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Liquorice protein nanoparticles and preparation method thereof

A licorice protein and nanoparticle technology, applied in the field of new dosage forms and preparations of protein drugs, can solve the problems that licorice protein nanoparticles have not been reported in literature, and achieve the effects of regular surface, uniform particle size distribution and low toxicity

Active Publication Date: 2014-04-23
研译(杭州)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] But so far, the research on licorice protein nanoparticles has not been reported in the literature at home and abroad.

Method used

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  • Liquorice protein nanoparticles and preparation method thereof
  • Liquorice protein nanoparticles and preparation method thereof
  • Liquorice protein nanoparticles and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: the extraction of glycyrrhizin

[0030]The licorice decoction pieces are pulverized by a small high-speed pulverizer. The licorice powder and the phosphate buffer (0.02 mol / L, pH 7.2) containing 0.1 mol / L NaCl were mixed evenly at a mass ratio of 1:5, placed at 4 °C, and stirred for 12 h. Filter the obtained licorice extract with two layers of gauze, and discard the filter residue. The filtrate was centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was collected. Place the supernatant at 4°C, adjust the pH to 10.0 with ammonia water, and slowly pre-cool absolute ethanol while stirring, so that the final concentration of ethanol reaches 40%. Let stand for 4 h, centrifuge at 10,000 g for 15 min, and collect the supernatant. Continue to slowly add pre-cooled absolute ethanol to the supernatant until the ethanol concentration is 50%, let it stand for 12 h, centrifuge at 12000 rpm for 15 min, and collect the precipitate. The precipitate was f...

Embodiment 2

[0034] Embodiment 2: Preparation of Glycyrrhizin Nanoparticles

[0035] The glycyrrhizin was prepared as 1 mg / mL Tris-HCl buffer solution (pH 7.9), heated at 60-100°C for 1 hour, and unreacted glycyrrhizin was removed by TSK gel G6000PW gel chromatography to obtain glycyrrhizin nanoparticles particles. See the attachment for electron microscope observation of glycyrrhizin nanoparticles Figure 5 .

[0036] The particle size and surface potential were measured with a laser particle size analyzer, and the particle size was measured to be 74.09±0.69 nm, and the surface potential was -23±2 mV. The particle size distribution diagram of licorice protein nanoparticles is attached Figure 6 .

Embodiment 3

[0037] Example 3: In Vitro Cytotoxicity Assay of Glycyrrhizin Nanoparticles

[0038] The in vitro cytotoxicity of glycyrrhizin nanoparticles was determined using normal liver cells (L-02), human hepatoma cells (Hep-G2), human colon cancer epithelial cells (Caco-2) and dog kidney epithelial cells (MDCK). The cells were cultured in RPMI1640 medium containing 20% ​​calf serum at 37°C in a 5% CO2 culture environment. During the measurement, the cells were divided into 4×10 4 Each sample / mL was connected to a 96-well plate, 200 μL / well, and 6 parallel wells were set up for each group. After culturing for 24 hours, the medium was discarded, and 100 μL of the diluted sample was added to each well. At the same time, 100 μL of medium without serum was added as a blank control. After continuing to culture for 24 h, the cell proliferation rate was measured by the MTT method, and the calculation formula was as follows:

[0039] Survival rate=(A590 sample group-A590 blank group) / A590...

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Abstract

The invention discloses preparation methods of liquorice protein and nanoparticles of the liquorice protein. The liquorice protein with molecular weight of 31.0 kDa and isoelectric point of 4.5 is obtained through separation and purification. A primary structure sequence of N-end of the liquorice protein is NPDGLIACYCGQYCW. The liquorice protein nanoparticles are prepared by heating a saline solution of the liquorice protein at 100 DEG C under the conditions that the protein concentration is 1 mg / mL, and pH is 7.9 for 60 minutes, and the average particle size of the liquorice protein nanoparticles is 74.09+ / -0.69 nm. Freeze-dried products of the liquorice protein nanoparticles are good in liquidity and re-solubility, and easy to stably store. The liquorice protein disclosed by the invention derives from food, and is high in safety. The preparation method of the liquorice protein nanoparticles does not involve any crosslinking agent, finished products of the liquorice protein nanoparticles widely exist in various decoction using liquorice, are taken by the majority of people for many years, and are high in safety. The liquorice protein nanoparticles disclosed by the invention are expected to be applied to in-vivo delivery of various medicines.

Description

technical field [0001] The invention relates to a new dosage form and preparation technology of protein medicine in the field of medical technology, in particular to a glycyrrhizin nanoparticle with drug delivery function in vivo and a preparation method thereof. Background technique [0002] According to the definition in the National Nanotechnology Initiative (NNI) of the United States, particles with a particle size in the range of 1-100 nm are called nanoparticles. In recent decades, nanoparticles have been widely used in the development and research of drug delivery systems, including nano-drug carriers prepared from various materials such as metal aggregates, inorganic particles, hyperbranched polymers, and polymer micelles. While various nano-drug carriers are gradually showing the advantages of targeting and penetrating, they also expose their shortcomings in biocompatibility, cytotoxicity and degradability. After nanomaterials, the physicochemical properties (optic...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/20C07K1/18A61K47/42
CPCA61K9/5169A61K47/42C07K14/415
Inventor 刘树滔髙观祯汪惠勤
Owner 研译(杭州)生物科技有限公司
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