Studies On The Preparation And Biological Activities Of Polysaccharides From Stigma Maydis

Stigma maydis(Corn silk),the dry style and stigma of Maize(Zea mays L.),has beneficial effects on inhibiting tumor,lowering blood sugar,diuresis,antioxida nt activities and so on.With the aim to gain the information on chemical structures and antioxidant activities of polysaccharides from stigma maydis(PSM),in this thesis,the separation,purification and structural analysis of PSM were firstly studied,then its in vitro antioxidant activity,as well as in vivo acute toxicity were conducted.Based on the results of previous work,the water extracting-alcohol precipitating method was used to prepare PSM,the extraction yield was 3.39%;the polysaccharides contents of PSM were determined using phenol-sulfate acid method,and found to be 9.19%.Then,the uronic acid contents were tested by m-hydroxydiphenyl method,and its analytical conditions were optimized using the single factor and orthogonal experiments.The optimal conditions for uronic acid determination were as following: the quantity of antipyonin solution was 5.0 mL,incubation time in hot water was 5 min,the quantity of m-hydroxydiphenyl solution was 80 ?L,and coloration time was 20 min.Under this condition,the uronic acid contents in PSM were found to be 5.9%,the average recovery of m-hydroxydiphenyl method was closer to theoretical values than that of carbazole-sulfuric acid method.In the following purification,crude PSM was firstly decolorized by AB-8 macroporous resin(decolorization rate 65.94%),then deproteinized using Sevage reagents(deproteinization rate 67.33%),after dialysis,the polysaccharides contents in PSM were raised up to 65.33%.Three monomers of PSM,namely,PSM-1,PSM-2 and PSM-3,were separated by column chromatography using DEAE-cellulose 52 as packing materials.After being precipitated by ethanol,the contents of polysaccharides in PSM-1 were found to be 80.67%(uronic acid was not detected);polysaccharides contents in PSM-2 were 79.26%(uronic acid contents were 71.56%);polysaccharides contents in PSM-3 were 77.45%(uronic acid contents were not quantified).The main components of PSM,i.e.PSM-1 and PSM-2,were further separated by Sephadex LH-20 column chromatography to gain 15.30 mg of PSM-1(purity 95.16%),and 29.22 mg of PSM-2(purity 96.57%).No noticeable peak was observed in UV spectrum both at 260 nm and at 280 nm,suggesting that nucleic acids and proteins had been removed thoroughly.In the IR scanning,correlative characteristic peak of uronic acid at 1745 cm-1 was found,indicating that uronic acid exists in PSM-2.In the following monosaccharide analysis by LC-MS/MS,we found that PSM-2 is composed of L-rhamnose,D-arabinose,D-xylose,D-mannose,D-glucose,D-galactose,and the molar ratio is 0.92: 2.55: 0.75: 1.38: 1.0: 2.61,however,uronic acid was not detected,which was contradicted against the results of IR analysis,needed to be studied further.In the in vitro antioxidant evaluation,DPPH and hydroxyl radicals,coupled with reducing power to Fe3+ were used to evaluate the antioxidant activities of crude PSM,PSM-1,and PSM-2.The results showed that the above-mentioned samples possess scavenging capacities against DPPH and hydroxyl radicals,and have reducing power to Fe3+ in a certain concentration range,respectively,and a dose-dependant manner was also observed.Based on the comprehensive effects mentioned above,the sequence of antioxidant activity was crude PSM > PSM-2 > PSM-1,indicating that the purification process may have adverse effects on activities,and some substances possessing antioxidant activities may be removed together during purification,which is deserved to be studied further.It also suggested that uronic acid contents may be positively correlated to the antioxidant activities.Finally,due to the excellent antioxidant activities of crude PSM,making it a promising potential to be developed as functional food,the acute toxicity was conducted to assess its safety preliminarily.Mice were randomly divided into four groups,i.e.normal control(NC,distilled water),low dose(7.5 g/kg BW of crude PSM),medium dose(15 g/kg BW of crude PSM),and high dose(20 g/kg BW of crude PSM)groups,tested samples were orally administered once in 24 h.Then,body weight,food intake,and clinical signs relating to toxic reactions were observed and recorded.On the 15 th day,after being fasted for 16 h,the blood and organ samples were harvested for the determined of biochemical indexes and relative organ weights examination.The results exhibited that no noticeable clinical signs and organ weight changes associated with toxicity were observed,and no significant differences(P > 0.05)in body weight,food intake,biochemical indexes,and relative organ weights were found compared with the mice in NC group,indicating the crude PSM possesses high safety,and its maximum tolerated dose may be up to 20 g/kg BW.