Studies On The Preparation And Characterization Of Auricularia Auricula Polysaccharide-iron Complex And Its Anti-iron Deficiency Anemia Activity

Iron is one of the most important minerals in human body and plays a vital role in maintaining the normal function of organisms.The World Health Organization report shows that10-30%of the world's population has different degrees of anemia symptoms,most of which are caused by iron deficiency.At present,ferrous salts such as ferrous sulfate,ferrofumarate and ferrous gluconate are commonly used as oral iron supplements.However,long-term administration of these oral iron supplements can cause gastrointestinal side effects and iron-induced oxidative stress.Some clinical studies have shown that long-term use of therapeutic ferrous sulfate oral agent can cause gastrointestinal side effects such as nausea,vomiting,abdominal pain and constipation.Polysaccharide-iron complex has the characteristics of high stability,good water solubility and less side effects.It is an ideal iron supplement.It has also become an excipient for the development of functional product and medicines with anti-iron deficiency anemia activity.Auricularia auricula is a kind of special black saprophytic fungus growing on rotten wood in China.It has been widely used as edible fungus with medicinal and edible homology since1000 years ago.The annual output of Auricularia auricula in China is about 6.5 million tons,some of which are wasted due to the confusion of strain management,unstable quality,inadequate intensive processing and other problems,which increase the pressure of industrial environmental protection and affect the sustainable development of the industry.Auricularia auricula polysaccharide is an important functional component of Auricularia auricula.It has antioxidant,anticoagulant,immune enhancement and anti-tumor biological activities,and can be used as a potential ligand for complexing iron.In this study,Auricularia auricula polysaccharides were extracted and purified by ultrasonic and microwave assisted extraction.Auricularia auricula polysaccharides and iron complex were prepared by improved ferric trichloride method,using Auricularia auricula polysaccharides and iron elements.Then the general physical and chemical properties,structural characterization and antioxidant activity of Auricularia auricula polysaccharide-iron complex were studied.Finally,an iron deficiency anemia rat model was established to evaluate the anti-anemia effect and acute toxicity of Auricularia auricula polysaccharide-iron complex.If the research results can be carried out smoothly in practical application,not only can the added value of Auricularia auricula be greatly increased,the industrial chain of intensive processing of Auricularia auricula be prolonged,and enormous economic,social and environmental benefits be generated,but also can help to improve the chaotic situation of iron supplement products in the market.It has remarkable application value and broad market prospects.The research contents and results are as follows:(1)Auricularia auricula was used as raw material degreased by supercritical extraction and extracted assisted by ultrasound and microwave.The effects of material-liquid ratio,ultrasonic power,microwave temperature and synergistic action time on the yield of Auricularia auricula polysaccharides were investigated by single factor test and response surface test.The results showed that the ultrasonic power had the greatest influence on the extraction rate of crude lecithin from maize,followed by the ratio of material to liquid,microwave temperature and synergistic action time.The optimum extraction conditions of Auricularia auricula polysaccharides were as follows:ratio of material to liquid(g/mL)1:80,ultrasonic power 240W,microwave temperature 85?,synergistic action time 20 min.Under these conditions,the yield of Auricularia auricula polysaccharides was 9.42+0.12%,Macroporous resin method was used to remove protein and pigments from Auricularia auricula polysaccharides.The removal rates of protein and pigments were 80.2±1.12%and 87.4±1.25%respectively,and the loss rate of polysaccharides was 16.6±0.45%.After purified by DEAE-52 cellulose column and Sephadex G-100 dextran gel column chromatography,purified polysaccharides from Auricularia auricula were obtained,named AAPS.The molecular weight of Auricularia auricula polysaccharide(AAPS)was determined by gel permeation chromatography(GPC).The weight average molecular weight(Mw)of the Auricularia auricula polysaccharide(Mw)was 1.42×10~6u and the mean molecular weight(Mn)was 3.57×10~5u.The monosaccharide composition of Auricularia auricula polysaccharide(AAPS)was analyzed by PMP Pre-column Derivatization High Performance Liquid chromatography.The results showed that AAPS was mainly composed of mannose(Man),glucuronic acid(GlcA),glucose(Glc)and xylose(Xyl)with different molar ratios of 3.62:7.23:12.57:4.24.(2)Auricularia auricula polysaccharide iron complex was prepared from purified polysaccharide of Auricularia auricula.The effects of reaction temperature,reaction time,reaction system pH and trisodium citrate on iron content of Auricularia auricula polysaccharide iron complex were investigated by single factor test and response surface test.The results showed that the effect of four factors on the iron content of Auricularia auricula polysaccharide-iron complex was in turn pH>trisodium citrate>reaction time>reaction temperature.The optimum technological conditions for the preparation of Auricularia auricula polysaccharide-iron complex were as follows:reaction temperature 75?,reaction time 75min,reaction system pH 9 and adding 30mg of trisodium citrate.Under these conditions,the iron content of Auricularia auricula polysaccharide-iron complex was 28.42+0.16%.AAPS-iron(III)is a red-brown amorphous powder,odorless,odorless,soluble in water,insoluble in organic solutions such as ethanol,methanol,etc.It has a certain stability in air and water,but it is easy to absorb moisture and must be preserved under sealed drying conditions.The results of iron ion identification test showed that there was no free iron ion in AAPS-iron(III).Auricularia auricula polysaccharides combined with iron instead of mixing physically.(3)The morphology of AAPS and AAPS-iron(III)was analyzed and characterized by scanning electron microscopy(SEM)and laser particle size distribution instrument.The results showed that the surface of AAPS-iron(III)changed from flocculent morphology to flaky structure,and from roughness to smoothness,the particle size increased significantly.The thermal stability of AAPS and AAPS-iron(III)was analyzed by thermogravimetric analysis(TG).The results showed that the thermal stability of AAPS-iron(III)was enhanced,and the thermal decomposition process changed from severe to moderate after heating.AAPS-iron(III)has good stability in the temperature range of 50-256?.The molecular structure and properties of AAPS and AAPS-iron(III)were identified by FT-IR,CD and NMR-1H.The results showed that AAPS and iron formed a structure of?-FeOOH,and the combination of iron and hydroxyl did not destroy the active functional groups of Auricularia auricula polysaccharide.There were no obvious resonance absorption peaks on the NMR spectra of AAPS-iron(III)except for the heavy water peak.It is further explained that Auricularia auricula polysaccharides and iron are combined rather than physically mixed.The dissolution rate of iron in vitro of Auricularia auricula polysaccharide-iron complex showed that the dissolution rate of iron in simulated gastrointestinal environment could reach 93.1%.This indicated that AAPS-iron(III)exhibited good water solubility in gastrointestinal environment,was conducive to iron absorption and had good biological utilization ability.The antioxidant activity of AAPS-iron(III)showed that the DPPH free radical scavenging activity and ABTS free radical scavenging activity of AAPS-iron(III)reached 39.5%and 35.9%respectively at the concentration of 1.0 mg/mL,and the inhibition rate of liver lipid peroxidation reached 43.1%.The results showed that AAPS-iron(III)had good antioxidant activity.(4)The high-dose group of AAPS-iron(III)can gradually restore the contents of HB,RBC,HCT,MCV,MCH and MCHC in the blood of rats with iron deficiency anemia,which indicates that the high-dose group of AAPS-iron(III)has obvious auxiliary therapeutic effect on anemia rats.The high dose group of AAPS-iron(III)could significantly reduce the content of free protoporphyrin in erythrocyte compared with the model group,which indicated that AAPS-iron(III)could improve the iron deficiency in anemia rats.The high dosage group of AAPS-iron(III)could significantly increase the saturation of serum iron and blood iron,and decrease the total iron binding capacity of AAPS-iron(III).The high-dose group of AAPS-iron(III)could increase the body weight of animals significantly,improve the general situation,and gradually restore the appearance,behavior and coat color of animals to normal.By analyzing the content of SOD and MDA in rat liver,the high dose group could significantly reduce the content of MDA in rat liver and increase the level of SOD in liver,which indicated that AAPS-iron(III)could inhibit lipid peroxidation in liver tissue and enhance the antioxidant capacity of liver tissue.Acute toxicity test in mice showed that the maximum dosage of AAPS-iron(III)administered by intragastric administration was 13g/(kg d),which was 205 times the clinical dosage.After intragastric administration,the weight gain of mice belonged to the normal growth range,no poisoning or death occurred,and no obvious changes in tissues and organs were found after dissection.Compared with the blank group,there was no significant difference between the two groups,indicating that AAPS-iron(III)was safe and reliable,and belonged to non-toxic grade.