Method for tracing absorbable proteins in traditional Chinese medicine

It is a technology of absorption and traditional Chinese medicine, which is applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc. It can solve the problems of antibody preparation and detection, short half-life, and inability to reflect structural changes, etc., to eliminate the interference of homologous background, mass spectrometry data fast, Good application value and promotional effect

Active Publication Date: 2013-09-18
EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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Problems solved by technology

However, antibody detection and analysis requires the preparation of single-molecule specific antibodies, which is costly and time-consuming. Moreover, it is impossible to prepare and detect antibodies before the active ingredients of the macromolecules are identified. Therefore, it is difficult to specifically identify the macromolecules of traditional Chinese medicine.
The isotope labeling method is dangerous to operate, has poor specificity, short half-life, is unstable, and cannot reflect the structural changes of Chinese medicine macromolecules in the metabolic process.

Method used

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  • Method for tracing absorbable proteins in traditional Chinese medicine
  • Method for tracing absorbable proteins in traditional Chinese medicine
  • Method for tracing absorbable proteins in traditional Chinese medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, trace the protein that can be absorbed in velvet antler

[0058] 1. Preparation of deer antler extract

[0059] 1. Extraction

[0060] Fresh deer antler 500g, washed, sawed into small pieces, added acetic acid aqueous solution (0.006M, pH3.5), homogenized at 4°C; then centrifuged (2-4°C, 8500r / min, 20min) to take the supernatant, put on Add 99.9% (volume ratio) ethanol to the supernatant until the ethanol concentration reaches 65% (volume ratio), stir at 4°C for 4h; Vacuum rotary evaporation at 55°C, the dry matter obtained is the crude antler extract.

[0061] 2. Separation

[0062] The crude antler extract was subjected to molecular sieve chromatography (to remove unlabeled EITC and small salt molecules) in a chromatographic cabinet at 4°C, and the specific parameters were as follows:

[0063] Use Sephadex G-25 to pack a chromatographic column (Pharmacia, 17-0360-01), 100cm×1cm;

[0064] Elute with distilled water at a flow rate of 0.5ml / min;

[00...

Embodiment 2

[0122] Embodiment 2, the protein that can be absorbed in earthworm is traced

[0123] Earthworm fibrinolytic enzyme: purchased from Shanghai Guoyuan Biotechnology Co., Ltd., batch number: 20080312; fibrinolytic enzyme activity is greater than 12000U / mg, and the appearance is freeze-dried powder.

[0124] 1. Fluorescein-labeled earthworm protein

[0125] Same as Step 2 of Example 1.

[0126] The eluate collected after passing through the column is the solution containing the labeled fluorescent protein (FITC-LK).

[0127] Freeze-dry the collected eluate after passing through the column and store at -20°C.

[0128] 2. Composition determination and molecular weight analysis of PE and FITC-LK

[0129] LK and FITC-LK were subjected to non-denatured protein electrophoresis, respectively, with a loading volume of 100 μg and a loading volume of 20 μL. After the electrophoresis, first use the G-box to observe the fluorescent bands at 488nm (see Figure 13 B), then observe the prot...

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Abstract

The invention discloses a method for tracing absorbable proteins in a traditional Chinese medicine. The method comprises the following steps: 1, marking a traditional Chinese medicine protein extract product by using a fluorophore; 2, carrying out intragastric administration of the marked traditional Chinese medicine protein extract to an experiment mode animal; 3, acquiring the serum of the experiment mode animal, concentrating, carrying out native protein electrophoresis , cutting the obtained gel in direction vertical to the electric field direction to form a plurality of gel blocks, and detecting the fluorescence intensities and diffusion coefficients of all the gel blocks; 4, respectively carrying out in-gel enzymatic hydrolysis of screened gel blocks by using trypsin, carrying out real-time fluorescence monitored molecular sieve chromatography of the obtained enzymatic hydrolysis products, recovering enzymatic hydrolysis products having fluorescence signal peaks, and carrying out mass spectrum determination; and 5, determining the absorbable proteins according to mass spectrum determination results. The method will make great contributions to the disclosure of the action mechanisms of traditional Chinese medicinal macromolecules, and lays a theoretic foundation for promoting the development of the traditional Chinese medicine and pharmacy.

Description

technical field [0001] The invention relates to a method for tracing the absorbed protein in traditional Chinese medicine. Background technique [0002] Traditional Chinese medicine is a natural chemical treasure house. It not only contains small molecular compounds of various structures, but also contains macromolecular components such as proteins, polysaccharides, and glycopeptides. Under the guidance of TCM theory, multiple components work together. However, due to the limitations of detection methods, it is very difficult to trace the activity of macromolecules of traditional Chinese medicines. Even some extremely effective traditional Chinese medicines cannot be developed because their main active ingredients are macromolecules that are difficult to analyze and detect, such as velvet antler and scorpion venom. [0003] Compared with small molecular compounds, the current understanding of macromolecular traditional Chinese medicine components is not deep enough. Existing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 王毅张倩马淑骅雷洪涛侯燕鸣于友华
Owner EXPERIMENTAL RES CENT CHINA ACAD OF CHINESE MEDICAL SCI
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