Study Of The Determination Of Soybean Isoflavones In Health Foods By HPLC

1. ObjectiveISO is a secondary metabolite in soybean growth. The basic carcass of its chemical construction is 3-phenyl group dihydrocoumarin. There are 12 natural ISO which including daidzein, genistein, glycitein and their glycoside.The main active component contenting in soybean is daidzein, genistein and its β-glycoside. The study of biologos shows ISO is a only safe estrogen of the natural plants in the world at present and it has a two-way adjusting role for the estrogen level in the body of female. ISO has a molecular structure and weight similar to estrogen and has a role for replenishing estrogen to the body; when the estrogen level is too high in the body, ISO can also stop excessive estrogen to affect the target organs, thus keeping the balance of the estrogen in the female s body. So ISO is also called regulator for the estrogen level in the female body. Phenol hydroxy contained in the ISO molecule can be used as a oxygen feeder. ISO can react with the free radical in the body and reduce the free radical of oxygen to harm the biological micromolecule and reduce the tumor to occur, thus it has a great role for prevent tumor. Daidzein can reduce cholesterol to synthesis and cholesterol concentration as wellas to prevent arteriosclerosis. Atthe external of the body, is a PTK active inhibitor;it can stop many growth factors and platelet source growth factors(PDGF) related with thrombus forming. Genistein can also inhibit the proliferation for the cell of vascular smooth muscle and the activation of leucocyte adhesive molecule; limit the enlarging of atherosclerosis and inhibit some enzyme activity related with DNA amputating. At the same time, ISO is a protector of the central nervous; it has a rule of anti-nervous disease; it can also adjust cell circle, activate the signal transmiting route of the cell death; inhibit the proliferation for the cell of vascular smooth muscle and it has effect of anti-thrombosis, anti-senile dementia, antibiosis and anti-inflammatory , antihemolysis, trengthenning immunocompetence of human body, delaying the senility of human body and prevent osteoporosis. Soybean and its productions are the traditional food of the people in easten asia all along. The ISO in the diet is related the low incidence of cancer and cardiovascular disease in the people in eastn asia ( Chinese; Japanese). Physiological activity of ISO is paid more and more attention by society and research workers in recent years, the food and health food taking ISO as an additives is more popular in Japan and there are such food in Europe. Even Hendrich S etc. suggest to take the ISO as a new nutrient. In the home and abroad, the determinating methods of ISO include GC, HPLC, GC-MS, HPLOMS, CE, TR-FIA, ELISA and so on. The methodadopting at present mostly determines ISO content in soybean and its productions. This method can only determine diadzein and genistein, it can not determine their glycoside. In order to determine the total content of glucoside, it also needs to hydrolyze the substance taked from the sample, it needs or use a lot of time and great effort as well as operating overelaboratly. This study probes into the extracting and determining condition of ISO in the health food, not hydrolyzing the substance taked from the sample. The four substance above are determined methanol-pure water by FLD, UV-VIS, ELSD at the same time and compare the merits and demerits of three above. 2. Methods and resultsMethod 1. To establish a method for the determination of soybean isoflavones including D, De, G, Ge in health foods by HPLC. After sonicated for 30 min and filtered through the 0. 45 u m filter, the samples were separated on a C18 reversed -phase colum(200X4. 6 mm 5 u m) using methanol +water (60+40 v/v) as the mobile phase, with time programe and detected by time programe with ultraviolet detector. Good separation of D, G, De, Ge was achieved in about 15min , D r=0.9999, G r=0. 9999, De r=0.9999, Ge r=0.9996. The recoveries of the added D, G, De, Ge was 90. 2%—108. 4%, RSD was between 0. 93%—5. 2%. The in-day relative standard deviations were 1. 2-2. 5%. With it saccuracy, sensitivity, reproducibility as well as thesimplicity of sample preparation, the method was feasible for the determination of 4 soybean isoflavones in health foods. Method 2. To establish a method based on RP-HPLC with ELSD for determination of 4 soy isoflavones in health foods. The method was developed with the condition as follows: Cromasil C18 column (200X4.6 mm, 5y m), Gradient elution was employed with the mobile phase of methanol-pure water at the speed of 1.0 ml/min. ELSD rimpactor :off, gas flow:2. 7 ml/min, temperature of the drift tube :104 °C . Grinded sample was ultrasonically attracted in 80% methanol for 30min, centrifuged and filtered through the 0. 45 um filter. Good separation of the daidzin> genistin >, daidzein> genistein was achieved in about 15 min. The base line of the chromatogram was straight with gradient elution. The four contents were all linear over the range 2.6 u-g/mL—150 ug/mL. D: r=0. 9999 , G: r=0. 9994, De: r=0. 9999, Ge: r=0. 9996 and recovery of the added sample D> G^ De> Ge was 88. 7%—103. 2%, RSD was between 1. 2%-5. 5%. The in-day relative standard deviations were 1.1 % -3.0 % .With its accuracy ,sensitivity reproducibility as well as the simplicity of sample preparation, the method is feasible for the determination of 4 soy isoflavones in health foods. Method 3. To establish a method for the determination of soybean isoflavones including D De G Ge in health foods by HPLC. After sonicated for 30 min and filtered through the 0. 45 urn filter, the samples were separated on a C18 reversed-phase colum(200X4. 6 mm 5 urn) using methanol+water (60+40 v/v) as the mobile phase, with time programe and detected by fluorescence detector. Ex=366nm, Em=417nm, The content was linear over the range 0.5 Mg/mL-30 Mg/mL. Y=13924X-8625, r=0. 9995. The recovery of the added sample De was 90. 2%—108. 4%, RSD was between 0. 93%-5. 2%.3. ConclusionDetermining ISO by compared and used FLD, UV-VIS and ELSD shows that UV-VIS has a superiority, its using extent is very wide and can determine four standard substances for ISO at same time. Its linear relation is good and has a highly sensitive, the limiting level can reach to 0.6ng. Because using methanol-pure water as the mobile phase , methanol has ultraviolet absorption, so its base line is not smooth and has drift phenomenon in gradient elution analysis. When using ELSD, it is not effected by mobile phase changing, so the base line is smooth, but its detecting limit is higher. Its detectin limit can meet the needs of the detecting for most samples. Because of its price is higher, it is not to be popularized. FLD can only detect glycitein and its sensitivity is not high, but its base line is smooth and it has a demerit of high price and it is notto be used widely. Setting up the HPLC method to determine ISO in health food provides a analysis method to study and control the ISO contents in the health food and provides protection for further study the molecular mechanism actting on the human body and sanitation monitor; detects and investigates the health food contenting ISO and its quality in the market at the same time.