| Allium macrostemon bge. is dry bulb of lily branch plant Allium macrostemon Bunge or A. chinese G. Don. Allium macrostemon bge. has the characteristic of spicy, bitter, warm and the fuction of regulating the flow of vital energy, relieving chest, passing positive, relieving congestion.It is used for the treatment of chest pain,lung diseases such as asthma in traditional Chinese medicine for a long time. Allium macrostemon bge. is a kind of the extensive style of economic crops having dual purpose of medicine and food, which has good resistance for drought, tightness, low temperature and strong adaptability.It is rich in resources in China, and has the important development future. Modern research has shown, Allium macrostemon bge has the functions of anticancer, anti-aging and so on. Chemical components such as the volatie oil, soap glucoside, the prostaglandin, compounds containing nitrogen and so on have been separated from Allium macrostemon bge., but there are few reports on polysaccharide from Allium macrostemon bge.In this paper, we study mainly the enzymatic extraction, separation, purification, molecular weight determination and physico-chemical properties of polysaccharide from Allium macrostemon bge., and study the interaction of Allium macrostemon polysaccharide with DNA by fluorescence probe method. The results were as follows:This study, which was investigated through single factor test and L9(34) orthogonal experiment, has researched the influence of factors including the temperature, time, pH, and dosage of enzyme on the extraction rate of Allium macrostemon bge. polysaccharides. The results showed that the factors having effect on extraction rate were enzyme dosage (D)> pH value(C)> temperature (A)> time (B) successively. Enzyme dosage and pH was the main factors. The optimum extraction technology was A2B3C2D3, namely the optimum temperature is50℃, time is2.5h, pH is5.0, enzyme dosage is0.4%. Under these conditions, the extraction rate of soluble polysaccharides from Alluim macrostemon Bge. was17.27%. The extraction rate of soluble polysaccharides from Alluim macrostemon Bge was12.78%by water extraction. So the cellulose enzyme extraction is better than the water extraction.After removing the protein by protein enzymes and Sevag method, discolor by H2O2, penetration by dialysis bag from Allium macrostemon bge, we got part-pure product of polysaccharide (AMP). And the optimum extraction technology about discolor by H2O2is concentration10%, temperature60℃, time4h.AMP was separated by ultrafiltration, DEAE-32cellulose chromatography and Sephadex G-100gel column chromatography sequencely. It was separated into two fractions (AMP-Ⅰ, AMP-Ⅱ) by ultrafiltration. Then AMP-Ⅰ was separated into AMP-Ⅰ a, AMP-Ⅰ b and AMP-Ⅱ was separated into AMP-Ⅱa, AMP-Ⅱb, AMP-Ⅱe by DEAE-32cellulose chromatography. Lastly, AMP-Ⅰa、AMP-Ⅱb、AMP-Ⅱ c was further purified by Sephadex G-100. The identification of purity implies that AMP-Ⅰ a and AMP-Ⅱb are homogeneous components by HPLC.Molecular weight of the polysaccharides is determined by HPLC. The average molecular weights of AMP-Ⅰ a, AMP-Ⅱb are respectively1.86×105Da,6.43×104Da. AMP-Ⅰ a is white powder, AMP-Ⅱb is light yellow powder, and both of them are soluble in water.The interaction of Allium macrostemon bge. polysaccharide with DNA was investigated through ethidium bromide(EB) as a sensitive fluorescence probe. The results indicated, in the same concentration (0.1000mg/ml), that the interaction sequence of different polysaccharides with DNA is as follows:AMP-Ⅱ b24.49%> AMP-Ⅰ a20.90%> crude polysaccharide10.38%> crude polysaccharide (Not removed protein)5.31%, and the higher purity of the polysaccharide is, the stronger the interaction of AMP with DNA is, so Allium macrostemon bge. polysaccharide is a active component which has interaction with DNA.The concentration of AMP and the degree of interaction of polysaccharide with DNA are directly relative, and the better concentration of AMP-Ⅱ b is0.2975mg/ml. |
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