Study On Hepatoprotective Effects And Liposome Preparation Of Rhdiola Rosea Polysaccharides

Liver is an extremely important organ responsible for the metabolism of harmful and noxious substances from the human's metabolites and some exogenous chemicals.However,toxic and harmful substances produced in the process of metabolism and detoxification of the liver may cause acute and chronic liver damage at the same time.If the damage cannot be repaired,liver function will be affected,causing serious consequences.Liver disease has become one of the most common diseases that affect human's health includes liver necrosis,hepatitis,fibrosis,liver cirrhosis,and other pathological changes are causing pain to patients.In recent years,plant polysaccharides have received more and more attention from scientific researchers due to their multibioactivity,a wealth of sources and non-toxic.The rhizome and roots of Rhodiola rosea also called the “golden root” or “roseroot” have been used for thousands of years with a wide range of functions.Therefore,the main content of this study is extract,isolate and purify the natural active product polysaccharide from the Rhdiola rosea.Importantly,this is the first time to investigate the hepatoprotective effect of Rhodiola rosea polysaccharide(RRP1 and RRP2),and the liposome nano preparation was prepared by the reverse phase evaporation method to improve its hepatoprotective effect.Chapter ? Review This chapter is divided into two parts.The first part summarizes the extraction,separation and purification technology of polysaccharides,and the method of structural analysis.It focuses on the research progress of polysaccharide's structure analysis methods in the past five years.The second part mainly introduces the research progress on the structure,biological activity and application of Rhodiola rosea polysaccharides.This study provides a theoretical support and research direction for the single polysaccharide components were isolated,purified from Rhodiola rosea,structural analysis and hepatoprotective effect evaluation and preparation technology.Chapter ? Isolation,Purification and Physicochemical Properties of Polysaccharide from Rhodiola rosea In this chapter,the dried rhizome and roots of Rhodiola rosea was extracted with hot distilled water and crude polysaccharides were obtained after ethanol precipitation.The precipitate was deproteinized via Savage method and decolorized with macroporous resin D315.The rate of protein removal is 81.07% and the decolorization effect was also good.The polysaccharide of Rhodiola rosea was further separated and purified by cellulose DEAE-52 and Sephadex G-100 column chromatography.The first fraction(the water eluted fraction of cellulose DEAE-52)as a single peak was named RRP1.The second is cellulose 0.1 M salt elution fractions further gelation Sephadex G-100 elution components,which was named RRP2.The basic physical and chemical properties of RRP1 and RRP2 polysaccharides were determined.The results showed that RRP1 contains 89.31 ± 1.18% polysaccharide,4.97 ± 0.54% protein and 0.90 ± 0.21% uronic acid.The polysaccharide content of RRP2 was 79.33%,with the uronic acids content and protein being 13.98% and 2.18%,respectively.Ultraviolet wavelength scanning shows that the two polysaccharide components contain trace amounts of proteins and nucleic acids.The molecular weights of RRP1 and RRP2 were determined by HPLC-ELSD.The HPLC chromatogram showed that both RRP1 and RRP2 are single symmetrical peaks.And the average molecular weights of RRP1 and RRP2 were estimated to be 5.5 k Da and 425.7 k Da,respectively.Chapter ? Structural analysis of Polysaccharide RRP1 and RRP2 from Rhodiola rosea In this chapter,the structure of Rhodiola rosea polysaccharides(RRP1 and RRP2)was characterization.Chemical analysis and instrumental analysis were used to determine the monosaccharide composition,glycosidic bond position,characteristic functional groups,glycosidic bond types and surface morphology,etc.of Rhodiola rosea polysaccharides.The main results are as follows: the monosaccharide composition of RRP1 and RRP2 were analyzed by PMP-HPLC method.HPLC analysis suggested that RRP1 and RRP2 were composed of Mannose,Rhamnose,Galacturonic acid,Glucose,Galactose and Arabinose at the molar ratio of 0.69:0.11:0.15:1:0.51:7.5 and ratio of 0.15:0.19:1.01:0.18:0.47:1,respectively.FT-IR spectra showed pyranose ring and uronic acid in both RRP1 and RRP2.At the same time,RRP1 is linked by ?-type and ?-type glycosidic bonds,while RRP2 only contains ?-glycosidic bonds.NMR analysis further confirmed the composition of the five monosaccharides and the type of glycosidic bonds.Analysis of periodate oxidation and Smith degradation speculated that arabinose and glucose in RRP1 are mainly connected by 1 ?3,and mannose,rhamnose,and galactose mainly consist of 1?2,6,1?6,1?2,or 1?4 connection.In RRP2,rhamnose,glucose and galactose are predominantly linked by 1?3.A small amount of glycerol and erythritol indicate that mannose and arabinose in RRP2 are mainly 1?2,1?6,or 1?4 connection.X-ray diffraction results indicated that both RRP1 and RRP2 are semi-crystalline materials.The information obtained from the Congo red experiment is that RRP1 possessed a triple helical conformation and the conformation of RRP2 was not triple-helical.SEM observation showed that the surface of RRP1 exhibited a small lamellar or irregular dendritic structure and RRP2 had a smooth surface topography with characteristic large wrinkles and drop-shaped bulges on the edges.AFM tests showed that RRP1 is a chain structure with a width or length ranging from 1.2 ?m to 2.5 ?m;its height is 1-3.5 nm,indicating that the molecular chains were branched and entangled.RRP2 exhibits particles of different sizes(40-200 nm)with a height range of about 1-4.5 nm and similar branched and entangled.Chapter ? Antioxidant and hepatoprotective effect of RRP1 and RRP2 This chapter first measured the antioxidant activity of polysaccharide RRP1 and RRP2 in vitro.The results showed that the fraction RRP1 had superior antioxidation than RRP2.The scavenging effects of RRP1 on DPPH radical,hydroxyl radical,and superoxide anion radicals reached 90.54%,83.55%,and 72.50%,respectively and presented dose correlation.Then,we established acute liver injury model in mice by CCl4 administration.The hepatoprotective effects was evaluated by administering different amounts of RRP1 and RRP2.The experimental results showed that the polysaccharide RRP1 decreasing ALT and AST levels in serum and MDA in the liver tissue,as well as increasing the levels of CAT,SOD,and GSH the liver tissue homogenates compared with the model group.And each RRP1 dose group achieved good hepatoprotective effect.Histopathological observations revealed that CCl4 can cause hepatocellular necrosis and inflammatory cell infiltration,while polysaccharide RRP1 can reduce liver cell damage and inflammatory cell infiltration in mice.And all the experimental results showed that RRP1 dose group showed better liver protection than the same dose of RRP2.In vitro and in vivo experiments have indicated that RRP1 fraction exhibited good antioxidant activity and hepatoprotective effect.Chapter ? Preparation of Rhodiola polysaccharide RRP1 liposome and evaluation of its hepatoprotective effects In this chapter,the polysaccharide fraction RRP1 liposome(RRP1L)was prepared by reverse-phase evaporation method.On the basis of single-factor experiments,the formulation of RRP1 L optimized by the response surface method(Box-Behnken model).In addition,the entrapment efficiency,particle size,morphology and stability of RRP1 L were investigated.The CCl4-induced acute liver injury model in mice was successfully established.The hepatoprotective effect of RRP1 L was examined.The same dose of fraction RRP1 was used as the positive control groups and continuously administered(gastric administration)for 7 days.The experimental results showed that the optimal formulation of RRP1 L is that the phospholipid and polysaccharide ratio is 29.17:1,the phospholipid and cholesterol ratio is 6.98:1,the phospholipid and Tween 80 ratio is 9.20:1,and the rotary evaporation temperature is 60 °C.The entrapment efficiency of RRP1 L was 73.94 ± 1.16%.The quality evaluation results showed that the shape of liposome RRP1 L was spherical,the average particle size was 137.32 ± 7.28 nm,and the dispersibility coefficient(PDI)was 0.198 ± 0.006.Zeta potential was-28.50 ± 0.98 m V and the RRP1 L was uniformly distributed with good stability.The stability test showed that there was no significant precipitation of RRP1 L within 15 days,and the entrapment efficiency was not significantly reduced.The results of pharmacological experiments show that,the liposome RRP1 L administration group reduced serum ALT and AST levels and liver tissue MDA content,and increased CAT,SOD activities and GSH content in the liver tissue compared with the model group.Moreover,compared with the same dose of polysaccharide RRP1 group,the results of various indicators showed that the RRP1 L administration group showed better hepatoprotective effect than the direct administration of polysaccharide RRP1,which has potential application value.