| Alfalfa saponins is a kind of secondary metabolite by alfalfa and has a great high biological activity. In some papers show that alfalfa saponins can reduce the cholesterol of eggs and the piglets’ diarrhea, enhance antioxidant ability of animals. The objectives of the experiments, therefore, were to study the influence of adding alfalfa saponins to feeds on the angiogenesis, and metabolism of fat for chick embryo. Alfalfa saponins was separated by thin layer chromatography(TLC) and high performance liquid chromatography(HPLC), and then the single alfalfa saponins were identified by HPLC-MS. The anti-oxidation of alfalfa saponins was further researched in vitro. Ckick chorioallantoic membrane(CAM) was carried for studying on the angiogenesis of chicken embryo and we also discussed the effect of alfalfa saponins on the metablism of fat during the egg hatching.1.Alfalfa saponins was extracted with methanol by ultrasonic wave. Alfalfa saponins were separated by TLC(silica gel plate 5 cm × 20 cm) with the developing solvent of chloroform: methanol: water( 65:35:10, the under layer), the plats were observed at 254 nm and collected the separate samples; Five alfalfa saponins compounds were obtained by TLC, the Rf values were I(0.08)、II(0.18)、III(0.49)、IV(0.63) and V(0.80), respectivly. All the separate samples were determined by HPLC. Venusil Mp C18(2)(4.6 mm×250 mm) column was used with mobile phase: methanol- water(15:85) at a flow rate of 0.15 m L/min、0.2 m L/min and methanol- water(20:80) at a flow rate of 0.3 m L/min, detecting wavelength: 200 nm, column temperature: room temperature. Thirteen single alfalfa saponins were detected by HPLC and identified by HPLC-MS. A compound with m/z=475 in the LC-MS experiments was due to soyasapogenol A(MW=474). The indicated that the antioxidant capacity of alfalfa saponins was increased in vitro at ranges from 0.09 mg/m L to 0.72 mg/m L, while the rates of scavenging effects on hydroxyl radical(?OH) was increased from 17.46% to 70.29%, for superoxide radical(O2-?) was from 9.23% to 46.38%, and for DPPH was increased from 26.86% to 76.12%, respectively.2. Ckick chorioallantoic membrane(CAM) model was carried for the research the effects on the angiogenesis by alfalfa saponins at the ranges of 0.00~0.36 mg/m L. 150 fresh and healthy eggs were prepared for the experiment. 10 eggs were hatched for natural status, 140 eggs were divided for 4 groups with 3 mm alfalfa saponins paper pills on its ckick chorioallantoic membrane(CAM) blood, the contents of 4 groups were 0.00 mg/m L(0-dose), 0.09 mg/m L(low-dose), 0.18 mg/m L(median-dose) and 0.36 mg/m L(high-dose), respectively. The results showed that the number of the first and second grade vascular in medial-dose group were more than control group, the increasing number is 34 and 42(P < 0.05), respectively. The high-dose group enhanced 63 and 69(P < 0.05), respectively. Comparing with control group, the number of small vascular in median-dose and high-dose groups were increased 26 and 46(P < 0.01), respectively, but for great wessels, of all the three experimental groups were enhanced 3、3 and 2(P > 0.05), respectively. It indicated that the small vascular was increased by alfalfa saponins from 0.00~0.36 mg/m L, the neovascularization was improved by alfalfa saponins and its growth promoting effect was positive correlation with the content of alfalfa saponins.3. Experiment 3 was focused on the effects of alfala saponins on chick embryonic development by micro hole injection at three key time. 220 fresh and healthy eggs were selected for the experiment. 100 mL alfalfa saponins(0.32 mg/m L) was injected in the eggs on 0 d, 7 d and 18 d, the control group was normal incubated without micro hole. The result showed that:(1) Alfalfa saponins had no influence on chick birth weight at different injection day(P > 0.05), but the hatching rate was increased. The highest hatching rate was on d 18, 12.5% and 10.7% was increased respectively comparing with d 0 and d 7.(2) Serum lipid was lowest on d 7. Comparing with the control group, T-CHO and TG was extremely significant decreas ed 74.45% and 27.5%, respectively(P < 0.01), LDL-C was significantly reduced 48.5%(P < 0.05). The antioxidant ability was the highest on d 18. T-AOC was increased significantly by 42.25%(P < 0.01) and T-SOD was enhanced 35.56%(P < 0.05). But, the HDL-C in serum were not different from all the treatments(P > 0.05).(3) The 7th d injection group’s alfalfa sapoins changed the rate of sarurated fatty acid and unsarurated fatty acid in the muscle and abdominal fat of the chicks. On d 7 injection treatment, the content of saturated fatty acid in the muscle was 66.41%, and decreased 4.37% than control group, the content of unsaturated fatty acid was 26.47%, and increased 3.55%. The saturated fatty acid in abdominal fat was 36.33%, and decreased 1.24% than control group, the content of unsaturated fatty acid was 63.36% and increased 2.64%. The results show that alfalfa sapoins should be changed the rate of saturated fatty acid and unsaturated fatty acid in the muscle and abdominal fat of the chicks. |
PDF Full Text Request