Study On Extraction Of Laminaria Polysaccharides And Its Effect On Macrophage Inflammation And Alleviation Of Liver Injury Induced By CCL₄ In Mice

Kelp is a kind of natural food which is widely used by people and has high medicinal value.Kelp contains polysaccharides,peptides,?-3 fatty acids,carotenoids,phenols and other bioactive substances.Among them,Laminaria polysaccharides have attracted much attention because of their physiological functions such as antibacterial,anti-tumor,antioxidant,lowering blood lipid and so on.In this paper,firstly,water extraction method and ultrasonic method were used to extract Laminaria polysaccharides.By determining the extraction rate,the content of total sugar,sulfuric acid group and the scavenging rate of free radicals,the optimal extraction method was selected.Subsequently,the immunomodulatory effect of LPS was measured by adding LPS in the process of cultured RAW264.7 macrophages.Finally,taking Kunming mice with acute liver injury induced by CCl₄ as the experimental model,Laminaria polysaccharides was added to clarify the effects of Laminaria polysaccharides on the inflammatory response and oxidative stress of mice with liver injury induced by CCl₄,and to evaluate the protective effect of Laminaria polysaccharides on liver injury.The experimental results include the following aspects:(1)Water extraction and ultrasonic extraction were used to extract polysaccharide from kelp,and the best extraction method was selected.This experiment:the polysaccharide was extracted by water first,adopts the single factor experiment method to study the temperature,p H,and solid-liquid ratio and extraction times on the effect of seaweed polysaccharides yield,and then through the response surface experiment to optimize the best extraction conditions:temperature of 87°C,p H of 5,material liquid ratio of 1:48(mg/m L),extraction times for 3 times,seaweed polysaccharide extraction yield of 14.53%.The extraction of seaweed polysaccharides by ultrasonic method,explores the temperature,ultrasound power,solid-liquid ratio and extraction times of polysaccharides of the four main factors influence on the yield of polysaccharide,and then through the response surface experiment to optimize the best extraction conditions:material liquid ratio of 1:64(mg/m L),temperature of68°C,the ultrasonic power is 89 W,extraction times for 3 times,the extraction yield of polysaccharide was 17.73%.The results showed that the total sugar content of polysaccharides extracted by water extraction method and ultrasonic extraction method were 3.01%and 3.49%,and the sulfuric acid content was 1.06%and 1.30%,respectively.Finally,the antioxidant activity of polysaccharides extracted by the two methods was tested in vitro,and the scavenging ability of polysaccharides on DPPH,ABTS,hydroxyl radical and superoxide anion was determined.The experimental results showed that the antioxidant activity of polysaccharides extracted by ultrasonic method was higher than that extracted by water method.Therefore,the Laminaria polysaccharides extracted by ultrasonic method was used for subsequent experiments.(2)In order to investigate the effects of Laminaria polysaccharides on adhesion ability,proliferation activity and release of NO and ROS of RAW264.7 macrophages,we added Laminaria polysaccharides into RAW264.7 macrophage culture.By observing the adhesion experiment of Laminaria polysaccharides on macrophages,it can be seen that Laminaria polysaccharides has no effect on the adhesion of macrophages.After adding Laminaria polysaccharides,macrophages differentiated from the original round or oval shape to the typical fission shape,and there will be a certain degree of concentration dependence.The results of MTT assay showed that polysaccharides had no significant inhibitory effect on the proliferation of macrophages.The results showed that when the concentration of polysaccharide was500 g/m L,the release of NO was 7.16 times that of the normal control group.By measuring the release of ROS,it was found that Laminaria polysaccharides could significantly up-regulate the release of ROS in RAW264.7 macrophages,indicating that Laminaria polysaccharides could activate macrophages and improve the non-specific immune function.(3)Sixty Kunming mice were fed for 7 days and randomly divided into 6 groups,which were blank control group,negative control group(model group),positive control group(200 mg/kg biphenyl diester),LPS low-dose group(200 mg/kg),LPS medium-dose group(400 mg/kg)and LPS high-dose group(600 mg/kg).Every day should be in accordance with the relative concentration of lavage,blank and the negative control group with the same amount of normal saline instead of,for seven days in a row,the last delivery after 1 h,blank control group according to the volume of 1 m L/g injection amount of peanut oil,the rest of the four groups in the same amount of volume concentration of injection of 0.2%CCl₄ vegetable oil solution(including CCl₄ peanut oil was used as solvent)to build mould,then make mice eat,give plenty of water,after 24 h,will all eyeball blood of mice were put to death.After the experiment,the serum and liver of the mice were analyzed.Results show that when the damage by CCl₄ in mice,mice liver tissue homogenate of SOD,CAT and GSH-Px,T-AOC content decreased,the content of MDA increased,after adding Laminaria polysaccharides,SOD,CAT and gsh-px in mice,T-AOC content with polysaccharide concentration rises,MDA content and decreased with the increase of polysaccharide doses,shows seaweed polysaccharide can reduce oxidative stress to protect liver from CCl₄ hepatic toxicity.When mice were damaged by CCl₄,the contents of IL-1?,IL-6 and IL-12 in serum increased,while the contents of IL-2 and IL-10 decreased.The addition of Laminaria polysaccharides could significantly increase the contents of IL-2 and IL-10,and reduce the contents of IL-1?,IL-6 and IL-12,suggesting that the addition of Laminaria polysaccharides could effectively improve the release of inflammatory cytokines in mice.Therefore,this study showed that Laminaria polysaccharides has a certain protective effect on acute liver injury of CCl₄ mice.