Preparation Of Eucheuma Low-molecular-weight Seaweed Polysaccharides As Well As Study On Absorption And Bioactivities

Objective:To establish a method taking Eucheuma as raw materials to prepare low-molecular-weight seaweed polysaccharides and detect its characteristics such as viscosity, gel strength and sulphate mass fraction. To determine its molecular weight with high performance liquid chromatography-mass spectrometry (HPLC-MS) and high performance gel permeation chromatography(GPC). To study on the absorption of low-molecular-weight seaweed polysaccharides,as well as its effect on food absorption and bioactivities such as anti-oxidant and immunomodulation functions.Methods:There are the preparation and analytical methods, as well as experimental methods on absorption and bioactivities.1Preparation of Eucheuma low-molecular-weight seaweed polysaccharides:1.1Clean and cut:Eucheuma was cut into slice form after cleaning by running water and open-air drying.1.2Twice hydrolyze in acid liquor:The collected Eucheuma slice was placed and hydrolyzed in the solution of hydrochloric acid (pH2.5). Then the collected slice was filtrated, placed and hydrolyzed in the solution of hydrochloric acid (pH4.5),too. The collected slice was filtrated and washed by distilled water once more time。1.3Saponification in lixivium:The collected Eucheuma slice was placed and saponified in alkaline solution (pH8). The collected slice was filtrated and washed by distilled water again.1.4Dry and ground:After the collected Eucheuma slice was dried in the dehydrator, it was placed in disintegrator and grounded to small particles. The powder was Eucheuma low-molecular-weight seaweed polysaccharides.1.5Extraction yield of sample: Extraction yield(%)=sample(g)÷Eucheuma dry weight(g)×1002Characteristic detection and analysis of sample:2.1Detection and analysis of oranoleptic indicator, viscosity, gel strength, sulphate mass fraction, humidity and total ash.2.2Determination of its molecular weight with high performance liquid chromatography-mass spectrometry (HPLC-MS) and high performance gel permeation chromatography. 3Study on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in fasting mice:Thirty Kunming mice were randomly divided into normal control group, carrageenan control group and experimental group (Eucheuma low-molecular-weight seaweed polysaccharides group). After fasting and drinking for12h, the carrageenan control group and experimental group were intragastrically given0.09g/mL carrageenan and Eucheuma low-molecular-weight seaweed polysaccharides,1mL each time, once every3.5hours,4times continuously. An equivalence of distilled water was administered in the normal control group. Wet fecal weight and dry fecal weight were measured and food absorbtivity was calculated. The blood was obtained in venous plexus behind the eyeball to detect the content of serum D-xylose.4. The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in mice fed a normal diet:Thirty Kunming mice were randomly divided into three groups:normal control group, carrageenan control group and experimental group(Eucheuma low-molecular-weight seaweed polysaccharides group).The mice in all of groups were fed normal diet.The rats in carrageenan control group and experimental group were intragastrically given0.05g/mL carrageenan and Eucheuma low-molecular-weight seaweed polysaccharides,1mL each time, twice one day,7days continuously. An equivalence of distilled water was administered in the normal control group. The body weight, food consumption, wet fecal weight and dry fecal weight were measured. Food availability and food absorbtivity were calculated according to the below formula:food availability (%)=(the final body weight-the initiate body weight)÷food consumption in experimental session×100; food absorbtivity=(food consumption in experimental session-dry fecal weight in experimental group)÷food consumption in experimental session×100.5The effect on digestion and absorption, anti-oxidant and immunomodulation function of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:Fifty Wistar rats were divided into five groups:normal control group, high-fat diet control group, carrageenan control group, low dose and high dose Eucheuma low-molecular-weight seaweed polysaccharides groups. All of the rats were continuously fed for30days.Body weight, food consumption,wet fecal weight and dry fecal weight were measured. Food availability and food absorbtivity was calculated. Na+-K+-ATPase activities were measured by spectroscopic analysis. Blood glucose, triglycerides(TG), total cholesterol(TC) and the activity of glutathione-peroxidase (GSH-Px), the content of malanydiadehyde (MDA), oxidized low-density lipoprotein (ox-LDL), interleukin-2(IL-2),tumor necrosis factor-a(TNF-a) and the proliferation of splenic lymphocyte were detected in each group.Results: 1Preparation of Eucheuma low-molecular-weight seaweed polysaccharides:The method to prepare low-molecular-weight seaweed polysaccharides was established and the sufficiency experimental sample was prepared. Extraction yield of the Eucheuma low-molecular-weight seaweed polysaccharides was48.31%.2The oranoleptic indicator and the main characteristic of Eucheuma low-molecular-weight seaweed polysaccharides:The sample of Eucheuma low-molecular-weight seaweed polysaccharides was sandy beige powder. It was dissolved in water.Its viscosity was10mPa·s and gel strength was below10g/cm2.The sulphate mass fraction of it was limited in15%-40%which was national standards of carrageenan.3The molecular weight of Eucheuma low-molecular-weight seaweed polysaccharides:Its number-average molecular weight which was determined by high performance gel permeation chromatography was1199Da.The most molecular weight of Eucheuma low-molecular-weight seaweed polysaccharides which was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) was under1000Da.The molecular weight was far below carrageenan.4The digestion and absorption condition of Eucheuma low-molecular-weight seaweed polysaccharides in fasting mice:The dry fecal weight and wet fecal weight of the experimental group were (0.168±0.029)g and (0.218±0.031)g, respectively. The food absorbtivity was (57.78±6.06)%. The conten of serum D-xylose was (2.71±0.42) mmol/L. The dry fecal weight and wet fecal weight of the Eucheuma low-molecular-weight seaweed polysaccharides group were lower than the carrageenan group,but the food absorbtivity and the conten of serum D-xylose were higher than the carrageenan group, significantly (P<0.05). These show that the food absorbtivity of Eucheuma low-molecular-weight seaweed polysaccharides is higher than that of carrageenan,but feces and fecal moisture capacity are lower than those of carrageenan. The cause is relevant with the lower molecular weight and hydrophilia of Eucheuma low-molecular-weight seaweed polysaccharides than carrageenan.5The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in mice fed a normal diet:The dry fecal weight and wet fecal weight of the experimental group were (2.19±0.29) g/d and (3.10±0.31) g/d, respectively. All of them were significantly higher than those of the normal control group,but lower than those of the carrageenan group (P<0.05).The food availability and food absorbtivity were (15.52±0.91)%and (39.17±3.45)%. All of them were significantly lower than those of the normal control group,but higher than those of the carrageenan group(P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on food availability and food absorbtivity of a normal diet in mice, but the action degree is lower than carrageenan. The cause is relevant with the lower molecular weight and hydrophilia of Eucheuma low-molecular-weight seaweed polysaccharides than carrageenan, too.6The effect on digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:The food availability and food absorbtivity of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (45.37±2.24)%and (52.13±4.49)%. The postprandial blood sugar of the Eucheuma low-molecular-weight seaweed polysaccharides group was (6.16±0.70) mmol/L, the TG and TC were (2.62±0.55)mmol/L and (1.57±0.35)mmol/L, All of them were significantly lower than those of the high-fat control group,but higher than those of the carrageenan group(P<0.05). The Na+-K+-ATPase activity was (12.27±1.43) μmol pi·mg pro-1·h-1, which significantly lower than those of the high-fat control group and the carrageenan group(P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on high-fat diet,but the action degree is lower than carrageenan.The possible mechanism is not only the molecular weight and hydrophilia is below carrageenan but also concerned with the effect of enzymic activity in mucous membrane of small intestine.7The effect of anti-oxidant and immunomodulation functions of Eucheuma low-molecular-weight seaweed polysaccharides in rats fed a high-fat diet:The content of serum ox-LDLand MDA of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (3.87±0.31) ng/ml and (5.63±0.63) nmol/ml. All of them were significantly lower than those of the high-fat control group (P<0.05). The GSH-Px activity of Eucheuma low-molecular-weight seaweed polysaccharides high dose group was (1377.17±82.53)U/ml which was significantly higher than those of the high-fat control group(P<0.05).These show that Eucheuma low-molecular-weight seaweed polysaccharides can decrease the content of serum ox-LDL and MDA, increase the GSH-Px activity, strengthen anti-oxidant ability and reduce the lipid peroxidation level.The content of serum IL-2of Eucheuma low-molecular-weight seaweed polysaccharides high dose group were (814.17±25.53) pg/ml and the OD value of the proliferation of splenic lymphocyte was (0.264±0.019). All of them were significantly higher than those of the high-fat control group (P<0.05). The TNF-α of Eucheuma low-molecular-weight seaweed polysaccharides high dose group was (141.70±43.76) pg/ml which was significantly lower than those of the high-fat control group (P<0.05). These show that Eucheuma low-molecular-weight seaweed polysaccharides can increase the content of serum IL-2and the proliferation of splenic lymphocyte, decrease the content of serum TNF-a, strengthen immunomodulation activities.Conclusion:The experimental results show the conclusion as follow: 1Our research has established the preparative method of Eucheuma low-molecular-weight seaweed polysaccharides taking Eucheuma as raw materials. Compared to the method of taking carrageenan as raw materials, the method have some characteristic such as convenient technology, making the best of resources and so on. The results of characteristic detection and analysis of preparative sample show that the molecular weight, viscosity, gel strength and so on are coincident with the same kind of product which is made from carrageenan.2The digestion and absorption of Eucheuma low-molecular-weight seaweed polysaccharides is easier than those of carrageenan. Eucheuma low-molecular-weight seaweed polysaccharides has the inhibitory action on food availability and food absorbtivity of a normal diet, but the action degree is lower than that of carrageenan.3Eucheuma low-molecular-weight seaweed polysaccharides can inhibit the digestion and absorption of a high-fat diet, decrease blood glucose and blood fat because of a high-fat diet, but the action degree is lower than carrageenan. Eucheuma low-molecular-weight seaweed polysaccharides can reduce Na+-K+-ATPase activity, the action degree is higher than carrageenan.4Eucheuma low-molecular-weight seaweed polysaccharides can strengthen the anti-oxidant ability, reduce the lipid peroxidation level, increase immunomodulation activities.5The action mechanism of Eucheuma low-molecular-weight seaweed polysaccharides like carrageen can take into water to expand, impede the contact of intestinal mucosa and food to affect digestion and absorption. Because its molecular weight is low and easy to be absorbed, the Eucheuma low-molecular-weight seaweed polysaccharides can produce some bioactivity.