| Hyperlipidemia is a common lipid disorder which is mainly characterized by increased total cholesterol(TC),triglyceride(TG),low density lipoprotein-cholesterol(LDL-c)and decreased high density lipoprotein-cholesterol(HDL-c).Recently,statin and fibrate drugs are widely used in clinical settings to regulate blood lipids levels,but these chemical drugs are usually accompanied by adverse side effects including liver and kidney injury,drug dependence,drug antagonism,the emergence of drug resistance and so on.Polysaccharides which widely exist in animals,plants and microorganisms have shown a series of bioactivities such as anti-tumor,antioxidant,antidiabetic,anti-fatigue,hypolipidemia,immunomodulatory activities and gene therapy.As a kind of medicine and edible fungi,Pleurotus eryngii(P.eryngii)is rich in nutrients and bioactive substances,one of its main active ingredient,P.eryngii polysaccharide,has various pharmacological activities.It has a very positive significance for the application of edible mushroom polysaccharides to in-depth study the physical properties,the chemical structure and its active constituents of P.eryngii polysaccharide.This paper systematically studied P.eryngii polysaccharides,including the extraction,isolation,purification,structure identification and activity evaluation of P.eryngii polysaccharides,attempting to reveal the relationship between the conformation of Pleurotus eryngii polysaccharides and its biological activity,and studied the preparation of P.eryngii polysaccharides effervescent tablets.Chapter One:ReviewThis part reviewed the research the polysaccharides from edible fungi,at the same time,cited the research object of this article,providing theoretical support for the experimental design of this subject.Chapter Two:Isolation and purification of P.eryngii polysaccharidesOn the basis of single-factor experiments,this chapter used orthogonal design experiments to optimize the polysaccharide extraction process of P.eryngii fruit body and determined the optimum extraction conditions were the solid-liquid ratio was 1:40,time was 2 h,temperature was 100?and the concentration of ethanol was 90%,the yield of crude P.eryngii polysaccharide was 17.2%under this condition.By comparing the three deprotein methods of Sevag method,trichloroacetic acid method and enzymatic method,the Sevag method was determined as the best deprotein method.The protein content in PEP was 0.4%by Sevag method.For the first time,this chapter separated two components from PEP by D101macroporous adsorption resin and DEAE-52 cellulose,namely PPEP-1 and PPEP-2.By high performance liquid chromatography,both PPEP-1 and PPEP-2 showed a single symmetrical peak with molecular weights of 223 kDa and 12 kDa,respectively;The total sugar content of PPEP-1 and PPEP-2 were 98.23%and 97.59%;The uronic acid content in PPEP-1 and PPEP-2 were 0.42%and 0.37%;UV spectrophotometer scanning showed that PPEP-1 and PPEP-2 had no absorption peaks at 280 nm and 260nm,indicating that neither of them contained proteins and nucleic acids.The above results demonstrated that both PPEP-1 and PPEP-2 were purified homogeneous polysaccharides.Chapter Three:Structural characterization of PPEP-1 and PPEP-2This chapter further characterized the structure of PPEP-1 and PPEP-2.The analysis of monosaccharide composition showed that PPEP-1 consisted of mannose,rhamnose,glucose,galactose and xylose in approximate mole percentages of 23.04%,4.34%,64.16%,5.99%and 6.14%,respectively.Besides,PPEP-2 was composed of mannose,rhamnose,glucose,xylose and fucose in the mole percentages of 34.67%,0.81%,56.81%,7.38%and 0.33%,respectively.PPEP-1 and PPEP-2 both had pyranose configuration and?-configuration by IR and ~1H NMR.The results of Congo red reaction showed that there was a triple helix conformation in the structure of PPEP-2,while PPEP-1 not.The results of atomic force microscopy showed that PPEP-1 has a more highly aggregated sugar chain configuration than PPEP-2.The observation of Scanning electron microscopy and transmission electron microscopy showed that the tangles between the polysaccharide chains of PPEP-1 were closer than PPEP-2.Chapter Four:Study on the hypolipidemic effects of PPEP-1 and PPEP-2In this chapter,we established two mice hyperlipidemic models by high-fat diet and intraperitoneal injection of Poloxamer-407(P-407)to induce hyperlipidemia to study the hypolipidemic effects of PPEP-1 and PPEP-2.The experimental groups were orally given PEP,PPEP-1,and PPEP-2 at a dose of 400 mg/kg.Kits were used to determine the serum levels of TC,TG,LDL-C,and HDL-C,and pathological examination was performed on the liver tissues slice of mice.The results showed that compared with the model group,the serum levels of TC,TG,and LDL-C significantly decreased and the serum levels of HDL-C increased in PEP and PPEP-1(P<0.05 or P<0.01),and the reduction rate of serum TC,TG,and LDL-C by PPEP-1 was higher than that of PEP.The results of liver tissues slice showed that after the treatment of PPEP-1 and PPEP-2,the liver tissue structure of hyperlipidemia mice basically returned to normal,fat particles were significantly decreased,liver cell morphology was relatively neat,and liver cell damage was significantly improved.In hyperlipidemia mice model induced by P-407,compared with the model group,PEP,PPEP-1,and PPEP-2 all significantly reduced the serum levels of TC,TG,and LDL-C(P<0.05 or P<0.01),among which PPEP-1 had higher reduction rates of serum TC,TG,and LDL-C than PEP.The results of the two models showed that PPEP-1 may be the main hypolipidemic substance in P.eryngii polysaccharides.This chapter explored the hypolipidemic effects of P.eryngii polysaccharides possibly due to their chain conformation rather than the helical conformation.Chapter Five:Preparation and evaluation of P.eryngii polysaccharide effervescent tabletsIn this chapter,the P.eryngii polysaccharide effervescent tablets were prepared and their quality was analyzed.Based on the determination of acid sources,alkali sources,lubricants,and fillers in the previous experiments,the disintegration time was used as an inspection index to design an orthogonal test to screen the additive amount of P.eryngii polysaccharide,disintegrates,and lubricants in effervescent tablets prescriptions.The results showed that the best addition ratios of P.eryngii polysaccharide,sodium bicarbonate and citric acid,lubricant(PEG6000)and filler(mannitol)were:15%,25%,25%,10%,25%,respectively.Under the conditions,the weight difference error of the effervescent tablets prepared were within 5%,the disintegration time is less than 5 minutes,the pH value varies within the range of 5.9~6.3,the average hardness is greater than 4 kg.The tested quality evaluations all met the requirements of the Pharmacopoeia 2015 edition,indicating that the formula had good reliability and feasibility. |
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