CN111195233A - Preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles - Google Patents
Preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles Download PDFInfo
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- CN111195233A CN111195233A CN201811370160.XA CN201811370160A CN111195233A CN 111195233 A CN111195233 A CN 111195233A CN 201811370160 A CN201811370160 A CN 201811370160A CN 111195233 A CN111195233 A CN 111195233A
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Abstract
The invention relates to a preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles, belonging to the technical field of food biology. The key point of the process is that a folic acid coupling chitosan solution with the concentration of 1-2mg/ml is prepared, folic acid coupling chitosan is dissolved in a 1% glacial acetic acid solution, a certain amount of EGCG is added, the pH value is adjusted to 3-5 by a 1% NaOH solution, and the folic acid coupling chitosan solution with the EGCG concentration of 0.5-1mg/ml is prepared. Dissolving a certain amount of TPP (tetrapropylene phosphate) by using pure water, respectively passing the TPP solution and the EGCG solution of the folic acid coupled chitosan through 0.45 mu m filter membranes, dropwise adding the TPP solution into the folic acid coupled chitosan EGCG solution which is stirred at a constant speed, wherein the ratio of the folic acid coupled chitosan to the TPP is 4-6: 1, finally obtaining the EGCG-loaded folic acid chitosan nano-microsphere.
Description
Technical Field
The invention relates to a preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles, belonging to the technical field of food biology.
Background
Epigallocatechin gallate (EGCG) is a polyphenol substance, and has special physiological functions of reducing blood lipid, resisting oxidation, and scavenging free radicals in vivo. However, because EGCG is unstable, EGCG is easily degraded or generates byproducts by external environment changes, and the application of EGCG in various fields is limited.
At present, few researches on the embedding of the folic acid coupling chitosan into the EGCG are carried out at home and abroad, the folic acid is coupled with the chitosan by utilizing the targeting property of the folic acid, and the folic acid-chitosan-EGCG nano microspheres are prepared by utilizing an ion gel method, so that the stability of the EGCG is improved, and the bioavailability of the EGCG is improved.
Worldwide, malignant tumors have become a major disease that seriously threatens human health over the history of human disease development. The current main treatment method has low drug selectivity, kills tumor cells and damages certain normal cells in vivo, so that obvious toxic reaction is often generated in treatment to influence the curative effect. Therefore, the targeting preparation has obvious advantages and is imperative to be used for tumor chemotherapy.
Disclosure of Invention
The invention mainly aims at the prevention and treatment of malignant tumors and prepares a link of a novel molecular targeted drug and a drug delivery carrier.
The invention provides a preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles, which comprises the following specific contents:
A. preparation of active folic ester: weighing 200-300mg of FA, dissolving in 5-10ml of anhydrous DMSO, adding 200-300mg of DCC and 100-200mg of NHS for dissolving, filtering out DCU after overnight, distilling the filtrate under reduced pressure to remove part of solvent, dropwise adding the filtrate into ice-cold anhydrous ether solution containing 30-40% acetone while stirring to obtain yellow precipitate DCU, and performing rotary evaporation and vacuum freeze drying on the filtrate to obtain folic acid active ester;
B. synthesis of chitosan: weighing 100-200mg of CS, dissolving in 30-60ml of acetic acid-sodium acetate buffer solution (PH 4.6) to obtain chitosan acetic acid aqueous solution, slowly adding 8-15ml of DMSO solution (20 mg/ml) of folic acid active ester into the solution at the speed of 2-3ml/min under magnetic stirring, reacting at 30 ℃ for 18-20h, centrifuging, washing the precipitate with distilled water for several times, dissolving the precipitate in 2-4% acetic acid aqueous solution again to obtain 1-2mg/ml folic acid coupling chitosan solution;
C. separation and purification of folic acid chitosan: taking a proper amount of folic acid coupling chitosan solution, centrifuging at low speed, taking supernatant, separating by using a SephadexG-25 sephadex chromatographic column, eluting by using 2-4% acetic acid solution, and enabling the flow rate to be 1.5 ml/min. Detecting and eluting at 363nm wavelength, collecting the first light absorption peak value, and freeze-drying to obtain folic acid coupled chitosan pure product;
D. preparing an EGCG solution of folic acid coupled chitosan: dissolving folic acid coupled chitosan in 1-3% glacial acetic acid solution, weighing a certain amount of EGCG, adding into the solution, adjusting pH with NaOH solution to obtain folic acid coupled chitosan solution with EGCG content of 0.5-1 mg/ml;
E. preparing nano particles: dissolving a certain amount of TPP (Tetranitroglycerin) by using pure water, and keeping the ratio of folic acid coupled chitosan to TPP between 4-6: 1, respectively filtering the TPP solution and the EGCG solution of folic acid coupled chitosan through 0.45 mu m filter membranes, and dropwise adding the TPP solution into the EGCG solution of folic acid coupled chitosan which is stirred at a uniform speed to obtain the EGCG-loaded folic acid chitosan nano-microspheres.
The main advantages and positive effects of the invention are as follows:
1. the targeting property of folic acid is utilized to couple folic acid with chitosan.
2. The folic acid-chitosan-EGCG nano-microspheres are prepared by an ion gel method, so that the stability of the EGCG is improved, and the bioavailability of the EGCG is improved.
Fourth, detailed description of the invention
Example 1
Weighing FA223mg, dissolving in 5ml of anhydrous DMSO, adding DCC200mg and NHS133mg for dissolving, filtering out DCU after overnight, distilling the filtrate under reduced pressure to remove part of solvent, dropwise adding the filtrate into ice-cold anhydrous ether solution containing 30% acetone while stirring to obtain yellow precipitate DCU, and performing rotary evaporation and vacuum freeze drying on the filtrate to obtain folic acid active ester; weighing CS160mg, dissolving in 40ml acetic acid-sodium acetate buffer solution (PH 4.6) to obtain 4mg/ml chitosan acetic acid aqueous solution, slowly adding 8ml DMSO solution (20 mg/ml) of folic acid active ester into the solution at the speed of 2ml/min under magnetic stirring, reacting at 30 ℃ for 18h, centrifuging, washing the precipitate with distilled water for several times, dissolving the precipitate in 2% acetic acid aqueous solution again to obtain folic acid coupling chitosan solution with the concentration of 2 mg/ml; taking a proper amount of folic acid coupling chitosan solution, centrifuging at low speed, taking supernatant, separating by using a SephadexG-25 sephadex chromatographic column, eluting by using 2% acetic acid solution, and enabling the flow rate to be 1.5 ml/min. Detecting and eluting at 363nm wavelength, collecting the first light absorption peak value, and freeze-drying to obtain folic acid coupled chitosan pure product; dissolving folic acid coupled chitosan in 1% glacial acetic acid solution, weighing a certain amount of EGCG, adding into the solution, adjusting pH to 5 with 1% NaOH solution, and making into folic acid coupled chitosan EGCG solution with a certain concentration; dissolving a certain amount of TPP by using pure water, wherein the ratio of folic acid coupled chitosan to TPP is 6: 1, respectively filtering the TPP solution and the EGCG solution of folic acid coupled chitosan through 0.45 mu m filter membranes, dropwise adding the TPP solution into the EGCG solution of folic acid coupled chitosan which is stirred at a uniform speed, and finally obtaining the EGCG-loaded folic acid chitosan nano-microspheres.
Example 2
Weighing FA248mg, dissolving in 6ml of anhydrous DMSO, adding DCC210mg and NHS140mg for dissolving, filtering out DCU after overnight, distilling the filtrate under reduced pressure to remove part of solvent, dropwise adding the filtrate into ice-cold anhydrous ether solution containing 30% acetone while stirring to obtain yellow precipitate DCU, and performing rotary evaporation and vacuum freeze drying on the filtrate to obtain folic acid active ester; weighing CS180mg, dissolving in 30ml acetic acid-sodium acetate buffer solution (PH 4.6) to obtain 6mg/ml chitosan acetic acid aqueous solution, slowly adding 10ml DMSO solution (20 mg/ml) of folic acid active ester into the solution at the speed of 3ml/min under magnetic stirring, reacting at 40 ℃ for 18h, centrifuging, washing the precipitate with distilled water for several times, dissolving the precipitate in 2% acetic acid aqueous solution again to obtain folic acid coupling chitosan solution with the concentration of 2 mg/ml; taking a proper amount of folic acid coupling chitosan solution, centrifuging at low speed, taking supernatant, separating by using a SephadexG-25 sephadex chromatographic column, eluting by using 2% acetic acid solution, and enabling the flow rate to be 1.5 ml/min. Detecting and eluting at 363nm wavelength, collecting the first light absorption peak value, and freeze-drying to obtain folic acid coupled chitosan pure product; dissolving folic acid coupled chitosan in 1% glacial acetic acid solution, weighing a certain amount of EGCG, adding into the solution, adjusting pH to 3 with 1% NaOH solution, and making into folic acid coupled chitosan EGCG solution with a certain concentration; dissolving a certain amount of TPP by using pure water, wherein the ratio of folic acid coupled chitosan to TPP is 5: 1, respectively filtering TPP solution and EGCG solution of folic acid coupled chitosan through 0.45 mu m filter membrane, dropwise adding TPP solution into the EGCG solution of folic acid coupled chitosan which is stirred at a uniform speed, and finally obtaining the EGCG-loaded folic acid chitosan nano-microsphere.
Claims (1)
1. A preparation method of folic acid-chitosan-epigallocatechin gallate (EGCG) nanoparticles is characterized by comprising the following steps:
A. preparation of active folic ester: weighing 200mg of Folic Acid (FA), dissolving the Folic Acid (FA) in 5-10ml of anhydrous dimethyl sulfoxide (DMSO), adding 300mg of Dicyclohexylcarbodiimide (DCC) and 200mg of N-hydroxysuccinimide (NHS) to dissolve the Folic Acid (FA) and the anhydrous dimethyl sulfoxide (DMSO), filtering out Dicyclohexylurea (DCU) after the overnight, distilling the filtrate under reduced pressure to remove part of the solvent, dropwise adding the filtrate into an ice-cold anhydrous ether solution containing 30-40% of acetone while stirring to obtain a yellow precipitate DCU, and performing rotary evaporation and vacuum freeze drying on the filtrate to obtain active folic ester;
B. synthesis of chitosan: weighing 100 mg of Chitosan (CS), dissolving the Chitosan (CS) in 30-60ml of acetic acid-sodium acetate buffer solution (PH 4.6) to obtain a chitosan acetic acid aqueous solution, slowly adding 8-15ml of DMSO solution (20 mg/ml) of folic acid active ester into the solution at the speed of 2-3ml/min under magnetic stirring, reacting for 18-20h at the temperature of 30-40 ℃, centrifuging, washing the precipitate with distilled water for several times, and dissolving the precipitate in 2-4% acetic acid aqueous solution again to obtain 1-2mg/ml folic acid coupling chitosan solution;
C. separation and purification of folic acid chitosan: taking a proper amount of folic acid coupling chitosan solution, centrifuging at low speed, taking supernatant, separating by using a SephadexG-25 sephadex chromatographic column, eluting by using 2-4% acetic acid solution, and enabling the flow rate to be 1.5 ml/min; detecting and eluting at 363nm wavelength, collecting the first light absorption peak value, and freeze-drying to obtain folic acid coupled chitosan pure product;
D. preparing an EGCG solution of folic acid coupled chitosan: dissolving folic acid coupled chitosan in 1-3% glacial acetic acid solution, weighing a certain amount of EGCG, adding into the solution, adjusting pH to 3-5 with NaOH solution, and making into folic acid coupled chitosan solution with EGCG content of 0.5-1 mg/ml;
E. preparing nano particles: dissolving a certain amount of TPP (Tetranitroglycerin) by using pure water, and keeping the ratio of folic acid coupled chitosan to TPP between 4-6: 1, respectively filtering the TPP solution and the folic acid coupled chitosan EGCG solution through 0.45 mu m filter membranes, and dropwise adding the TPP solution into the folic acid coupled chitosan EGCG solution which is stirred at a uniform speed to obtain the EGCG-loaded folic acid chitosan nano-microspheres.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114948935A (en) * | 2022-03-28 | 2022-08-30 | 厦门大学 | Gallic acid derivative nano-drug, preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114948935A (en) * | 2022-03-28 | 2022-08-30 | 厦门大学 | Gallic acid derivative nano-drug, preparation method and application |
| CN114948935B (en) * | 2022-03-28 | 2024-05-17 | 厦门大学 | Gallic acid derivative nano-drug, preparation method and application |
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