| Allium macrostemon Bunge is a lily branch plant, or called purple root garlic, root garlic. Chinese herbalist doctor use it for the treatment of diseases such as chest tightness, lung asthma commonly. It is tasted with slightly bitter, acrid, warm. It has good resistance for tightness and drought, distribution in many places of China which is used also as food, so it shows the extensive prospect of development and application. Allium macrostemon has antioxidant, antitumor, antibacterial and other activities. Many chemical components include the prostaglandin, soap glucoside, volatie oil and so on have been separated from it, but the polysaccharide of Allium macrostemon is rarely reported.This paper focuses on the water extraction, purification, the purity and molecular weight determination, composition analysis, sulfation, biological activities of Allium macrostemon polysaccharide. The results are as follows:(1) Allium macrostemon polysaccharide was extracted by water.The optimum extraction process of the polysaccharide are90℃extracting temperature,80min of extraction time,1:14of ratio of solid to liquid. Under these conditions, we get the crude polysaccharide (cAMP), the extraction rate of was14.26%.Then the extract was treasted by protein enzymes and Sevag method to remove the protein of cAMP, discolor by3%H2O2,60℃temperature,4hours. The results show a good protein removal and decolorization, and we got part-pured Allium macrostemon polysaccharide (AMP) by dialysis lastly.(2) AMP was separated and purified by both column chromatography and graded ethanol precipitation method, and the purity and molecular weight were determined by gel permeation chromatography.Through DEAE-cellulose and Sephadex G-100column chromatography,we got three polysaccharides as AMP-I’(1.50×104Da), AMP-Ⅱ’(8.95×105Da), AMP-Ⅲ’(1.51×10Da), which were homogeneous components,.and the final yield is9.36%,2.23%,5.68%respectively. By the graded precipitation, we got two weight-uniform polysaccharides namely AMP-I (1.57×104Da) and AMP-Ⅲ (1.55X106Da),which yield is17.31%,7.81%. Comparison of the two methods, the graded precipitation was choosen for the further purification, AMP-Ⅰ and AMP were selected for the chemical modification, and then together with AMP-III’for biological activity research.The structural analysis of homogeneous polysaccharides was determined by thin layer chromatography and high efficiency liquid chromatography, the results showed that AMP-I is composed of glucose and galactose, AMP-Ⅲ is composed of glucose, fructose and galactose. The principal chain of AMP-I is composed of glucose and galactose. The core polysaccharide is glucose. The terminal residues of branched chain or principal chain are composed of glucose and galactose. The principal chain of AMP-Ⅲ is composed of glucose, fructose and galactose. The core polysaccharide is glucose and galactose. The terminal residues of branched or principal chain are composed of glucose and fructose.(3) AMP and AMP-I were sulfated by the means of chlorosulfonic acid-pyridine, sulfurtrioxide-pyridine, concentrated sulfuric acid respectively.Through the analysis of physicochemical properties, ultraviolet and infrared scanning, the results showed that all of the polysaccharide and esterification reagent reacted to generate the ester. The degree of substitution (DS) was determined by BaSO4turbidimetric method, the results indicated that DS of AMP-I is higher than the one of corresponding AMP. The degrees of substitution were different among the three esterification methods. DS of the polysaccharide modified by chlorosulfonic acid is the highest; DS by sulfuric acid is the lowest.Allium macrostemon polysaccharides were sulfated with chlorsulfonic acidy-pyridine. Reagent proportion, reaction time and reaction temperature were selected with substitution degree and yield as index by L9(34) orthogonal design. The optimal technical parameters were pyridine/chlorosulfuric ratio5, temperature80℃and reaction90min. The degree of substitution of AMP was1.624, the yield was81.24%, and DS of AMP-I was1.735, the yield was83.13%.(4) The ability of the Allium macrostemon polysaccharides to scavenge hydroxyl radi-cals was tested by phenanthroline-Fe2+-H202system. The results indicated that, both the ori-ginal polysaccharides and sulfated polysaccharides have scavenging effects on hydroxyl free radical, and the effect enhances along with the increase of concentration basically. The scavenging rate of un-modified polysaccharide ranges as follow:AMP-Ⅲ> AMP> AMP-I. The highest scavenging effect is AMP-Ⅲ’, which has reached51.32%as8mg/mL. The modified polysaccharide decreases on the scavenging effect of hydroxyl free radical.(5) The ability of the interaction of polysaccharides with DNA was tested by ethidium bromide (EB) as a fluorescent probe. Different polysaccharides have different interaction constants (D) and a maximum in a certain range. The maximum constant of un-deproteined polysaccharide (cAMP) is the smallest (11.07%, an half of AMP), and the corresponding concentration is the highest (0.3mg/ml).This showed that the main active components with DNA are polysaccharides, rather than protein. The role of sulfated polysaccharide has a larger increase than the original one, the most is sAMP-I’, as38.25%, the concentration is0.01mg/ml. The interaction sequence of the polysaccharides by three sulfated methods with DNA is as follows:chlorosulfonic acid> sulfurtrioxide-pyridine> concentrated sulfuric acid, and the sulfated method by chlorosulfonic acid is superior to other two methods. |
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