| The inability of normal metabolism of lipids in human body is an element of arterial disease.When atherosclerotic plaques are produced and hinder circulation,cardiovascular and cerebrovascular diseases follow.Hyperlipidemia is a metabolic disease in which lipids in the body cannot be normally metabolized,and is characterized by an increase or decrease in certain specific indicators in serum.At present,some drugs for the treatment of hyperlipidemia are clinically effective,but they are often accompanied by many adverse reactions.These drugs are mainly statins,niacin and fibrates,except for nausea and dizziness.The serious side effects they cause are liver damage and myopathy.Natural drug polysaccharides are widely found inanimals,plants and even microorganisms in nature.In more and more studies,polysaccharides have excellent biological activity and have natural and non-toxic advantages.Pueraria lobata(willd.)ohwi is a kind of plant which can be used for the consumption of arrowroot powder and has rich medicinal value.The remarkable biological activity of flavonoids is well known and widely used,and its other main component is Pueraria lobata polysaccharide.It also has good pharmacological effects.In this paper,Pueraria lobata polysaccharides were extracted from traditional Radix Puerariae and their structure identification and activity evaluation were carried out.Attempts were made to analyze the relationship between the results of structural identification of Pueraria lobata polysaccharide and the results of its activity evaluation,and then choose a new dosage form of liposome.Formulation optimization was carried out,and the research included quality and activity evaluation.Chapter ? ReviewThis chapter mainly reviews the recent research progress of natural Pueraria lobata polysaccharides,and focuses on the separation and purification of polysaccharides,structural analysis and various methods of formulation research.The biological activity,physicochemical properties and other characteristics of Pueraria lobata polysaccharides were summarized.At the same time,the development and characteristics of new polysaccharide formulations were briefly summarized,which laid a theoretical foundation for the subsequent discussion and research.Chapter ? Isolation,Purification and Physicochemical Propertiesof Polysaccharide from Pueraria lobataIn this chapter,the crude polysaccharide of Pueraria lobata was obtained from the root of the genus Pueraria lobata by water extraction and alcohol precipitation.The polysaccharide yield after removal of impurities such as protein was 5.68%.The crude polysaccharide of Pueraria lobata was separated and purified by macroporous adsorption resin D101 and DEAE-52 cellulose column to obtain two purified polysaccharide components PLR1 and PLR2.The first one is the 10% alcohol eluting component of the macroporous adsorption resin D101,which is named PLR1,and the second is the water eluting component of cellulose named PLR2.The physical and chemical properties of PLR1 and PLR2 were tested.The results showed that PLR1 contained 97.32±1.18% polysaccharide,3.53±0.54% protein and 1.22±0.39% uronic acid.PLR2 contains 95.79 ± 1.68% polysaccharide,0.22 ± 0.08% uronic acid and 1.46± 0.37% protein.The UV full-wavelength scanning of the two has no obvious absorption peak at a specific wavelength band,and shows that it contains only a very small amount of impurities such as proteins and nucleic acids.The molecular weight was determined by HPLC-ELSD.The spectra showed that both PLR1 and PLR2 were single symmetric peaks with molecular weight concentrations of 19.7 kDa and 2.3 kDa,respectively.Chapter III Structural analysis of Polysaccharide PLR1 andPLR2 from Pueraria lobataIn this chapter,a preliminary structural study was carried out on the purified Pueraria lobata polysaccharides PLR1 and PLR2.The contents of the study included composition,bond analysis,and spatial conformation.In addition to chemical methods such as periodic acid oxidation test and Congo red test,nuclear magnetic resonance was also used.Analytical methods such as NMR,scanning electron microscopy,and atomic force microscopy.PLR1 and PLR2 were hydrolyzed and derivatized by1-phenyl-3-methyl-5-pyrazolone(PMP).The results of high performance liquidchromatography for the determination of monosaccharide components showed that PLR1 consisted of glucose and rhamnose.The molar ratio is 93.65: 6.34,while PLR2 is a single glucan.The results of periodate oxidation experiments indicated that PLR1 was mainly composed of 1?3 and 1?6 glycosidic bonds,and the ratio was 44.99%and 39.75%,while PLR2 was mainly composed of 1?2 or 1?4 glycosidic bonds with a ratio of 67.41%.Analysis of Congo red results showed that neither had a triple helix.Infrared and nuclear magnetic spectroscopy analysis of PLR1 and PLR2 showed that both glycosidic bond configurations were ?-configuration and there was no uronic acid absorption peak.This indicates that both PLR1 and PLR2 are neutral polysaccharides,which are consistent with the determination of uronic acid content.Scanning electron microscopy(SEM)observations show that the surface of PLR1 exhibits an irregular antennae structure and is surrounded by circular structures of different sizes.The shape is scattered and partial stacking occurs.This may be because PLR1 has a large molecular weight and strong attraction between molecules.Lead to its complex structure and amorphous.However,PLR2 has a small molecular weight and is easy to form an irregular geometric shape,so it is gathered in a crumb-like state,and the surface is smooth and a small amount of pleats appear as pores in the middle of the emulsion-like structure.Atomic-grain microscopy AFM measurements showed that PLR1 is a complex chain structure with many branches,and the height of the chain ranges from 1.2 nm to 6.1 nm,indicating that molecular aggregation has occurred.PLR2 two-dimensional image display points are randomly arranged.The distance between the points is not tight,that is,the chain and the chain are not connected.The height of the three-dimensional image structure exceeds the height of a single polysaccharide molecule,indicating that there is inter-molecular aggregation but the intensity is not as good as PLR1.Chapter IV Study on the hypolipidemic activity of Puerarialobata polysaccharideThis chapter induces hyperlipidemia in mice by long-term feeding of a fixed amount of high-fat diet.In this model,the physiological activities of Pueraria lobata polysaccharide PLR1 and PLR2 in the treatment of hyperlipidemia were evaluated.The TC,TG,LDL-C,and HDL-C levels in the serum of the obtained mice were determined by kits.After 30 days of continuous administration(100,200,300mg/kg/d),Pueraria lobata polysaccharides PLR1 and PLR2 significantly reduced serum TC,TG,and LDL-C levels compared with the model group.And serum HDL-C levels were increased(P<0.05 or P<0.01).The pathological examination of mouse liver tissue showed that after oral administration of PLR1 and PLR2,the hepatocyte damage of the hyperlipidemic model mice was fine and the morphology of the cells was relatively neat,and no fatty granule vacuolar lesions,hepatocytes were observed.Inflammatory damage has also been alleviated.These phenomena suggest that Pueraria lobata polysaccharide can improve liver disease in mice with hyperlipidemia to some extent.This chapter also discusses that the hypolipidemic effect of Pueraria lobata polysaccharides may be attributed to their glycosidic bond configuration and branched structure.Chapter V Preparation of Pueraria polysaccharide PLR1 liposomeand evaluation of hypolipidemic activityIn this chapter,the liposome of Pueraria lobata polysaccharide PLR1 was prepared by reverse phase evaporation method.The single factor test was used to investigate the effects of four factors on the encapsulation efficiency and drug loading.The Box-Behnken model was designed to obtain the best prescription of PLR1 Pueraria lobata polysaccharide liposome,and the Pueraria lobata polysaccharide liposome prepared according to the optimal prescription process was evaluated.Physical and chemical properties evaluation includes various indicators such as encapsulation efficiency,average particle size,apparent morphology and stability.The specific method for activity evaluation was to induce the acute hyperlipidemia model in mice by using poloxamer P-407.The mice were orally administered with the same dose of PLR1 Pueraria lobata polysaccharide and PLR1 Pueraria lobata polysaccharide liposome to examine the hypolipidemic effect of PLR1 liposomes.The experimental results show that the mass ratio of soybean lecithin to polysaccharide,cholesterol and Tween 80 is 24.32:1,7.07:1 and 9.68:1 at 60 °C,respectively.The encapsulation efficiency of liposome prepared according to this method is reached.77.29 ± 1.12%.PLR1 liposomes were spheroidal under TEM,with an average particle size of 117.32±5.36 nm.The particle size range was narrow and normal distribution,and the particle size was uniform.The Zeta potential analysis showed that the value was-19.37±0.74 m,and the absolute value was large,indicating that it might have good stability.The preliminary stability test results confirmed this and left at room temperature for one month,and no visible deposits of the liposomes occurred.The encapsulation efficiency was slightly reduced but not significant,and the particle size value did not fluctuate greatly indicating that the prepared puerarin liposome had good stability.An acute hyperlipidemia model in mice was induced by P-407.The lipid-lowering effect of liposome PLR1 L was observed by comparing the same dose of Pueraria lobata polysaccharide drug substance group.The experimental results showed that the PLR1 liposome group showed better hypolipidemic activity on the TC,TG,and LDL-C than the PLR1 drug group.However,both the PLR1 drug substance group and the PLR1 liposome group did not significantly increase serum HDL-C,thatis,for P-407-induced hyperlipidemia mice,Pueraria lobata polysaccharide is not suitable for treatment of low HDL-C symptom.Liposomes contribute to the hypolipidemic effect of Pueraria lobata polysaccharides in vivo,which broadens new ideas for the study of Pueraria lobata polysaccharide preparations. |
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