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IRPS extraction and separation of research and its molecular weight determination

Author: HuangXiaoFang
Tutor: LiuMoXiang
School: Yangzhou University
Course: Of Pharmacy
Keywords: polysaccharide from Isatis root extraction process uniform design separation and purification High performance gel filtration chromatography(HPGFC) evaporative Light scattering detector(ELSD)
CLC: R284
Type: Master's thesis
Year: 2007
Downloads: 622
Quote: 4
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Abstract


Radix Isatidis is one of the most common used traditional Chinese medicines, the root of Isatis indigotica Fort. is always used as the medicine of Radix Isatidis. It’s bitter in flavor and cold in nature, and is attributive to heart and stomach channels according to Chinese medical theory, usually used for seasonal febrile diseases, pestilence mumps, eruptive diseases, inflammatory diseases with redness of skin, sore throat, etc. In modern clinic application, it is usual prescribed to prevent and treat the disease coursed by virus or bacteria, such as influenza, fever, epidemic encephalitis, hepatitis, pneu- monia, tonsillitis,parotitis, eruptive conjunctivitis,herpes,and so on. Radix Isatidis has strongly antivirus activity, recognized as one of the most efficient antivirus Chinese traditional medicine. It also has other effictions such as antisepsis and antiphlogosis, immunoloregulation, antiendotoxin, anticancer.Polysaccharide is one of the important components in Radix Isatidis, and the content of polysaccharides in Radix Isatidis (RIP) is high. The exiting research evidences show that RIP has the efficacy of antivirus, reducing blood fat and immunoloregulation. Therefore, it has the great meaning in theory and practice to go deep to the research of RIP. It can also be applied in the quality control and exploitation of functional foods and drugs for Radix Isatidis.In order to optimize the best condition for the extraction process of Polysaccharides from Isatis root, the experiment was arranged with uniform design, and determined the polysaccharides with vitriol-phenol colorimetry, the effect of temperature, time and the added water on the pick-up rate of polysaccharide was considered. As far as concerned, the optimal condition for the extraction process is in the case of temperature at 100℃, time about 2.5h, and the mass of the added water is 10 times of the materials. The pick-up rate of polysaccharides is in the range of 13.8%~20.44%. The optimization of extraction process is reasonable, can be taken as the reference for the industrial production of Polysaccharides from Isatis root. On the basis of the best conditions for the extraction process, the research makes a further study on the technology of separation prepartion process of RIP. After edulcoration with alcohol and water extraction-alcohol deposition process, the crude polysaccharides from Radix Isatidi(sRIPⅠ) was redissolved with flash extraction. The extracted solution was standing in cold circumstance, subsequently, the solution was separated into two parts. One was the indissoluble deposition, named as RIPⅡ, the other was the dissoluble solution, named as RIPⅢ. And further separation and purification was processed for the constituent RIPⅢ. In the step of deproteinization for the RIPⅢ, it is found that the Sevage improved with flash extraction is more efficient than the traditional one.The sample of RIPⅢwas added on the chromatographic column of DEAE, and eluted with salt of different concentration, so that we get six constituents named as RIP A,RIP B,RIP C,RIP D,RIP E and RIP F respectively. Each constituent was purified with hollow-fibre membrane and Sephadex G100 gel column.In the research of polysaccharides, the determination of the molecular weight is the key to the discovery of its property. Our research takes the ELSD as the detector for the determination of the molecular of RIP.The standard polysaccharides and the samples were injected in the HPLC to detect the molecular weight. The condition is like that: the column is OHpad (G2000SW, 7.5mm×300mm); the eluent is super purified water; the flow rate is 1.0ml/min; temperature is 25℃. We get the standard linear relation is lgMw=-0.5788RT+8.53572, r=-0.9969,linear range is in 5900~404000.The result shows that: RIP A1 contains the polysaccharides with its molecular weight is 314889; RIP A2 contains the polysaccharides with its molecular weight is 222970,215664 and 165203; RIP B1 contains the polysaccharides with its molecular weight is 566771,512859 and 311134; RIP C1 contains the polysaccharides with its molecular weight is 566771,512859 and 311134; RIP D1 contains the polysaccharides with its molecular weight is 252057,220606,336251 and 55682; RIP E1 contains the polysaccharides with its molecular weight is 272312; RIP F1 contains the polysaccharides with its molecular weight is 321676.

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