption experiments,and the best purification conditions were determined as follows:the wet sample,the upper sample flow rate was 2BV?h-1,the sample liquid concentration was0.866mg?mL-1,the sample liquid was 88mL,the elution flow rate was 2BV?h-1,the first water was washed to the eluent colorless,then 5BV（10mL）20%ethanol was used to elute the residual liquid of the partial isochlorogenic acid,and then the 6BV（12mL）was eluted with 60%ethanol to obtain a higher purity Kudingcha saponin fraction.The total saponin enrichment efficiency of Kudingcha was expressed by these five saponin contents,the purity before purification was 11.80%,the purity after purification was 81.51%,the enrichment factor was 6.91,and the overall recovery was 69.76%.Finally,five Kudingcha Saponins（kudinoside G,C,A,F and D）were separated by semi-preparative HPLC.Their structures were identified by high resolution time of flight mass spectrometry and nuclear magnetic resonance techniques.In this experiment,high fat diet was used to establish experimental hypercholesterolemia model in mice.By comparing the indexes of hypercholesterolemia and hypercholesterolemia,the effect of kudinoside A was evaluated.The results are as follows:Histopathologic examination showed that the control group of mice liver was red and bright,the liver of model group was gray,dull,volume was significantly increased compared to controls.The liver of positive group was light yellow,larger than that of the control group.The liver of Kudinoside A high and low dose group compared with control group,color white color,gloss poor,volume decreased compared with the model group.Compared with the blank control group,model group,the liver fat accumulation was the most serious,as the red area large,and the cell gap,arranged irregularly,liver cells decreased significantly.After intragastric administration of four week to the treatment of kudinoside A group,high dose group and low dose group can significantly reduce the accumulation of lipid in mice liver.And kudinoside A high dosage group had the best effect.It shows that kudinoside A group has a significant protective effect on the liver and can effectively alleviate the lipid lesion of the liver of the experimental hyperlipidemia mice.The results of serum biochemical indexes in mice showed that compared with normal group,serum TC,LDL-C and MDA levels in model group increased significantly（p<0.01）,and SOD level decreasedsignificantly（p<0.01）,indicating that the experimental hyperlipidemia mice model was successful.Compared with the model group,positive group,Kudinoside A high dose group serum TC,LDL-C and MDA were significantly decreased（p<0.01,p<0.01,p<0.05）,serumSODlevelwas significantly increased（p<0.05）.Kudinoside A low dose for serum TC,LDL-C,MDA,SOD group and the level of compared with the model group,no statistical significance.In this study,the extraction,separation and purification process of Kudingcha saponin was the main research content,and the higher purity Kudingcha saponin part was finally obtained.Then,fivepuresaponincompoundswereisolatedby semi-preparative HPLC,and the structures were identified by liquidchromatographyquadrupoletimeofflight Mass spectrometer/Mass Spectrometry（LC-QTOF-MS/MS）and nuclear magnetic resonance（NMR）. |
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