Study On The Extraction, Purification And Determination Of Isoflavones In Rapeseed Meal

Isoflavones are the plant secondary metabolic products, they contain isoflavones which are mainly soybeans, Gegen and Trifolium pratense in the common plants. Within this total, the largest and most in-depth study is the soybean isoflavone research in legumes. The melting point of most of pure isoflavones is above 100℃, with the yellow-white colour, and it tastes bitterness slightly. The isoflavones can dissolved in acetone, Ethanol, methanol, ethyl acetate and other polar solvents, but they cann't dissolved in petroleum ether, n-hexane and other non-polar solvents. Isoflavones have a variety of physiological functions,which are anti-tumor effect, the weak estrogen and anti-estrogen effect, the cardiovascular effect, the bone metabolism effect, and it also has anti-oxidation, enhancing non-specific immune function and anti-inflammatory effects. So it's a hot research in the physiological function of isoflavones.At the present time, the main routes of extracting isoflavones from plants is that: extraction—separating protein—purification—determination. The main extraction is using solvent, ultrasonic method.The main separating protein is isoelectric point method, centrifugation and freeze-isoelectr -ic point-centrifugation, ethanol precipitation, ultrafiltration method and so on. Purification mainly contains solvent extraction, ultrafiltration, resin adsorption and column chromatography. the meth -ods of Determination are UV spectrophotometry (UV), thin layer chromatography (TLC), High performance liquid chromatography (HPLC), gas chromatography (GC), HPLC a mass spectrum -etry (HPLC-MS)..In this paper, it chooses the most commonly method for rapeseed's isoflavones extraction by solvent.usingTLC and HPLC, we can make sure that it has isoflavones or not,and then make clear the type and the content of isoflavones in Rapeseed meal. On this basis, by using high-performance liquid chromatography method as a standard, comparing the accuracy and reliability of different UV wavelength (254nm, 259nm,260nm,262nm, three wave -length) ,we can choose a simplier and rapider method of determining isoflavones; by comparing different extraction condition (solvent, concentration, temperature, time, weight ratio,number) ,we can also select the optimalitical extracting condition;by comparing the isoelectric point method, centri -fugation, freeze-isoelectric point-centrifugation, we can make clearly of the best protein separation condition; by comparing solvent extraction and resin adsorption, we can also choose the best puri -fication methods; and then we do a text between the standard sample and the purified isoflavone for physiological activity.The main results are as follows:(1) In this paper, we found that Rapeseed Meal does contain genistin which is one of the isoflavones by extraction of rapeseed meal. and the content range of genistein is 0.100%-0.290%. (2) Rapid determination of isoflavones Research shows : Using UV to Determinate the isoflav -one's content which extracted from the rapeseed meal is feasible, angd its best colorimetric wavele -ngth is 259nm, the calibration coefficient is 1.67. 3 wavelength can not be used to determinated the isoflavone's content.(3) By comparing the rate of isoflavone and impurities under different solvents (methanol, ethanol), the extraction time (1,2 and 3 hours), different numbers(1,2,3) and different tweight ratio (1:5,1:10. 1:15,1:20), the different temperature (50℃, 60℃, 70℃, 80℃) ,it conclude the optimizatial condition: 60%ethanol, the temperature is 60℃, and weight ratio is 1:15, the extraction time is 3 hours, extraction 2nd,.(4) By using isoelectric point precipitation, centrifugation and freeze—isoelectric point—centrifugation, it conclude the best method for protein separation : chilled for 24 hours, after HC1, make sure pH between at 3-4. Then at 2000r/min, centrifugal 20min..(5) Two method of parification which is ethyl acetate extraction, macroporous resin adsorption method results showed that: using macroporous resin adsorption purified isoflavones better than extraction with ethyl acetate. using macroporous resin adsorption,the content of genistin in dry matter is up to 77%. And the extraction line of isoflavones is that: Ethanol extraction—Freeze-isoelectric point-Centrifugal Separation of protein—macroporous resin extract—Determination.(6) The isoflavone's anti-oxidant capacity from rapeseed meal showed : Genistin can signify -cantly reduce the lipid substance's POV and TBA value.