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Method for using fluorescence to label hedysarum polybotrys saccharide and hedysarum polybotrys saccharide labeled through method and application of hedysarum polybotrys saccharide

A technology of fluorescent labeling and Radix Astragalus polysaccharides, which is applied in chemical instruments and methods, luminescent materials, pharmaceutical formulations, etc., can solve problems such as difficult direct detection, slow progress in research on molecular mechanism of action and metabolic kinetics, and lack of luminescent groups

Inactive Publication Date: 2018-02-23
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the lack of luminescent groups in the polysaccharide structure, it is difficult to directly detect, which makes the research on the molecular mechanism of action and metabolic kinetics of polysaccharide compounds slow.

Method used

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  • Method for using fluorescence to label hedysarum polybotrys saccharide and hedysarum polybotrys saccharide labeled through method and application of hedysarum polybotrys saccharide
  • Method for using fluorescence to label hedysarum polybotrys saccharide and hedysarum polybotrys saccharide labeled through method and application of hedysarum polybotrys saccharide
  • Method for using fluorescence to label hedysarum polybotrys saccharide and hedysarum polybotrys saccharide labeled through method and application of hedysarum polybotrys saccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of ANTS-labeled Astragalus polysaccharide HPS-3

[0038]Pulverize the Radix Radix Radix Radix Radix, add 6 times the amount of ethyl acetate at 80°C for reflux extraction 3 times, each time for 1.5h, evaporate the solvent; then add 6 times amount of ethanol for 80°C reflux extraction for 3 times, each time for 1.5h, evaporate Dry the solvent; finally add 10 times the amount of water and soak at 60°C for 3 times, each time for 1h, filter, and combine the filtrates. Add ethanol to the filtrate to make the final concentration of ethanol 50%, let it stand for centrifugation, add ethanol to the supernatant to make the final concentration of ethanol 70%, let it stand and centrifuge to get the precipitate as crude HPS-3, and use the Sevage method to prepare the crude HPS-3 Deproteinization, H 2 o 2 The pigment was removed by method, dialyzed, and freeze-dried to obtain purified HPS-3.

[0039] Dissolve 500mg of HPS-3 in water, add 58.5ml of 0.02mol / l A...

Embodiment 3

[0049] Example 3 Preparation of FITC-labeled Astragalus polysaccharide HPS-3

[0050] Take 400mg HPS-3 and dissolve in 100ml 0.2mol L -1 Phosphate buffer (pH8.0), add 400mg tyramide (Tyr) to react at room temperature for 24h. After the reaction, add 150 mg of sodium cyanoborohydride (solid), and react in a water bath at 30° C. for 60 h, shaking occasionally. After the reaction is completed, centrifuge, take the supernatant, pass through the Sephadex G-25 column, elute with distilled water, collect 10ml of the eluate in each tube, measure the sugar content in each tube by the phenol-sulfuric acid method, and measure the 280nm ultraviolet absorption value with a UV-visible spectrophotometer. The elution curve of HPS-3-Tyr was drawn, and the eluents were combined according to the elution curve, and freeze-dried to obtain HPS-3-Tyr.

[0051] The experiment finds that adding solid sodium cyanoborohydride and adding sodium cyanoborohydride dissolved in dimethyl sulfoxide has littl...

Embodiment 5

[0062] Example 5 Study on tissue distribution of ANTS-labeled Astragalus polysaccharide HPS-3

[0063] 48 Kunming mice were randomly divided into 8 groups, with 160mg·kg -1 The dose of HPS-3-ANTS was administered through the tail vein, and the cervical vertebrae were sacrificed at 0, 0.25, 0.5, 1, 1.5, 2, 3, and 4 hours after administration, and the heart, liver, spleen, lung, kidney, and stomach were quickly removed. , intestine, brain and other tissues, rinse the surface blood and contents with normal saline, weigh the wet weight of each organ respectively, put them into a centrifuge tube, add 9 times the volume of normal saline, prepare a homogenate, centrifuge, and take the supernatant Freeze at -80°C. Thaw at room temperature immediately before use, and use a fluorescence photometer (λ ex =366nm,λ em = 500nm) to measure the fluorescence intensity, put the fluorescence intensity into the corresponding standard equation to obtain the drug concentration, draw the tissue c...

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Abstract

The invention provides a method for labeling the reduction terminal of hedysarum polybotrys saccharide (HPS) with a fluorescent substance being a probe and application of HPS labeled through the method in studying the aspects of quantitative analysis, cell locating, tissue distribution, oral taking absorption, metabolism and excretion, pharmacology and the mechanism of the pharmacology and the like of HPS. Properties of the reduction terminal of HPS are utilized, and through a reductive amination reaction, the reduction terminal is directly or indirectly reacted with the fluorescent substance,so that HPS is connected to fluorophores. By means of the method, HPS without luminophores becomes hedysarum polybotrys saccharide which is easy to detect and has fluorescence, and the labeling method does not obviously influence both the internal structure and the biological activity of HPS. HPS prepared and subjected to fluorescence labeling through the method can be used for basic study in theaspects of quantitative analysis, cell locating, tissue distribution, oral taking absorption, metabolism and excretion, pharmacology and the mechanism of the pharmacology and the like.

Description

technical field [0001] The present invention relates to a method for fluorescently labeling Heqi polysaccharide (HPS), HPS marked by the method, and a method for studying quantitative analysis, cell location, tissue distribution, absorption, metabolism, pharmacology and mechanism of HPS. Background technique [0002] Plant polysaccharides have a variety of biological activities and have received extensive attention in recent years. A large number of studies have shown that plant polysaccharides participate in and mediate the regulation of various life activities in cells, and have pharmacological activities such as immune regulation, hypoglycemia, antioxidant, hypolipidemic, anti-tumor, and anti-aging. Therefore, the research and development of drugs based on plant polysaccharides has become a frontier topic in the field of medicine. Due to the lack of luminescent groups in the polysaccharide structure, it is difficult to detect directly, which makes the research on the mol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00C09K11/06A61K49/00
CPCC08B37/00A61K49/0054C09K11/06C09K2211/145
Inventor 赵良功封士兰师志强
Owner LANZHOU UNIVERSITY
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