Study On The Extraction, Separation And Detection Methods Of Effective Components In Stevia

Stevia rebaudiana（Bertoni）Hemsl.is a perennial herb in the Asteraceae genus zelam,Wei-gan and Xin-ping.And it belongs to the two chnnels of lung and stomach.It has the functions of nourishing and moistening lung,eliminating phlegm and stopping cough,lowering blood sugar and so on.Many pharmacological activities including modulation of glucose metabolism,attenuation of vasodilation,antagonism of the negative effect of epinephrine,inhibition of bacterial growth,anti-inflammation and prevention of cancer have been reported for the in vivo metabolites steviol and isosteviol from Stevia leaf extract.However,there are currently incomplete and high-performance standards for the extraction and purification of Steviol Glycosides SGs and the isolation of each component,than this research has been established an auxiliary method for high performance liquid chromatography.Therefore,use the easily purchased dried leaf powder of Stevia rebaudiana as the original material,and seven Stevia glycoside standards were used as the control samples,which were evaluated by extraction technique of Stevia glycosides and isolation and purification products of impurities,resin separation of monomeric constituents of Stevia rebaudiana,simultaneous qualitative identification and quantitative deter mination of multiple constituentslts efficient use,large-scale production and rapid identification of its active constituents,Steviol Glycosides,provide experimental data and theoretical references.The main research content and conclusions are as follows:1.Establishment of HPLC assay for Steviol Glycosides compounds.HPLC conditions were optimized using a single factor test,column Luna C₁₈（2）,250*4.6,5μm,（00G-4252-E0）;32%acetonitrile（mobile phase B）+68%water（mobile phase A）;1.25 mg/m L concentration of sample in water;DAD detector,210 nm detection wavelength;0.5 m L/min flow rate;20μL injection volume;and column temperature at 30℃.The Resolution（R）,Theoretical Plates Number（N）and Keep Factor（K）of each component peak in the liquid phase profiles were in accordance with the pharmacopeial regulation,and a standard curve was established with seven steviol glycosides’standards to exa mine the linearity,detection range,precision,repeatability solution stability and loading recovery of the method.2.Establishment of thin layer chromatography for rapid detection of four steviol glycoside monomers.Using n-butanol:acetic acid:water=4:1:5（upper liquid）,chloroform:methanol:water=65:35:10（lower liquid）as the unfolding agent,one-way unfolding is performed versus two-dimensional vertical unfolding,our method has good repeatability in intra-day precision and within-day precision,high durability and practicability for impurities.It is suggested that high performance liquid chromatography should be the main detection method and thin layer chromatography should be the auxiliary detection method for steviol glycosides in the future.3.Optimization of extraction and purification technology of steviol glycosides’compounds.The TSG95 process for SGs as theory based for this research.simplified and optimized the extraction and purification procedures for Steviol Glycosides SGs,which were hot-water extraction at 75℃,for 2 h,1 h/time,use gauze filtration,flocculation of iron sulfate and barium hydroxide to remove hybrids,macroporous adsorption resin adsorption,then washing with acid-base cleaning agent,desalination and desalting with anions and dextran gel resin refinement,concentration by rotary evaporation,pre freezing at–80℃,lyophilization in vacuum lyophilizer to powder,Finally,the purified SGS were obtained.Compared with the samples prepared by the original TSG95 process,the yield of the optimized as prepared pure product rose by3.54%,it’s shows that the method has significant economic significance.4.Research on innovative isolation techniques.Two new resins,LX-T28 and LX-T81,were selected as the research candidates,and the best perfor ming resin was selected to be LX-T81 by exa mining the static adsorption equivalents,static adsorption time curves,and desorption kinetic curves of seven steviol glycosides inside the two resins,respectively.And the single column separation pre-test with six column tandem experiment was carried out,the experimental results showed that the resin separation was not only low cost but also effective and feasible,which can be further intensively studied for establishing more efficient separation methods.