CN112006288A - Method for preparing double-layer modified reduced glutathione nano-liposome - Google Patents
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- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides a preparation method of a double-layer modified reduced glutathione nano-liposome, belonging to the technical field of health food. Because the reduced glutathione has poor stability, is difficult to permeate cell membranes and has low bioavailability, the reduced glutathione can permeate cell membranes by being embedded by liposome, so that the bioavailability of the reduced glutathione is improved; however, the problem of poor stability is still difficult to completely solve only by using the liposome for embedding, and the invention provides a preparation method of the sodium alginate-chitosan double-layer modified liposome aiming at the problem, and the prepared liposome has the advantages of high embedding rate, uniform particle size distribution, good stability and the like.
Description
Technical Field
The invention relates to a preparation method for double-layer modification of nanoliposomes, in particular to a method for preparing double-layer modified nanoliposomes embedded with free reduced glutathione, and belongs to the technical field of health food.
Background
Reduced Glutathione (GSH) is a biologically active tripeptide compound. GSH has unique physiological function, is called as human endogenous active polypeptide, can not only clear excessive free radicals in human body, but also inhibit the activity of tyrosinase and the formation of melanin in vivo, and can play the roles of spot lightening and whitening. It is also a prosthetic group of a plurality of enzymes, can protect the sulfydryl of biological molecular protein, and has the functions of detoxifying, protecting liver, delaying senility and the like. Glutathione as a bioactive factor can be added into different types of functional foods, such as beverages, candies, fermented foods, dairy products, oral health-care foods specially designed for some special crowds, and the like. However, GSH is extremely easily oxidized, and very difficult to permeate cell membranes, and has low bioavailability, thus limiting its applications.
Liposomes are an effective delivery system consisting of a phospholipid bilayer. The bilayer structure is mainly composed of soybean lecithin or egg yolk lecithin, the hydrophilic head of the lecithin forms the inner and outer surfaces of the vesicle, and the lipophilic tail end forms the hydrophobic region of the bilayer. The biomembrane-like bilayer structure of the liposome can embed hydrophilic substances and lipophilic substances, can improve the stability of the embedded substances and directionally transfer the embedded substances to corresponding tissues. However, the structure of the liposome is easily damaged by light, acid, alkali and the like in the storage and digestion processes, and the problems of particle flocculation, particle size enlargement, drug release and the like are caused, so that the application of the liposome is severely restricted. Chitosan and sodium alginate are natural polysaccharides existing in nature, a large amount of amino groups on a chitosan molecular chain are positive charges, a large amount of carboxyl groups on a sodium alginate molecular chain are negative charges, a polyelectrolyte membrane is alternately formed through the electrostatic action of the positive charges and the negative charges and is modified on the surface of the liposome, the stability of the liposome can be greatly improved, and the application range of the liposome is widened. At present, partial scholars use the two polysaccharides to prepare delivery systems such as microcapsules, microspheres and the like, but the common modification of sodium alginate-chitosan on the surface of liposome is not common.
Aiming at the problems of the reduced glutathione in practical application, the reduced glutathione needs to be embedded and modified to prepare the sodium alginate and chitosan double-layer modified reduced glutathione nano-liposome, so that the stability and the bioavailability of the reduced glutathione nano-liposome are improved, and the wide application of the reduced glutathione nano-liposome in the fields of food, medicine and the like is promoted.
Disclosure of Invention
The invention aims to solve the technical problem of poor stability of the glutathione liposome prepared by the existing method, and thus provides a preparation method of a sodium alginate-chitosan double-layer modified reduced glutathione nano-liposome.
The invention provides a preparation process of sodium alginate-chitosan double-layer modified reduced glutathione nano-liposome, which comprises the following components: the weight ratio of soybean lecithin to cholesterol is 1:1-8:1, the weight ratio of tween 80 to lecithin is 0.1:1-1:1, the solid-to-liquid ratio (mg/ml) of free reduced glutathione to buffer solution is 1:1-8:1, and sodium alginate and chitosan with the weight percentage of 0.01-0.5% and 0.01-0.5% respectively are subjected to double-layer modification.
Aiming at the formula, the preparation method of the invention is as follows:
1) dissolving a certain amount of soybean lecithin, cholesterol and tween 80 in absolute ethyl alcohol, then placing the mixture in a round-bottomed flask, carrying out reduced pressure evaporation to remove an organic phase, and then carrying out elution and hydration by using a buffer solution containing glutathione.
2) Slowly and dropwise adding the glutathione nano-liposome into the chitosan solution while stirring, and standing for a period of time.
3) Dropwise adding the chitosan-modified glutathione liposome obtained in the step 2 into a sodium alginate solution with a certain concentration, stirring while adding, and standing for a period of time to obtain the double-layer modified reduced glutathione nanoliposome solution.
[ advantageous effects ]
The invention has the beneficial effects that: the invention provides a preparation method of a sodium alginate-chitosan double-layer modified reduced glutathione nano-liposome, which can solve the problems of poor stability, difficulty in cell membrane permeation and low bioavailability of reduced glutathione, and can be better applied to the food industry.
[ description of the drawings ]
FIG. 1 is a diagram showing the results of the relationship between chitosan concentration and embedding rate;
FIG. 2 is a diagram showing the results of the relationship between the concentration of sodium alginate and the embedding rate.
[ detailed description ] embodiments
Aiming at the formula, the preparation method of the invention is as follows:
example 1:
accurately weighing soybean lecithin, cholesterol and tween 80 according to the mass ratio of 4:1: 1.6.
Example 2:
the weighed materials were placed in a 25ml beaker, 15ml of absolute ethanol was added, and after complete dissolution, the mixed solution was poured into a round bottom flask. The round-bottom flask was connected to a rotary evaporator and the organic phase was evaporated at 50 ℃ under reduced pressure to form a uniform transparent film on the wall of the flask, and then the evaporation was continued under reduced pressure for 30min to completely evaporate the organic phase therein. Then phosphate buffer solution dissolved with reduced glutathione is quickly added to elute and disperse the membrane, the concentration of the phosphate buffer solution is 0.05mol/L, pH to be 6.0, and the solid-to-liquid ratio (mg/ml) of the reduced glutathione to the phosphate buffer solution is 4: 1. And carrying out rotary hydration in a water bath at 50 ℃ under normal pressure for 30min to obtain a crude liposome solution. And then carrying out ultrasonic treatment on the mixed solution in an ice bath for 10min (intermittent ultrasonic treatment, 1s/1 s), and standing for 2h to obtain a liposome solution.
Example 3:
putting the reduced glutathione nanoliposome into a syringe, respectively dropwise adding the reduced glutathione nanoliposome into chitosan solutions (0.01, 0.05, 0.1, 0.3 and 0.5g/100 ml) with different concentrations, wherein the volume ratio is 1:1, adding the reduced glutathione nanoliposome while stirring, and standing for 2 hours after the liposome solution is completely titrated. And determining the optimal chitosan coating concentration by taking the encapsulation efficiency, the particle size, the dispersion coefficient and the potential as investigation indexes.
And then placing the prepared chitosan modified liposome with the optimal concentration into a syringe, respectively slowly dropwise adding the prepared chitosan modified liposome into sodium alginate solutions (0.01, 0.05, 0.1, 0.3 and 0.5g/100 ml) with different concentrations in a volume ratio of 1:1, stirring while adding, and standing for 2 hours after the liposome solution is completely titrated. And determining an optimal scheme by taking the encapsulation efficiency, the particle size, the dispersion coefficient and the potential as investigation indexes.
Particle size, dispersion coefficient, potential measurement method: the mean particle size of the liposomes, the particle size distribution thereof, and the surface charge (zeta potential) were measured at 25 ℃ by a particle size-potentiostat.
The encapsulation efficiency determination method comprises the following steps: separating the liposome from free glutathione by using an ultrafiltration centrifugal tube, measuring the amount of the free glutathione, and then calculating the embedding rate of the liposome according to the following formula:
TABLE 1 relationship of chitosan concentration to average particle diameter, dispersion coefficient, zeta potential
As can be seen from Table 1, with the increase of the concentration of chitosan, the average particle size of the liposome is continuously increased, and the dispersion coefficient is gradually increased, but not very obvious; the zeta potential also increases, and a negative value from the beginning becomes a positive value. These changes suggest that they may occur because chitosan is successfully entrapped on the surface of the liposomes by electrostatic interactions. As can be seen from FIG. 1, the glutathione entrapment rate gradually increased with increasing chitosan concentration, but the change of the entrapment rate was small after 0.1%. When the concentration of the chitosan is 0.1%, the particle size of the chitosan modified liposome is small, the dispersion rate is good, the embedding rate of glutathione is high, and therefore 0.1% of chitosan is selected as the optimal concentration for subsequent sodium alginate modification.
TABLE 2 relationship between sodium alginate concentration, average particle diameter, dispersion coefficient, zeta potential
As can be seen from Table 2, the average particle size of the liposome is continuously increased and the dispersion coefficient PDI is also obviously increased with the continuous increase of the concentration of sodium alginate, and from FIG. 2, the coating rate of the liposome is also increased with the increase of the concentration, but after the concentration of the sodium alginate is 0.1%, the increase of the coating rate is not changed greatly, but from 0.1%, a large amount of precipitates are released. The interaction principle of chitosan and sodium alginate is that chitosan and sodium alginate are combined together through electrostatic interaction, and when the amount of a certain component is too high, a precipitate is generated and released. The charge of the liposome changes from positive to negative with the increasing concentration of sodium alginate, but the increase of the charge is little when the concentration exceeds 0.1%, which indicates that the encapsulation capacity of the liposome reaches the saturation level. The optimum concentration selected is 0.1% sodium alginate.
Example 4
Stability test
The unmodified liposome and the bilayer-modified liposome sample solution were stored at 25 ℃ for 7 days, and the leakage rate was measured on days 1, 3, 5, and 7, and the experimental results are shown in Table 3.
TABLE 3 leakage rates of unmodified liposomes versus bilayered modified liposomes
As can be seen from table 3, compared with the unmodified liposome, the stability of the liposome modified by the sodium alginate-chitosan bilayer is significantly improved, and the experimental result shows that the bilayer modification can improve the stability of the liposome.
Claims (5)
1. A bilayer modified reduced glutathione nanoliposome is characterized in that: the method comprises the following steps:
the method comprises the following steps: weighing lecithin, cholesterol and tween 80, and dissolving with anhydrous ethanol to obtain a mixed solution; evaporating under reduced pressure on a rotary evaporator to remove anhydrous ethanol, forming a layer of uniform transparent film on the wall of the bottle, continuing to evaporate under reduced pressure to completely evaporate the anhydrous ethanol, then adding a phosphate buffer solution dissolved with reduced glutathione for elution and dispersion, carrying out rotary hydration for a period of time under water bath and normal pressure at a certain temperature, carrying out ultrasonic treatment in an ice bath, and standing for a period of time to obtain a liposome solution;
step two: slowly and dropwise adding the reduced glutathione liposome into a 0.1% chitosan solution, stirring and adding the reduced glutathione liposome, and standing for a period of time;
step three: slowly and dropwise adding the liposome prepared in the step two into a 0.1% sodium alginate solution, stirring and adding simultaneously, and standing for a period of time to obtain the sodium alginate-chitosan double-layer modified reduced glutathione liposome solution.
2. The method for preparing the double-layer modified reduced glutathione nanoliposome of claim 1, wherein the method comprises the following steps: the concentration of the chitosan is 0.01-0.5%, and the concentration of the sodium alginate is 0.01-0.5%.
3. The method for preparing the double-layer modified reduced glutathione nanoliposome of claim 1, wherein the method comprises the following steps: firstly, adding unmodified liposome into chitosan for modification, and then adding the liposome modified by chitosan into sodium alginate for secondary modification.
4. The method for preparing the double-layer modified reduced glutathione nano-liposome of claim 1, wherein the liposome solution is slowly added into the chitosan solution drop by drop, and then the mixture is left for a period of time to completely wrap the liposome.
5. The method for preparing the bilayer modified reduced glutathione nanoliposome of claim 2, wherein the liposome solution modified with chitosan is slowly added into the sodium alginate solution drop by drop and left for a period of time.
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CN115191617A (en) * | 2022-07-13 | 2022-10-18 | 江南大学 | Preparation method of sodium alginate-stabilized hypoglycemic collagen peptide liposome |
CN115191617B (en) * | 2022-07-13 | 2023-10-24 | 江南大学 | Preparation method of sodium alginate stabilized blood glucose-reducing collagen peptide liposome |
CN116236417A (en) * | 2023-03-07 | 2023-06-09 | 大连理工大学 | Polymer multilayer skin sun-screening agent coated carrier material and preparation method thereof |
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