Study On The Purification And Primary Structure Of Water-Soluble Longya Lily Polysaccharide And Its Biological Activities

Lily is one of the first batch of edible and medicinal food approved by the Ministry of Health.To date,The research results home and abroad have demonstrated that Lily Polysaccharide has many bioactivities,such as lowering blood sugar and antioxidation,etc..But the purification of Lily polysaccharide was very complicated.At the present time,coarse extracts of Lily polysaccharide are mostly used in the research of its bioactivities and pharmacology.It is obvious that this could not elucidate ennough the bioactivities of Lily polysaccharide.Then,it isn't enough that is the research about structure and conformation of Lily polysaccharide.At the same time,the results about the research of Lily polysaccharide are different because different researchers chose different materials and methods.In my study,Longya Lily polysaccharide(LLP) that was extracted from Longya Lily bulbs was investigated by using a series of analytical chemistry and modern instrument analysis techniques,including establishment of a more reasonable content determination method and purification procedure,determination on molecule weight,determination on composition of LLP,indetification on primary structure of sugar chain and conformation of LLP,bioactivities tests based on purified LLP.The main research results obtained from my study are outlined as followed:1.Establishment of a more scientific method for Lily polysaccharide content determination and calculation.The anthrone-sulfuric acid method and conversion factor were used to determine and calculate the Lily polysaccharide content.The results determined by this new method are accuracy,reproducible that indicated this method is suitable for Lily polysaccharide content determination and calculation. The conversion factor of LLP is 2.718 determined by above method,and Average polysaccharide content of fresh Longya Lily bulbs is 28.18%.2.Establishment of Lily polysaccharide purification procedure,identification method of purity and determination of molecule weight.The purification procedure of Lily polysaccharide was established by using DEAE-Sepharose Fast Flow column (4.6cm×80cm),and two main components LLP₁,LLP₂(Longya Lily Polysaccharide) were obtained.The result of high performance gel permeation chromatography(HPGPC) indecated LLP₁ is a homogeneous polysaccharide component,its purity exceeds 98%and molecule weight(Mw₁) is 11756 Dal.. LLP₂'s maximum polysaccharide and protein absorption were overlapped implying that this component is a glycan protein complex,its purity is 98%and molecule weight(Mw₂) is 1038773 Dal..The results demonstrated that DEAE-Sepharose Fast Flow column(4.6cm×80cm) is compatible for the purification of Lily polysaccharide and this method is fast,simple,effective and fit to be magnified.3.The physical and chemical properties of LLP₁,LLP₂ were studied.The results show that LLP₁ is white flocculent solid,very easy absorption of moisture,and dissolvable in water and dimethyl sulfoxide,especially in hot water,and insoluble in organic solvent,such as high-concentration ethanol,acetone,etc..The water solution of LLP₁ is transparent and melicera,and the results of the Molish reaction, anthrone-sulfuric acid reaction and carbazole-sulfufic acid reaction show that LLP₁ contains sugar and uronic acid.Coomassie brilliant blue reaction indecated LLP₁ contains no protein.LLP₂ is light yellow flocculent solid,easy absorption of moisture,dissolvable in hot water and dimethyl sulfoxide,and insoluble in organic solvent,such as high-concentration ethanol,acetone,etc..The water solution of LLP₂ is translucent and has strong viscosity.The results of the Molish reaction, anthrone-sulfuric acid reaction and carbazole-sulfuric acid reaction show that LLP₂ contains sugar and uronic acid.Coomassie brilliant blue reaction indecated LLP₂ contains protein.At the same time,the conformation of two polysaccharides was studied.The results of I₂-KI reaction indecated that two polysaccharides contains relatively long side chain and many branches.LLP₁ and LLP₂ have no triplehelix structure difined by Congo red reaction.4.The primary structures of LLP₁,LLP₂ were obtained by using many chemical methods(sugar component analysis,uronic component analysis,periodate oxidation, Smith degradation,etc.) and many instrumental methods(such as UV,IR,GC,¹H NMR,¹³C NMR,etc.).Contents of uronic acid and neutral sugar in LLP₁ are 4.02%,94.08%,respectively.The primary structure of LLP₁ was outlined as below: LLP₁ consists of Glc:Man:Ara and their molar ratio are 18.60:25.14:1.00.It is acidic polysaccharide and doesn't contain methyl glucose.The core structure of the main chain consists of Glc,Man.The branch chain consists of Ara and uronic acid,Glc exists 1,4-linked Glcp and 1,6-linked Glcp forms and Glc exists at the terminal end. Man exists 1,4-linked Manp form.All monosaccharide linkages areα-forms.Contents of uronic acid and neutral sugar in LLP₂ are 77.13%,19.46%, respectively.The primary structure of LLP₂ was outlined as below:LLP₂ consists of Gal:Rha:Ara and their molar ratio are 3.58:1.00:1.09.It is acidic polysaccharide and doesn't contain methyl glucose.The core structure of the main chain consists of uronic acid linked in 1→4 form,The branch chain consists of Gal,Ara and Rha. LLP₂ doesn't exist 1,6-linked forms.All monosaccharide linkages areα-forms.5.The bioactivities of LLP₁ and LLP₂ were studied by four different systems (the inhibition effects toα-amylase,DPPH·,O₂^-·and·OH).The results indicated the inhibition effects of LLP₁ and LLP₂ toα-amylase increased with their concentration increase,and LLP₂'s effect is higher than LLP₁'s based the same concentration.LLP₁ has no effect of scavenging DPPH·and possesses lower inhibitory effect on the O₂^-·. However,LLP₁ has obvious scavenging action to·OH.LLP₂ has lower scavenging ability to O₂^-·,while it possesses distinctive scavenging ability to DPPH and·OH. The test results show that the bioactivities of LLP₂ is higher than those of LLP₁ at certain concentration in the four different systems,related to content of uronic acid, structure-activity relationship and whether it is a glycan protein complex.