Studies On The Chemical Constituents Of Alfalfa And The Influences Of Environmental Factors On Flavonoid

Alfalfa(Medicago sativa L.) is a major legume forage crop rich in good quality protein andfirber. It is distributed mainly in the north and southwest, of China. Alfalfa is a good source ofbioactive constituents,such as saponin, flavonoid, polysaccharides. It exhibited various biologicalactivities,for examples:lowering cholesterol levels, decreasing the risk of atheroma and coronaryheart disease, enhancing body immunity and controlling tumor.But now which is the mainbioactive constituent is still unknown.And while the method for determination of saponins hasbeen established, very little is done on the flavonoids. In this paper, based on the systemicchemical research of alfalfa, the chemical study on alfalfa,the quality study on the differentvarieties of the plant and the environmental influences on flavonoids were carried out so as toprovide foundation of the research on the quality control, seed breeding and artificial cultivated..1. Studies on the constituents of alfalfa extract12 compunds were isolated from the ethanol extracts of the aerial parts of alfalfa by variouscolumn chromatographies, and were identified on the basis of spectral analysis(including MS andNMR).There are 3 flavonoids(tricin, apigenin, 7,4′-dihydroxyflavone), 1 saponin(soyasapogenolB-3-O-β-glcA), 1 alkaloids(berberine), 2 anthraquinones(emodin, emodin 8-O-β-D-glucoside), 3organic acids(o-hydroxybenzoic acid, (E)-p-hydroxycinnamic, (Z)-p-hydroxycinnamic),1steroids(β-daucosterol) and 1 fatty alcohol (n-triacontanol), of which 4 were isolated from sativa L.for the first time.2. Studies on the chemical analysis method of flavonoidsSample preparing: 1g Sample was put in a 50ml measuring flask which was filled up with 70％ethanol, extracted for 30min at 45℃waterbath by ultrasonicate.HPLC chromatography: Samples were applied to Symmetry ODS2-C18 (4.6×250mm, 5μm),and eluted with 0.1％phosphoric acid with an increasing gradient of acetonitrile(0 to 5min,5％;5 to 10min,5％to 10％;10 to 25min, 10％to 19％;25 to 50min, 19％to 20％; 50 to 60min,20％;60 to100min,20％to 32％) at a flow rate of 1ml/min. The column temperature was 30℃.Detectionwavelength was set at 340nm.3. Studies on the influence factors to flavonoids of alfalfaThe content of flavonoids has carried on the research from three respects:harvest periods,varieties, and growing temperature. Main results were as follows:First,the influences of varieties on flavonoids were determined.The mean difference issignificant at the level of 0.05.The content of flavonoids is higher in "Fenart", "Avilain", andlower in "Reformer" through the comparison of flavonoids' content from 9 varieties of alfalfaplanted in the same area and harvested at the same time..Second, the total flavonoids'content was significantly different at different harvest period.It washigher in October, while the concertration of apigenin and tricin were higer in the same alfalfavarieties harvested in August. The concertration of 7,4′-dihydroxyflavon showed a differentchange from the other two flavonoids.Finally, The growing temperature has the most significant influence on the content of flavonoids.It'smean difference is significant at the level of 0.05.The concertration of 7,4′-dihydroxyflavon wasincreased instantaneously on the forth day when the testing plant was at 10℃and 30℃. Theapigenin level didn't change obviously.