| Coumestrol, a benzofurocoumarin, is the predominant plant estrogen in alfalfa. The objective of the present study was to determine that coumestrol has e has estrogenic and antioxidant properties. It also can act as a mitigating factor of cancer, osteoporosis, glycuresis and immune systems. In addition, coumestrol also can attenuate endothelium-dependent coronary vasorelaxation enhanced by beta estradiol and be able to suppress the agonist effects of SR12813 on human PXR activity. Through studying extraction, isolation, purification and detection of coumestrol in alfalfa, the paper aims to optimize the extraction and separation conditions of coumestrol and find a simple, reasonable and rapid extraction process line to provide material base for further research on the physiological activity. Meanwhile, the study made a series of statistics and analysis of the crowd intake of coumestrol to provide reliable basis for phytoestrogens risk assessment. The main research results are as follows:1. A spectrophotometry method was established for the determination of coumestrol in alfalfa. The methodology investigation confirmed that the method has good precision, linear relation, reproducible and recovery, can satisfy the realistic samples detection requirements. Response surface methodology (RSM) was employed to optimize extraction technology of coumestrol from Alfalfa. On the basis of single-factor experiments, a mathematical regression model was established for predicting extraction yield of crude coumestrol at different levels of three crucial extraction conditions for maximizing yield of crude coumestrol from Alfalfa. The analysis for this model by SAS V8.0 software indicated that the optimal extraction ratio of liquid to material, extraction temperature and extraction time were 27.28:1 mL/g,67.7℃and 13.77 h respectively. Under these conditions, the actual value of yield of crude coumestrol from Alfalfa was 2.64‰, in basic agreement with the predicted value.2. A high-speed counter current chromatography (HSCCC) has been used sueeessfully for the isolation and purification of coumestrol in alfalfa. Through the investigation of the layered time and distribution coefficient, four solvent systems suited for the isolation and purification of coumestrol in alfalfa Acetone- skellysolve B-ether-H2O(2:1:1:1),(1.5:1:1:1),(1:1:1:1)and H2O-DMF-acetone-CCl4(2:4:3:5) were selected. The chromatographic conditions of the solvent system H2O-DMF-acetone-CCl4 (2:4:3:5) and acetone- skellysolve B-ether-H2O (1.5:1:1:1) were optimized.A two systems-two steps method for purifying coumestrol in alfalfa was obtained by comparing the advantages and disadvantages of the two solvent systems above. The product obtained was identified with UV and LC-MS/MS as coumestrol, and the purity was over 97%.3. A method for identification and qualification of coumestrol in food by LC-MS/MS was developed. The chromatographic separation was achieved on a Agilent XDB-C18 column, the column temperature was 30℃, the mobile phase was Acetonitrile-H2O (55:45, V:V), the flow rate was 0.35mL/min, the analysis was realized by multiple reaction-monitoring anion modes. Due to the large difference of coumestrol content in food, under optimal conditions, four linear regression equations were established:Y=154.6389X+15.8700 (R2=0.991),Y= 102.3375X +134.3646 (R2=0.999),Y=116.9685X-10809.6435 (R2=0.999),Y=36.8953X+ 48246.5312 (R2=0.995); and the linear ranges of tyrosine and tryptophan were 0.4-4 ng/mL,0.8-28 ng/mL,100-600 ng/mL,500-1200 ng/mL. A nonlinear standard curve Y=225.187X3-0.233X2-385.180 (R2=0.997) was fitted by SPSS13.0. The limit of detection was 0.107 ng/mL and the quantitative limit was 0.357 ng/mL. The methodology investigation confirmed that the method has good precision, linear relation, reproducible and recovery, can satisfy the realistic samples detection requirements.4. The statistic analysis of the crowd dietary intake of coumestrol was made combining with the results of qualification of coumestrol in food and the crowd dietary survey. The range of the total crowd intake of coumestrol was 26.282-802.468μg/d, and the mean value was 113.182μg/d. The results of the statistic analysis provided the effective basis for the crows dietary risk assessment of phytoestrogen. |
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