The Study On Extraction, Purification And Biology Action Of Polysaccharides From Cephalanoplos Segetum(Bge.) Kitam

Cephalanoplos segetum(Bge.) Kitam called thorn vegetable, wild red flower, green vegetable, is one kind of feverfew and is extensively grow in China and rich in resource. As the traditional Chinese medicine, it can cool the blood and stop bleeding, and other pharmacological activities. Besides, this wild plant is fonded by people because of its abundant nutritional ingredient and can be consumed as food and medical materials as well. Cephalanoplos segetum(Bge.) Kitam is very active in pharmacological aspect while poses no poisonous effect to the body, so it is widely applied to clinical service and sold in health product market. This experiment studies its extraction, separation, components, and active functions.This experiment adopts dry Cephalanoplos segetum(Bge.) Kitam which grows in Zhouzhi within Shaanxi province as the raw material researchs its nutrition ingredients and studies the best way of extraction, separation, and also researches its purity, molecular weight, component. What's more, this paper also focuses on its antioxidative activities by the means of H₂O₂/Fe²⁺ system and polyunsaturated fatty acid(PUFA) of the lipoprotein induced by Fe²⁺ system. This experiment is carried out by putting the polysaccharides of Cephalanoplos segetum(Bge.) Kitam in animal's body to research its biology action. This study provides reference and gives evidence to exploit and utilize Cephalanoplos segetum(Bge.) Kitam.The main experiment result is as following:1. The content of ash in Cephalanoplos segetum(Bge.) Kitam was 8.760% , crude protein 4.470%crude fat 0.430%. The content of 17 determined total amino acids added up to 10.302%, which included 7 essential amino acids accounting for 42.526%of total amino acids. 15 mineral elements had been determined, including 9 essential elements among which the contents of Ca and Mg were higher. The contents of harmful elements Hg, As, Pb and Cd were lower.2. The best extracting technology is in hot water is: at 90℃, extracting 120min, and the ratio of material to liquid is 1:10.3. With the help of ultrasonic, the best technology parameter of extracting is: at the power of 250W, extracting 20 mins, and the ratio of material to liquid is 1:15.4. The best way of extracting protein is using enzyme combined with Sevag method, which can nearly extract the total protein in raw polysaccharide, while degrade the loss rate of polysaccharide. The protein extraction rate is 96.45%.5. Four kinds of polysaccharide as CSK-A', CSK-B', CSK-C', CSK-D' areseparated by DE-52 and Sephadex G-200 chromatography.6. Four kinds of purification identification for the refined products: no nucleic acid and protein absorption peaks appear in the UV spectrum at the places of 260nm and 280nm. After the electrophoresis, there emerges a sole ribbon in the acetic acid membrane. After extracting by SephadexG-200 then, peaks of the four polysaccharides are basically and evenly symmetrical.7. Composing and Structure analysis: their molecular weights are 20.7kD, 28.6kD^ 1481dX 501.2kD respectively measured by Sephadex G-200 chromatography. The result in the spectrum indicates that CSK-A' is mainly composed of xylose, galactose and glucose, with the mass ratio of 6.31:8.52:1.03;CSK-B' is mainly made up of rhamnose, sorbose and glucose, with the mass ratio of 6.97:1.52:0.98;CSK-C is chiefly composed of xylose, fructose, sorbose and glucose, with the mass ratio of 7.01:8.93:3.04:1.03;CSK-D' is formed mainly by xylose, fructose, sorbose and glucose, with the mass ratio of 2.10:9.85:1.05. Infrared spectrum reveals that the Infrared spectrums of these four polysaccharides are classical absorption spectrums, that is, at the place of 1400 cm"'-1200 cm"1 there is angle change vibration, at the place of 2930 cm" there appears C-H stretching out and drawing back vibration area, at the place of 3400 cm"1 there emerges absorption wide peak caused by O-H stretching out and drawing back vibration, at the place of 1200cm"'-1030 cm"1 there is absorption peak that is formed by C-0 stretching out and drawing back vibration. In addition, the absorption peak around the area of 1110 cm"1 shows that the monosaccharide in the polysaccharide is hexahydric heterocycle, with the point of 1640 cm"'-1600 cm"1 as the characteristical peak. At the place of 1075 cm"1-1000 in the Infrared spectrum there appears an absorption condition. So we can deduce that it is one type of B-pyrane polyoses that contains acetamide group.8. The test of antioxidative activities indicates:(1) CSK-B, CSK-C, CSK-D can remove OH that is produced by Fenton reaction through H2O2/Fe2+ system. With the increase of amount, the rate of reduction goes with the same trend, so there is a mutual relationship between reduction rate and quantity of polysaccharides, in which CSK-D poses the highest restraining effect, 1.8ml usage reaching 67.29%, with CSK-B coming as a close second. Although CSK-C has some reduction effect, it is comparatively weaker. While CSK-A almost has no such effect.(2) CSK-A, CSK-B, CSK-C has antioxidative effect to the polyunsaturated fatty acid (PUFA) of the lipoprotein induced by Fe2+ system. With the increase of amount, the rate of reduction goes with the same trend, so there is a mutual relationship between reduction rate and quantity of polysaccharides, in which CSK-C poses the strongest restraining effect, 2.5mg/ml usage reaching 53.64%, followed by CSK-A, CSK-B.While CSK-D almost has no such effect. 9. Animal experiment indicates:(1) The polysaccharides of Cephalanoplos segetum(Bge.) Kitam can help decrease the content of triglyceride(TG) and total cholesterol(TC), increase the ratio of high-density lipoprotein (HDL-C) in the blood serum of mouse, and obviously regulate its metabolization of lipid.(2) The polysaccharides of Cephalanoplos segetum(Bge.) Kitam can stop mouse from getting old that is caused by D-galactose: it could increase the activities of blood superoxide dismutas(SOD), glutathione peroxidase(GSH-PX), catalase (CAT) of mice, liver and brain organization, decreased the content of the malondialdehyde(MDA)and the activity of monoamine oxidase(MAO). Which indicate that it can reduce the speed of getting old from D-galactose.(3) The polysaccharides of Cephalanoplos segetum(Bge.) Kitam can improve the motion capability of mouse, lower the content of blood urea nitrogen(BUN) of post-exercise mice, decrease the consumption of liver glycogen and increase the activity of lactic dehydrogenase(LDH). Which prove that it can effectively ease the hurt to mouse that is caused by over-motion.