CN113855811A - Preparation method and application of food-grade nano-carrier targeting aged cells - Google Patents
Preparation method and application of food-grade nano-carrier targeting aged cells Download PDFInfo
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- CN113855811A CN113855811A CN202111237793.5A CN202111237793A CN113855811A CN 113855811 A CN113855811 A CN 113855811A CN 202111237793 A CN202111237793 A CN 202111237793A CN 113855811 A CN113855811 A CN 113855811A
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
-
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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 discloses a preparation method and application of a food-grade nano-carrier for targeting aged cells, belonging to the field of targeted delivery of bioactive factors. The invention utilizes the characteristic that senescent cells highly express senescent related beta-galactosidase (SA-beta-Gal), takes protein, reducing sugar and ethanol which can be identified and cut by SA-beta-Gal as raw materials, and prepares the nano-carrier loaded with the lipophilic anti-aging bioactive factor through Maillard reaction, molecular self-assembly and other steps, and the obtained nano-carrier can target senescent cells to delay senescence. Based on the property of the food-grade nano-carrier prepared by the invention, the food-grade nano-carrier has an anti-aging effect and can be applied to the fields of functional foods, biological medicines, cosmetics and the like.
Description
Technical Field
The invention relates to a preparation method and application of a food-grade nano-carrier for targeting aged cells, belonging to the field of targeted delivery of bioactive factors.
Background
Cell aging is the basis of individual aging and is closely related to aging and aging-related diseases. Research shows that the health can be obviously improved and the life can be prolonged by eliminating senescent cells. Bioactive factors (such as fisetin, quercetin, astaxanthin and the like) with anti-aging effect can play an anti-aging role by eliminating free radicals, promoting apoptosis of aging cells, inhibiting cell aging-related secretory phenotype and the like. However, these factors tend to be lipid soluble, insoluble in water and have low bioavailability, thus limiting their use in pharmaceuticals, cosmetics and especially in food products. In addition, they can only be targeted for delivery to senescent cells to maximize their effect while avoiding the effects on normal cells. Based on the characteristics of the senescent cells, a food-grade senescent cell targeted nano-delivery system is required to be constructed to promote the utilization of the lipophilic anti-aging bioactive factors.
To date, many strategies for constructing a vector targeting senescent cells have been omitted, and mainly utilize the characteristics of senescent cells, such as increased activity of senescence-associated beta-galactosidase (SA-beta-Gal) (SA-beta-Gal is a cellular senescence biomarker widely adopted at present), high expression of CD9, and the like, to construct delivery vectors based on mesoporous silica nanoparticles, polymer nanoparticles, calcium carbonate nanoparticles, and the like. However, the methods have the defects of inedibility of raw materials, complex chemical preparation process and the like, so that the application of the methods in food is limited, and therefore, the establishment of a food-grade senescent cell targeted delivery system by using the food-grade raw materials and green chemical reaction is urgently needed.
Disclosure of Invention
[ problem ] to
Bioactive factors (such as fisetin, quercetin, astaxanthin and the like) with an anti-aging effect are usually fat-soluble, insoluble in water and low in bioavailability, and need to be delivered to aging cells in a targeted manner to exert the effects to the maximum extent; the delivery carrier adopted at present has the defects of inedibility and complex preparation.
[ solution ]
In order to solve the problems, the invention utilizes the characteristic that senescent cells highly express SA-beta-Gal, takes protein, reducing sugar capable of being identified and cut by SA-beta-Gal, ethanol and the like as raw materials, and prepares the nano-carrier loaded with the lipophilic anti-aging bioactive factor through Maillard reaction, molecular self-assembly and other steps. The nano-carrier prepared by the invention can target senescent cells to delay senescence, and can be applied to the fields of functional foods, biological medicines, cosmetics and the like.
The first object of the present invention is to provide a method for preparing food-grade nanocarriers that target senescent cells, comprising the steps of:
(1) preparing mixed dry powder of protein and reducing sugar:
dissolving protein and reducing sugar with water or buffer solution to obtain mixed solution of protein and reducing sugar; then carrying out hydration and vacuum freeze drying to obtain protein-reducing sugar mixed dry powder;
(2) protein-grafted reducing sugar:
placing the protein-reducing sugar mixed dry powder obtained in the step (1) in an inner chamber of a closed container, placing a saturated salt solution in an outer chamber of the closed container, reacting for 6-96 hours at 40-80 ℃ in a constant temperature incubator, and stopping the reaction to obtain a product; dissolving and dialyzing the product or dissolving the product after ethanol precipitation and cleaning the product, and performing vacuum freeze drying to obtain a protein-reducing sugar graft;
(3) dissolving lipophilic anti-aging bioactive factors:
fully dissolving the lipophilic anti-aging bioactive factor by using an organic solvent to obtain a lipophilic anti-aging bioactive factor solution;
(4) loading lipophilic anti-aging bioactive factors:
taking the protein-reducing sugar graft obtained in the step (2), and adding water for dissolving to obtain a protein-reducing sugar graft solution with the final concentration of 0.1-10% (w/v); then dripping the lipophilic anti-aging bioactive factor solution obtained in the step (3) into the protein-reducing sugar graft solution for reaction; and after the reaction is finished, removing the organic solvent, and performing vacuum freeze drying to obtain the food-grade nano-carrier targeting the aged cells.
In one embodiment of the present invention, the buffer solution in step (1) is a buffer solution with a pH of 3-10, including 0.1MpH 7.0.0 PBS buffer solution.
In one embodiment of the invention, the protein in step (1) is a food or pharmaceutical water soluble protein, including food derived proteins and pharmaceutical proteins; wherein the food source protein comprises one or more of whey protein isolate, whey protein concentrate, soy protein isolate, soy protein concentrate and casein, and the medicinal protein comprises albumin; further preferably whey protein isolate.
In one embodiment of the present invention, the reducing sugar in step (1) is a reducing sugar capable of being recognized and cleaved by SA- β -Gal, and includes lactose, galacto-oligosaccharide, galactan, etc., and is more preferably galacto-oligosaccharide.
In one embodiment of the present invention, in the mixed solution of protein and reducing sugar in step (1), the ratio of protein, reducing sugar and buffer is 0.1-10 g: 0.1-10 g: 100mL, more preferably 2 g: 2 g: 100 mL.
In one embodiment of the invention, the hydration in step (1) is at a temperature of 2-8 ℃ until complete hydration.
In one embodiment of the present invention, the closed container in step (2) comprises a conway.
In an embodiment of the present invention, the saturated salt solution in step (2) is a salt solution capable of providing a certain relative humidity, and includes one or more of a saturated potassium carbonate solution, a saturated magnesium nitrate solution, a saturated potassium iodide solution, and a saturated potassium bromide solution, and further preferably a saturated magnesium nitrate solution, and the relative humidity of the saturated magnesium nitrate solution is 45% at 60 ℃.
In one embodiment of the present invention, the termination reaction in step (2) is performed by placing the reaction product on ice for 5-20min, preferably on ice for 10 min.
In one embodiment of the invention, the dissolving in the step (2) is performed by using water, and the dialysis is performed by using a 3000Da dialysis bag and dialyzing in deionized water to remove unbound small molecular impurities such as galactooligosaccharides; the ethanol precipitation cleaning is to clean the precipitation for 3 times by using 75% ethanol to remove impurities such as unbound galacto-oligosaccharides and the like.
In one embodiment of the present invention, the lipophilic anti-aging bioactive factor in step (3) includes one or more of fisetin, astaxanthin, quercetin, and curcumin, and more preferably fisetin.
In one embodiment of the present invention, the organic solvent in step (3) comprises ethanol, ethyl acetate; more preferably, it is anhydrous ethanol.
In one embodiment of the present invention, the concentration of the lipophilic anti-aging bioactive factor solution in step (3) is 0.5 mg/mL.
In one embodiment of the present invention, the dissolving in step (3) is performed by stirring, dissolving and mixing under the condition of keeping out of the light.
In one embodiment of the present invention, the volume ratio of the lipophilic anti-aging bioactive factor solution and the aqueous solution of the protein-reducing sugar graft in the step (4) is 1: 4-14, and more preferably 1: 9.
in one embodiment of the invention, the reaction in step (4) is carried out for 1-12h under stirring at room temperature (20-30 ℃) in the dark, and the lipophilic anti-aging bioactive factor solution is added dropwise under stirring, or after 5min under stirring at room temperature (20-30 ℃) in the dark, dispersed and emulsified by a high-speed disperser, and homogenized by ultrasound or high pressure.
In one embodiment of the present invention, the organic solvent removal in step (4) is performed by rotary evaporation.
In one embodiment of the present invention, the vacuum freeze-drying used in step (1), step (2) and step (4) is performed by completely drying the product.
The second purpose of the invention is to obtain the food-grade nano-carrier targeting the aged cells by the method.
The third purpose of the invention is the application of the food-grade nano-carrier for targeting the aged cells in functional foods, biomedicines and cosmetics.
[ advantageous effects ]
(1) The invention firstly utilizes food-grade raw materials (such as whey protein isolate and galacto-oligosaccharide) and food-grade reactions (such as Maillard reaction and molecular self-assembly) to green construct the aging cell targeted nano-carrier loaded with the lipophilic anti-aging bioactive factor, has the advantages of good water solubility, high stability, good biocompatibility and the like, and can be applied to the fields of functional foods, biomedicines, cosmetics and the like.
(2) The invention utilizes the characteristic of high expression of SA-beta-Gal by senescent cells, takes protein, reducing sugar and ethanol which can be identified and cut by SA-beta-Gal as raw materials, and prepares the nano-carrier loaded with the lipophilic anti-aging bioactive factor through Maillard reaction, molecular self-assembly and other steps, and the obtained nano-carrier can target the senescent cells to delay aging.
(3) The method is simple, easy to operate, low in preparation cost and mild in conditions.
Drawings
Fig. 1 is an appearance view of the food-grade nanocarriers prepared in example 1, wherein WPI represents whey protein isolate, GOS represents galacto-oligosaccharide, and FIS represents fisetin.
Fig. 2 is a scanning electron microscope image and a particle size characterization image (inset) of the food grade nanocarriers prepared in example 1.
FIG. 3 is a graph representing the storage stability of the food-grade nanocarriers prepared in example 1.
FIG. 4 is food grade nanocarrier treatment H prepared in example 12O2SA-beta-Gal staining pattern (A) and staining cell number statistical pattern (B) of induced aged human lung fibroblast MRC-5.
FIG. 5 is the food grade nanocarrier treatment H prepared with fluorescent dye CY5 instead of fisetin in example 12O2Cytofluorescence profile of induced aged human lung fibroblasts, MRC-5.
Fig. 6 is a cell viability characterization graph of the food grade nanocarriers prepared in example 1.
Fig. 7 is a scanning electron microscope image of freezing food grade nanocarriers prepared in example 4.
Fig. 8 is a graph representing the storage stability of the food-grade nanocarriers prepared in example 4, wherein AST represents astaxanthin.
Fig. 9 is a graph representing in vitro simulated digestive stability of the food-grade nanocarriers prepared in example 4 in artificial gastric fluid (a) and artificial intestinal fluid (B).
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
The vacuum freeze drying mentioned in the examples is all that is needed to completely dry the product.
Example 1
A method for preparing food-grade nano-carriers targeting senescent cells comprises the following steps:
(1) preparing mixed dry powder of protein and reducing sugar:
weighing Whey Protein Isolate (WPI) and galacto-oligosaccharide (GOS) 2g respectively, adding 0.1M PBS buffer solution with pH 7.0 100mL, stirring at room temperature for 3h to dissolve completely to obtain mixed solution of protein and reducing sugar; then, overnight hydration is carried out at 4 ℃, and vacuum freeze drying is carried out, thus obtaining protein-reducing sugar mixed dry powder;
(2) protein-grafted reducing sugar:
taking 1g of whey protein isolate-galacto-oligosaccharide mixed dry powder obtained in the step (1), placing the powder in an inner chamber of a Kangwei dish, adding a saturated magnesium nitrate solution in an outer chamber, placing the powder in a constant temperature incubator at 60 ℃ with the relative humidity of 45%, reacting for 24 hours, immediately placing the powder on ice for 10min, and stopping the reaction to obtain a product; adding 40mL of 75% ethanol into the product, washing, centrifuging at room temperature of 10000rpm for 10min, discarding the supernatant, repeatedly washing for 3 times to remove impurities such as unbound galactooligosaccharide, pigment and the like, dissolving with 20mL of water, and freeze-drying under vacuum to obtain a whey protein isolate-galactooligosaccharide graft (WPI-GOS), wherein the degree of grafting is 63.8% by using an OPA method;
(3) dissolving lipophilic anti-aging bioactive factors:
1mg of Fisetin (FIS) and 2mL of absolute ethanol are uniformly mixed to completely dissolve fisetin, so that fisetin solution with the concentration of 0.5mg/mL is obtained;
(4) loading lipophilic anti-aging bioactive factors:
taking 90mg of the protein-reducing sugar graft obtained in the step (2), and adding 18mL of water for dissolving to obtain a protein-reducing sugar graft solution with the final concentration of 0.5% (w/v); then, 2mL of the lipophilic anti-aging bioactive factor solution obtained in the step (3) is added dropwise into the protein-reducing sugar graft solution while stirring, and the mixture is stirred overnight at room temperature in a dark place to react; after the reaction is finished, placing the mixture at 40 ℃ and rotating and evaporating the mixture to 10mL at 40rpm, removing the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain the food-grade nano-carrier (WP1-GOS-FIS) for targeting the senescent cells.
The prepared food-grade nano-carrier (WP1-GOS-FIS) is tested, and the test result is as follows:
the aqueous solution of the food-grade nano carrier (WP1-GOS-FIS) is clear and transparent and is light yellow in appearance (figure 1), and is extracted by dichloromethane/methanol (volume ratio is 1:1) solution, and the encapsulation rate of the fisetin is 93.9% quantitatively obtained by a 364nm ultraviolet absorption method.
The food grade nanocarriers (WP1-GOS-FIS) were shown by cryo-scanning electron microscopy to be spheroidal with an average particle size of 87nm (FIG. 2), and by dynamic light scattering analysis to be 171 nm.
The storage stability experiment shows that no obvious aggregation and color change are observed after the carrier is stored for 28 days under the condition of being protected from light at 4 ℃, and the carrier has better storage stability (figure 3).
To verify the inhibitory effect of the vector on cell senescence, MRC-5 cells were inoculated into 12-well plates in a medium of EMEM containing 20% fetal bovine serum and 100U/mL streptomycin, and after pre-incubation for 24h with food-grade nanocarriers (loaded with fisetin at a concentration of 25. mu.M), 400. mu. M H2O2The treatment was carried out for 2h, washed twice with PBS buffer, and replaced with the medium containing nanocarriers (loaded with fisetin at a concentration of 25. mu.M) for further incubation for 4 days. SA-beta-Gal staining shows, H2O2The proportion of SA-beta-Gal positive cells after treatment is increased from 3.2 percent to 47.6 percent, and the SA-beta-Gal positive cells are food-grade nanoThe proportion of the carrier treated is reduced to 19.0 percent, which shows that the food-grade nano carrier can obviously inhibit H2O2Induced cellular senescence (fig. 4).
In order to verify the targeting effect of the food-grade nano-carrier on aged cells, a fluorescent tracing carrier is prepared by using fluorescent dye CY5 to replace fisetin according to the method of example 1, and then MRC-5 cells are inoculated on a 12-well plate, 400 mu M H2O2The treatment was carried out for 2h, washed twice with PBS buffer, replaced with medium and incubated for 4 days. After incubating the fluorescent tracer carrier of 0.25mg/mL for 2h, washing twice by PBS buffer solution, directly fixing SA-beta-Gal staining on one part, replacing the other part with culture medium, continuing incubating for 4h, then fixing SA-beta-Gal staining, and observing by using an inverted fluorescent microscope. The results show that after the fluorescent tracing carrier is directly dyed after incubation, red fluorescent signals can be seen by normal cells and senescent cells, but the senescent cells have relatively strong signals, and the fluorescent signals of the normal cells are obviously reduced compared with 0h after the cells are incubated for 4h for dyeing, while the fluorescent signals of the senescent cells are not obviously changed (figure 5), namely: the senescent cells can promote the carrier to take in and inhibit the carrier from discharging outwards, which shows that the carrier has better senescent cell targeting property.
In order to verify the biological safety of the food-grade nano-carrier, MRC-5 cells are inoculated in a 96-well plate, and after the food-grade nano-carrier (loaded with fisetin with the concentration of 25 mu M) is incubated for 24 hours, the effect of the carrier on the cell viability is detected by a CCK8 method. The results show that the cell viability is 115.2% after 25 μ M free fisetin treatment for 24h and 122.9% after WPI-GOS-FIS treatment, which indicates that the food-grade nano-carrier has no obvious cytotoxicity and good biocompatibility (FIG. 6).
Example 2
Adjusting the saturated magnesium nitrate solution in the step (2) of example 1 to be a saturated potassium iodide solution, which has a relative humidity of 65% at 60 ℃; the other steps were identical to the steps (1) and (2) in example 1, and whey protein isolate-galactooligosaccharide grafts (WP1-GOS) were obtained in the same manner, and the degree of grafting was 52% by OPA method.
Example 3
The concentration of the fisetin solution in step (3) of example 1 was adjusted to 1 mg/mL; the rest of the steps are kept consistent with the steps in the example 1, and the food-grade nano-carrier is obtained.
And (3) carrying out performance test on the obtained food-grade nano-carrier, wherein the test result is as follows: the prepared water solution of the food-grade nano carrier (WP1-GOS-FIS) is clear and transparent and is light yellow in appearance, and the encapsulation rate of the fisetin is 87.4% quantitatively obtained by extracting the water solution with dichloromethane/methanol (the volume ratio is 1:1) through a 364nm ultraviolet absorption method. Dynamic light scattering analysis showed an average particle size of 262 nm.
Comparative example 1
The concentration of the fisetin solution in step (3) of example 1 was adjusted to 5 mg/mL; the rest of the steps are kept consistent with the steps in the example 1, and the food-grade nano-carrier is obtained.
And (3) carrying out performance test on the obtained food-grade nano-carrier, wherein the test result is as follows: the prepared water solution of the food grade nano-carrier (WP1-GOS-FIS) is opaque and yellow in appearance, is easy to aggregate and precipitate, and shows that the average particle size is 860nm by dynamic light scattering analysis.
Example 4
A method for preparing food-grade nano-carriers targeting senescent cells comprises the following steps:
(1) preparing mixed dry powder of protein and reducing sugar: same as example 1, step (1);
(2) protein-grafted reducing sugar: same as example 1, step (2);
(3) dissolving lipophilic anti-aging bioactive factors:
uniformly mixing 1mg of Astaxanthin (AST) with 2mL of ethyl acetate to completely dissolve the astaxanthin to obtain an astaxanthin solution with the concentration of 0.5 mg/mL;
(4) loading lipophilic anti-aging bioactive factors:
taking 90mg of the protein-reducing sugar graft obtained in the step (2), adding 18mL of ethyl acetate aqueous solution (8.3 g of ethyl acetate is added into 100mL of water) for dissolving to obtain a protein-reducing sugar graft solution with the final concentration of 0.5% (w/v); then, 2mL of the lipophilic anti-aging bioactive factor solution obtained in the step (3) is added dropwise into the protein-reducing sugar graft solution while stirring, the mixture is stirred at room temperature in a dark place for 5min, and is dispersed for 3min at 10000rpm by a high-speed disperser, and the mixture is subjected to ultrasonic treatment (200W, 5s for ultrasonic treatment and 5s for 5min in total); after the reaction is finished, placing the mixture at 40 ℃ and rotating and evaporating the mixture to 10mL at 40rpm, removing the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain the food-grade nano-carrier (WP1-GOS-AST) targeting the aged cells.
The prepared food-grade nano-carrier (WP1-GOS-AST) is tested, and the test result is as follows:
the aqueous solution of the food-grade nano-carrier (WP1-GOS-AST) is clear, transparent and red in appearance, and is extracted by a dichloromethane/methanol (volume ratio is 1:1) solution, and the encapsulation rate of the astaxanthin is obtained by a 478nm ultraviolet absorption method quantitatively and is 95.9%.
The nano-carrier was shown to be spheroidal by cryo-scanning electron microscopy (see fig. 7), and the average particle size was 158nm by dynamic light scattering analysis.
The storage stability experiment shows that no obvious aggregation and color change are observed after the carrier is stored for 60 days under the condition of keeping out of light at 4 ℃, and the carrier has better storage stability (figure 8).
In order to verify the in-vitro digestion stability of the food-grade nano-carrier, the nano-carrier is respectively incubated with artificial gastric juice (containing 3.2mg/mL pepsin) and artificial intestinal juice (containing 1mg/mL trypsin) for 2h, and sampling is carried out every 30min to detect the release rate of astaxanthin. The results show that the WPI-AST and WPI-GOS-AST carriers astaxanthin are gradually released along with the prolongation of the digestion time, the release rate of the WPI-AST astaxanthin is 26.5 percent after the gastric juice digestion is carried out for 2 hours, the release rate of the WPI-GOS-AST astaxanthin is 11.7 percent, the release rate of the WPI-AST astaxanthin is 16.2 percent after the intestinal juice digestion is carried out for 2 hours, and the release rate of the WPI-GOS-AST astaxanthin is 4.5 percent, which indicates that the nano-carrier modified by GOS has better gastrointestinal digestion stability (figure 9).
Example 5
Adjusting the protein-reducing sugar graft solution in step (4) of example 4 to a final concentration of 0.1% (w/v); the rest of the steps are kept consistent with the steps in the example 4, and the food-grade nano-carrier is obtained.
And (3) carrying out performance test on the obtained food-grade nano-carrier, wherein the test result is as follows: the prepared water solution of the food-grade nano-carrier (WP1-GOS-AST) is clear, transparent and red in appearance, is extracted by a dichloromethane/methanol (volume ratio is 1:1) solution, the encapsulation rate of the astaxanthin is quantitatively obtained by a 478nm ultraviolet absorption method and is 86.8%, and the average particle size is 158nm by utilizing dynamic light scattering analysis. The storage stability experiment shows that no obvious aggregation and color change are observed after the carrier is stored for 30 days at 4 ℃ in a dark condition, and the carrier has better storage stability.
Example 6
Adjusting the protein-reducing sugar graft solution in step (4) of example 4 to a final concentration of 2% (w/v); the rest of the steps are kept consistent with the steps in the example 4, and the food-grade nano-carrier is obtained.
And (3) carrying out performance test on the obtained food-grade nano-carrier, wherein the test result is as follows:
the prepared water solution of the food-grade nano-carrier (WP1-GOS-AST) is clear, transparent and red in appearance, is extracted by a dichloromethane/methanol (volume ratio is 1:1) solution, the encapsulation rate of the astaxanthin is 96.5% quantitatively obtained by a 478nm ultraviolet absorption method, and the average particle size is 115nm by utilizing dynamic light scattering analysis. The storage stability experiment shows that no obvious aggregation and color change are observed after the carrier is stored for 30 days at 4 ℃ in a dark condition, and the carrier has better storage stability.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing a food-grade nano-carrier targeting senescent cells is characterized by comprising the following steps:
(1) preparing mixed dry powder of protein and reducing sugar:
dissolving protein and reducing sugar with water or buffer solution to obtain mixed solution of protein and reducing sugar; then carrying out hydration and vacuum freeze drying to obtain protein-reducing sugar mixed dry powder;
(2) protein-grafted reducing sugar:
placing the protein-reducing sugar mixed dry powder obtained in the step (1) in an inner chamber of a closed container, placing a saturated salt solution in an outer chamber of the closed container, reacting for 6-96 hours at 40-80 ℃ in a constant temperature incubator, and stopping the reaction to obtain a product; dissolving and dialyzing the product or dissolving the product after ethanol precipitation and cleaning the product, and performing vacuum freeze drying to obtain a protein-reducing sugar graft;
(3) dissolving lipophilic anti-aging bioactive factors:
fully dissolving the lipophilic anti-aging bioactive factor by using an organic solvent to obtain a lipophilic anti-aging bioactive factor solution;
(4) loading lipophilic anti-aging bioactive factors:
taking the protein-reducing sugar graft obtained in the step (2), and adding water for dissolving to obtain a protein-reducing sugar graft solution with the final concentration of 0.1-10% (w/v); then dripping the lipophilic anti-aging bioactive factor solution obtained in the step (3) into the protein-reducing sugar graft solution for reaction; and after the reaction is finished, removing the organic solvent, and performing vacuum freeze drying to obtain the food-grade nano-carrier targeting the aged cells.
2. The method according to claim 1, wherein the reducing sugar of step (1) is a reducing sugar capable of being cleaved by recognition by senescence-associated β -galactosidase (SA- β -Gal), including lactose, galactooligosaccharides, galactans.
3. The method according to claim 1 or 2, wherein the protein of step (1) is a food or pharmaceutical water soluble protein, including food derived proteins and pharmaceutical proteins; wherein the food source protein comprises one or more of whey protein isolate, whey protein concentrate, soy protein isolate, soy protein concentrate and casein, and the medicinal protein comprises albumin.
4. The method according to any one of claims 1 to 3, wherein the saturated salt solution in the step (2) is a salt solution capable of providing a certain relative humidity, and comprises one or more of a saturated potassium carbonate solution, a saturated magnesium nitrate solution, a saturated potassium iodide solution and a saturated potassium bromide solution.
5. The method according to any one of claims 1 to 4, wherein the lipophilic anti-aging bioactive factor of step (3) comprises one or more of fisetin, astaxanthin, quercetin, and curcumin.
6. The method according to any one of claims 1 to 5, wherein the ratio of the protein, the reducing sugar and the buffer solution in the mixed solution of the protein and the reducing sugar in step (1) is 0.1 to 10 g: 0.1-10 g: 100 mL.
7. The method according to any one of claims 1 to 6, wherein the volume ratio of the lipophilic anti-aging bioactive factor solution and the aqueous protein-reducing sugar graft solution in the step (4) is 1: 4 to 14.
8. The method according to any one of claims 1 to 7, wherein the organic solvent in step (3) comprises ethanol and ethyl acetate.
9. The food-grade nano-carrier for targeting aged cells, prepared by the method of any one of claims 1-8.
10. The use of the senescent cell-targeted food-grade nanocarrier of claim 9 in functional foods, biomedicines, cosmetics.
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