Resrearch On Manufacture Process,Chemical Constitude And Quality Control Method Of Stevia Rebaudianum Bertoni Effective Fraction

Stevia rebaudianum Bertoni is also called "the sweet herb of Paraguay", in whichent-kaurene glucosides and flavonoids are the two main constituents. The former has gotgreat fame in food and medicine industries as the sugar substitutes, And the latter with strongpharmacological activities lacks of deep research. In order to make full use of Steviarebaudianum Bertoni research were carried out in this paper, which includes the manufactureprocess of ent-kaurene glucoside and flavonoids, ent-kaurene glucosides, flavonoids,constituents analysis on effective fraction of ent-kaurene glucoside and flavonoids andestablishment of the quality control system.Part one:Literatures and summarizationsDocuments about the research progresses in the chemical constituents andpharmacological activites of Stevia rebaudianum Bertoni were summed up and analyzed,thirty-five references were cited.Part two: Experiments about the effective fractions ofStevia rebaudianum BertoniChapter one: The observation of the extracted term of the effective fractions ofStevia rebaudianum BertoniOne: To optimize the technology of extracting chemical constituents from the leafs ofStevia rebaudianum Bertoni With Quercitrin, Stevioside and flavonoids as criterions,the water extraction and ethanol extraction technologies of extracting the constituents fromthe leafs of Stevia rebaudianum Bertoni were compared by HPLC and VISspectrophotometry, and the L₉(3⁴) orthogonal experimental design was used to optimize theconditions of the extracting technology. As a result, the conditions of the ethanol extractiontechnology of extracting the constituents from the leafs of Stevia rebaudianum Bertoni wereA₂% ethanol, C₁ hours of reflux, D₃ fold solvent and B₃ times of extraction. Observing theabsorption faction with the amount of the total flavonoids, Stevioside and Quercitrin, Wedraw the conclusion that the C resin has the best absorptive ability on the separation and enrichment of the effective fractions from the leafs of Stevia rebaudianum Bertoni. The besttechnology term is that the extraction substance with C₄ medicinal material/ml density, theratio of loading capacity and resin volume was E₂ passed the resin column that d/1 is L2 withthe D1 absorbed speed, firstly washed out with water B1 time of resin volume to wipe offimpurity, then S % ethanol for WBV of resin volume with F₁ eluted speed, and resin can beused repeatedly 7 times after refining with 95% ethanol and 5%NaOH。Two: To study the purification technology ofStevia rebaudianum Bertoni flavonoids fractionand ent-kaurene glucoside fraction. After investigated the adsorptive property ofpolyamide,We draw the conclusion that polyamide has the best absorptive ability on the separation andenrichment of the flavonoids effective fractions and ent-kaurene glucoside fraction, We Obs-erve the absorption effective faction with the amount of the total flavonoids and Quercitrin,stevioside. The best technology term is that the extraction substance with C₂ medicinal mat-erial/ml density, the ratio of loading capacity and resin volume was E₁ passed the resincolumn that d/1 is L₂ with the absorbed speed D₁, firstly washed out with water for B₁ times ofresin volume to get the ent-kaurene glucoside fraction, then washed out with P % ethanol forMBV of resin volume with F₁ eluted speed to get the flavonoids fraction, polyamide can beused repeatedly 5 times after refining with 95% ethanol and 5%NaOH.Chapter two: studies on the chemical constituents of effective fraction12 compounds were isolated from the effective fraction of the leafs of Stevia rebaud-ianum Bertoni by repeated silica colum chromatography, RP-18 silica colum chromatographypolyamide colum chromatography and Toyopeal HW-40, Pharmadex LH-20 colum chrom-atography. Based on the spectral data (1D, 2D NMR, MS), 12 of them were characterizedas: luteolin, quercetin, luteolin-7-O-β-D-glucoside, apigenin 7-O-β-D glucoside, quercetrinquercetin-3-O-β-D-arabinose, Quercetin-3-O-(4-O-trans-caffeoyl-α-L-rhamnopyranosyl-(1→6)-β-D-galacopyranoside, 4, 5-di-O-caffeoyl quinic acid,β-sitosterol, stimasterol,stevioside, rebaudiosideA, among them, luteolin and 4, 5-di-O-caffeoyl quinic acid were firstisolated from this plant, Quercetin-3-O-(4-O-trans-caffeoyl-α-L-rhamnopyranosyl-(1→6)-β-D-galacopyranoside) was a new compound.Chapter three: Establishment of the quanlity controlling methods of effective fractionOne: Establish method s of determinating the total flavonoids in the effective fractions ofthe leafs of Stevia rebaudianum Bertoni with the evaluation quota of Quercitrin; The contentsof the total flavonoids are 36.69 % (RSD= 1.53%) using resin and 85.52 % (RSD = 0.69%)using polyamide.Two: Establis.h the HPLC analysis methods of the content of the four main compositionsincludeing luteolin-7-O-β-D glucoside, quercitrin, 4, 5-di-O-caffeoyl quinic acid andQuercetin-3-O-(4-trans-caffeoyl-α-L-rhamnopyranosyl-(1→6)-β-D-galacopyranoside in effective fractions ofStevia rebaudianum Bertoni. the contents of luteolin-7-O-β-D glucosideare 0.40 % (RSD =1.42% )in the using resin effective fractions and 1.32 %(RSD = 0.44 %)in the using polyamide effective fraction; T he contents of Quercitrin are 1.01% (RSD=1.29%)in the resin effective fractions and 3.15% (RSD=1.09%) in the polyamide effectivefraction. T he contents of 4,5-di-O-caffeoyl quinic acid are 1.80%(RSD=0.84%) in theresin effective fractions and 7.04% (RSD=0.97%) in polyamide effective; the contents ofQuercetin-3-O-(4-O-trans-caffeoyl-α-L-rhamnopyranosyl-(1→6)-β-D-galacopy rano-side are 0.25%(RSD = 1.11%) in the resin effective fractions and 1.16% (RSD=0.26 %) inthe polyamide effective fractions.Three:Establish the HPLC analysis methods of the content of the main composition include-ing stevioside and rebaudioside A in effective fractions of Stevia rebaudianum Bertoni. Thecontents of stevioside is 34.98 % (RSD=1.92 % )in the resin effective fractions and 38.85%(RSD =0.63% ) in the polyamide effective fractions. The contents of rebaudioside A is 10.20%(RSD =1.98 %)in the resin effective fractions.and 16.91%(RSD=0.99%) in the polyamideeffective fractionsFour: HPLC finger print of the effective fractions of Stevia rebaudianum Bertoni is establishedprimarily. By analyzing and comparing chromatographic peaks, 10 peaks are found in theHPLC finger print.Part three: summary and discussionOne: summary and analysis of the whole experimentsTwo: point o ut the main innovation...