Study On The Blood Glucose Regulation Of Polysaccharides Originated From Tangerine Peel And Oral Liquid Preparation

Objective:As a kind of peel with a long history of medicinal and edible value in China,tangerine peel contains chemical components such as volatile oil,polysaccharides,flavonoids,alkaloids and trace elements.It has the functions of regulating qi,strengthening spleen,drying dampness and resolving phlegm.Modern pharmacology research shows that tangerine peel can resist oxidation,anti-inflammatory and bacteriostasis,reduce blood sugar,prevent and improve cardiovascular and cerebrovascular diseases.At present,there are few studies on the regulation of blood glucose of tangerine peel polysaccharides.The subject of present study is to explore the the blood glucose regulation effect of high-purity polysaccharides from tangerine peel,through the establishment of HepG2 cell model of insulin resistance and the animal model of alloxan induced hyperglycemia mice.In addition,the oral solution of tangerine peel polysaccharides was prepared and its quality standard was defined.So as to provide some experimental and theoretical basis for the comprehensive development and utilization of tangerine peel.Methods and Results:The first chapter of this topic focused on the extraction and purification of TPPS,to isolate the polysaccharides from tangerine peel.The optimal condition for extraction of TPPS was selected by ethanol subsiding method through single factor investigation and orthogonal test,the alcohol precipitation process was also investigated.Then purified by diethylaminoethyl cellulose?DEAE-52?ion exchange chromatography and Sepharose G-100 chromatography after removing the proteins and dialysis.The results showed that the optimal extraction process of the polysaccharides were at 95?,add 10 times distilled water to extract for 3 times,each time for 1.5 h.After combining the extract and reducing pressure to 20%,added 4 times volume of 90%ethanol and stood for 12 h.Then washed the precipitate with anhydrous ethyl alcohol and acetone for three times,freeze-drying to obtain the crude polysaccharides.Finally,5polysaccharides components were separated by DEAE-52 and the largest component of polysaccharide was TPPS,which extracted by Sephadex G-100 chromatography to obtain a single component with higher purity,and contained 84.96%of total sugar,33.91%of glucuronic acid and 0.62%of protein.The second part of this project aims towards analysising the structural characteristics and antioxidant capacity of TPPS in vitro.The monosaccharide composition and structural features of TPPS were determined by Gas chromatography-Mass spectrometry?GC-MS?,Irfrared Absorption spectroscopy?IR?,Ultraviolet spectrophotometry?UV?and Nuclear Magnetic Resonance spectroscopy?NMR?.In addintion,the antioxidant capacity in vitro was determined by reducing power,total oxidation capacity and scavenging effect of DPPH,ABTS and·OH radicals.The obtained results showed that TPPS had typical characteristics of carbohydrate compounds.It was composed of five monosaccharides,rhamnose,arabinose,xylose,mannose and glucose by GC-MS,with a molar ratio was 0.94:3.92:0.31:1.00:1.37.IR,¹H-NMR and¹³C-NMR showed that TPPS is mainly?-glycosidic bond,and may contain carbonyl,amino,pyran and furan ring structures.Moreover,TPPS has strong antioxidant capacity in vitro.When the concentration is 1.2 mg/mL,the scavenging rate of ABTS and·OH radicals reaches 85.23%,88.53%,and the reducing power reaches0.917.The third part of this work target for the effect of TPPS on blood glucose modulation from cell level and animal level.The insulin resistance model was established by inducing HepG2 cells with high concentration of insulin,and the insulin concentration and action time were optimized,then evaluated the improvement effect and cytotoxicity of TPPS on insulin resistance combining MTT method.In animal experiment,alloxan was injected into tail vein to induce hyperglycemia in mice.After administration of TPPS in different doses?200,400,600 mg/kg?,the effects of TPPS on blood glucose,body weight,glucose tolerance,oxidative stress ability of liver tissue,blood lipid level and other biochemical indexes of mice in each group were detected.And integrating histopathological observation of pancreas and liver and acute toxicity experiment to intire investigate the effect of TPPS on blood glucose regulation.The results showed that the model of insulin resistance HepG2 cells established by5.0×10^-77 mmol/L insulin for 24 hours was the best.TPPS could increase the glucose consumption of insulin resistance model cells in a dose-dependent manner,improve the glucose metabolism of cells,and had no cytotoxicity.In vivo animal experiments show that TPPS can reduce the blood glucose level of hyperglycemic mice,improve body weight and oral glucose tolerance,and regulate the blood lipid level of hyperglycemic mice.Simultaneously,TPPS can increase the activity of SOD,cat,T-AOC,GR and GSH-PX in liver tissue,reduce the content of MDA and improve the injury of pancreas and liver tissue,which suggested that TPPS can improve hyperglycemia symptoms by reduce liver oxidation and protect the pancreas and liver.The fourth part concentrates on the preparation,quality standard and stability of tangerine peel polysaccharide oral liquid.According to the optimized extraction conditions,the crude polysaccharide of tangerine peel was prepared,and the oral liquid of tangerine peel polysaccharide was prepared with the polysaccharide as the main drug.Through single factor investigation and L₉?3³?orthogonal test,the clarification process was optimized,and the kinds and dosages of preservatives and flavoring agents were screened.Finally,the quality standard and stability of the oral liquid of tangerine peel polysaccharide after the treatment of potting and sterilization was investigated.The results are mainly divided into the following parts:?1?The best clarification process was:under the condition of flocculation temperature of 60?and pH of 4.5,0.32%chitosan was added to the solution.And 0.2%xylitol and 0.1%potassium sorbate were used as the flavor modifier and preservative respectively.?2?The prepared oral liquid of tangerine peel polysaccharide has a clear orange yellow color,sweet taste,good antiseptic effect and meeting the requirements of the oral liquid quality standard of Chinese Pharmacopoeia 2015.?3?The stability inspection found that it should be preserved in a dry place,the influence of light and temperature on its stability was not significant,and in good condition within 9 months.Conclusion:In this study,we used tangerine peel as the research object to separate homogeneous and high purity TPPS by water extraction,alcohol precipitation,Sevag method,dialysis,DADE-52 fiber column and Sephadex G-100 dextran gel column.Structure analysis showed that TPPS was composed of five kinds of monosaccharides,which mainly?-glycosidic bond,possibly containing carbonyl,amino,pyran and furan ring structures.Moveover,it had good antioxidant properties in vitro.On this basis,the effects of TPPS on the insulin resistance model of HepG2 cells and the hyperglycemia mice induced by alloxan were explored.The results showed that TPPS could effectively promote the glucose metabolism of insulin resistant HepG2 cells,improve the insulin and no cytotoxicity,regulate the blood glucose,and reduce the oxidative stress of liver through the protection of pancreas and liver,so as to improve the diabetes Symptom.Finally,the tangerine peel polysaccharide oral liquid was prepared,and the quality standard and stability were investigated,which met the requirements of Chinese Pharmacopoeia 2015.The above results are expected to provide ideas and reference for the structural analysis,clinical application and new drug development based on the traditional Chinese medicine.