Establishment Of Stevia Rebaudiana Standard And Study On Improving Sugar And Lipid Metabolism Activity

Stevia rebaudiana(Bertoni)Hemsl,a compositae plant,is native to the alpine grassland on the border between Paraguay and Brazil in South America.It was introduced from China in the late 1970s and spread to all parts of the country.Since 1980s,Stevia rebaudiana has been cultivated artificially in Jilin Province.Its dried leaves,also known as STEVIAE REBAUDIANAE FOLIUM,also known as sweet tea,have the effects of promoting fluid production to quench thirst,inducing diuresis and low ering blood pressure,and are mainly used to treat thirst and hypertension,and are widely used in food,medicine and health care products.However,due to the short localization time,Stevia rebaudiana has not been included in People’s Republic of China(PRC)Pharmacopoeia,but has been listed in the standards of Chinese herbal medicines in Hunan,Beijing,Heilongjiang,Fujian,Zhejiang,Gansu and other provinces and cities,resulting in different quality evaluation standards for stevia rebaudiana production,sale and product processing,and confusing procedures,which hindered the development of stevia rebaudiana-related pharmaceutical industries in Jilin Province.In order to solve the above problems,this paper takes Stevia rebaudian a from Jilin Province and its surrounding areas as the research object,intends to evaluate its quality and establish local medicinal material standards suitable for Jilin Province’s production status,and studies its kaurane diterpenoid derivatives represented by stevioside on improving sugar and lipid metabolism.The experimental contents and results are as follows:1.Quality evaluation and standard establishment of Stevia rebaudiana: Taking 21 batches of Stevia rebaudiana samples from Jilin Province,Inner Mongolia Autonomous Region,Heilongjiang Province and other regions as the research objects,the methods of character identification,microscopic identification and TLC identification were established,and the indexes such as moisture,total ash,acid-insoluble ash,heavy metals and pesticide residues were detected,and the HPLC method for the determination of stevioside and rebaudioside A was established,and then the quality evaluation of the above Stevia rebaudiana samples was carried out.In this chapter,experiments were carried out on the selection of chromatographic column,mobile phase and preparation of test solution.Finally,amino column was used as the chromatogr aphic column,and acetonitrile-water(85: 15)was selected as the mobile phase for is ocratic elution,and the standard curve was drawn up.The methodology of precision,stability,detection limit and quantitative limit,repeatability and accuracy were in vestigated.Results:(1)Establishment of qualitative identification and quantitative method: description of character identification,microscopic identification,TLC identification method and HPLC method;(2)Quality evaluation results:Identification result:The inspection results and standard parameters stipulate that the moisture content of Stevia rebaudiana in different areas is between 7.14% and 10.68%,with an avera ge of 8.78%,and the specified moisture limit is not more than 12.0%;The total ash content is between 5.44% and 12.74%,with an average of 7.20%,and the specified limit does not exceed 9.0%;The acid-insoluble ash content is between 0.30% and 7.22%,with an average of 1.65%,and the specified limit is not more than 4.0%;The content of alcohol-soluble extract(using 70% ethanol as solvent)is between 39.62% and 56.01%,with an average of 47.83%,and the specified limit shall not be less than 38.0%;Heavy metal accumulation pollution,Cucontent in Stevia rebaudiana in different areas is 3.00～16.78 ppm,with an average of 5.71ppm;The content of As is 0.04 ～ 0.50 ppm,with an average of 0.26ppm;The content of Cd is 0.08 ～ 0.42 ppm,with an average of 0.19ppm;.Pb content is 0.18 ～ 0.49 ppm,with an average of 0.31ppm;The content of Hg is 0.01 ～ 0.04 ppm,with an average value of 0.02 ppm.The safety risk of Stevia rebaudiana caused by pollutants is low,so it is not listed in the standard alone.Pesticide residues,among the 33 prohibited pesticides specified in China Pharmacopoeia,except for three batches of stevia rebaudiana samples(TYJ-5,TYJ-15 and TYJ-20),the corresponding pesticide varieties of other medicinal materials samples were not detected,and the safety risk of stevia rebaudiana caused by pesticide residues was low,so it was not included in the standard separately;Stev ioside content in Stevia rebaudiana samples from different habitats is between 0.76% a nd 2.94%,with an average content of 1.54%,which is not less than 0.7%.The con tent of rebaudioside A is between 4.83% and 10.38%,with an average content of 7.85%,which is not less than 4.8%.2.Study on improving the metabolic activity of glucose and lipid: The 3T3-L1 in sulin resistance cell model(IR-3T3-L1)was established by dexamethasone,and the positive drug(rosiglitazone)and stevioside,rebaudioside A,steviol and isosteviol with different doses were given separately.The glucose consumption and intracellular triglyceride content were detected,and the size of intracellular lipid droplets in each group was observed after oil red Ostaining.The normal group was given the best concentration of drugs,glucose consumption and triglyceride content were detected,and the changing trend of intracellular lipid droplets was observed.The results showed that stevioside,rebaudioside A,steviol and isosteviol all enhanced the glucose uptake of IR-3T3-L1 cells,and stevioside also enhanced the glucose consumption of normal 3T3-L1 cells.Stevioside and isosteviol can reduce lipid droplets in IR-3T3-L1 cells and 3T3-L1 cells,reduce adipocyte hypertrophy and enhance the sensitivity to insulin.