CN110075313B - Protein/polypeptide-loaded lipoid composition and preparation method thereof - Google Patents
Protein/polypeptide-loaded lipoid composition and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a lipoid composition carrying protein/polypeptide and a preparation method thereof. The preparation method comprises the following steps: extracting natural polyphenol in edible plants or synthetic polyphenol obtained by processing, and assembling and constructing a polyphenol-protein/polypeptide compound (VPN) by utilizing the affinity effect of polyphenol and protein/polypeptide; then extracting lipoid from organism, coating and stabilizing VPN, and constructing lipoid composition loaded with functional protein/polypeptide component. The invention has simple preparation process, lower cost and good reproducibility, can solve the problems of stability and safety of protein/polypeptide components in storage, transportation and in-vivo use processes, better retains the functionality of the components, and has better application prospect in the fields of relevant medicines, foods, cosmetics and the like.
Description
Technical Field
The invention relates to a lipoid composition carrying protein/polypeptide and a preparation method thereof, belonging to the field of pharmaceutical food compositions.
Background
The protein/polypeptide is an indispensable nutrient for human bodies, and besides having a nutritional function, some bioactive peptides also participate in regulating various important physiological functions of organisms, such as antioxidation, immunoregulation, neuromodulation, antithrombotic, antihypertensive, cholesterol reduction, antivirus, anticancer and the like, and are one of the most popular fields of current research in the food and pharmaceutical sciences. However, such ingredients have poor stability, high side effects, short half-life in vivo, low oral bioavailability, susceptibility to protease degradation, etc. [ Chan ys. Et al, appl Microbiol Biotechnol,2016,100 (14): 6165-6181, increasing the research and development difficulty, especially the problem of in vivo and in vitro stability becomes an important factor for restricting the research and development of protein/polypeptide health food and drugs. Therefore, it is necessary to adopt appropriate means for improving the stability and safety of the protein/polypeptide composition.
There have been many reports of improvements to proteins/polypeptides, such as physicochemical modifications of proteins/polypeptides [ Werkmeister JA. Et al, biochim Biophys Acta,1993,1157 (1): 50-54], coupling the protein/polypeptide to an antibody [ Zhao X.et al, biotechnol Appl Biochem,2011,58 (6): 405-411], construction of protein/polypeptide loaded nano delivery systems [ Woo sy.et al, phys Chem Phys,2017, 19 (10): 7195-7203], these strategies, while solving to some extent the above-mentioned problems of proteins/polypeptides, still have the disadvantages: the activity of the protein/polypeptide is reduced after the structure is modified; drug loading in surface adsorption or covalent form cannot completely shield the hemolysis of the polypeptide; the load capacity of the vector and the safety of the vector itself are mutually restricted, and the like. In order to avoid the above-mentioned drawbacks of proteins/polypeptides, a safer and more effective method is needed to reduce the toxic side effects and increase the in vivo and in vitro stability.
It has been reported that ingredients contained in some edible plants include both primary metabolites such as lipids, sugars, and the like, and secondary metabolites such as polyphenols [ Soleti r.et al, arch Biochem biophysis, 2018, 644:57-63], and the like. Modern studies have confirmed that polyphenols have good physiological activities of antioxidant, anti-inflammatory, antibacterial, anticancer and antiviral [ nassii-Asl m.et al, phytother Res,2016,30 (9): 1392-1403; estela J m.et al, J Med Chem,2017,60 (23): 9413-9436; calabriso N.et al, molecules,2016,21 (9): 1 to 18; et al, J Food Drug Anal,2017,25 (1): 3-15]. In addition, polyphenol also has strong protein/polypeptide precipitation effect, which suggests that polyphenol-protein/polypeptide complex (VPN) can be constructed through the affinity of protein/polypeptide and polyphenol so as to realize the structural stability and functional synergy of the two.
The subject group reports that a large number of vesicle structures (graft-derived vesicles) composed of lipids and the like exist in grape pulp, melittin (Mel) is used as a model drug, and polypeptide-loaded grape recombinant vesicles are constructed by utilizing the aggregation property of Melittin and polyphenol and the loading capacity of the vesicles, and are uniformly distributed and good in stability, and the in vitro cytotoxicity proves that the grape recombinant vesicles have a better tumor inhibition effect. However, due to the structural limitation of the vesicle, the constructed recombinant vesicle has low encapsulation efficiency, slow in vitro release and high residual rate of protein/polypeptide.
In order to better develop the application of protein/polypeptide in medicines, foods, health products and cosmetics, how to design a novel protein/polypeptide-carrying composition is a problem to be solved urgently by scientists.
Disclosure of Invention
The invention aims to provide a lipoid composition carrying protein/polypeptide, which aims to solve the problems of in-vitro instability, nutrient loss and biological safety of protein/polypeptide components; and the protein/polypeptide has high encapsulation rate, high release effect and good utilization rate and efficacy.
Accordingly, it is another object of the present invention to provide a method for preparing a lipid body composition of proteins/polypeptides which is cost effective and does not rely on complex test equipment.
In order to achieve the purpose, the invention adopts the following conception:
the lipoid composition carrying protein/polypeptide consists of protein/polypeptide, polyphenol and lipoid, and features that the polyphenol and the protein/polypeptide form nanometer compound through affinity reaction to stabilize their structure. Through the loading effect of the lipoid, a lipoid system carrying protein/polypeptide is constructed. The lipoid as carrier can raise the stability of protein/polypeptide in-vivo and in-vitro transport, maintain the self function of protein/polypeptide, shield the positive charge of cationic polypeptide and raise biological safety.
A first object of the invention is to provide a protein/polypeptide loaded lipid composition comprising a protein/polypeptide, a polyphenol and a lipid.
Furthermore, the dosage proportion (by weight) of the protein/polypeptide, the polyphenol and the lipoid is 1 to (0.1-2.0) to (0.2-5.0). Furthermore, the dosage ratio of the protein/polypeptide, the polyphenol and the lipoid (by weight parts) is 1: 0.5-1.5: 1.0-3.0. Furthermore, the dosage ratio (by weight parts) of the protein/polypeptide, the polyphenol and the lipoid is 1: 2.
Further wherein the polyphenol interacts with the protein/polypeptide by affinity to form a nanocomplex; then, a lipoid system carrying protein/polypeptide is constructed through the load effect of the lipoid.
Further, wherein the protein/polypeptide is a functional food material or an active ingredient; or may be a single component or a mixture of components. For functional food materials, including but not limited to various types of nutritional supplements; for the active ingredient, compounds having antioxidant, antitumor, antithrombotic, anti-inflammatory, etc. properties are included, but not limited thereto. <xnotran> , / , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , EGF, GLP-1, PYY36, , GLP-2, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ; </xnotran> Wherein, the milk protein is preferably alpha-lactalbumin, beta-lactoglobulin, lactoferrin or casein. Such proteins/polypeptides may be isolated from natural sources, synthesized or semi-synthesized, or may be purchased commercially.
Further wherein the polyphenol is a natural plant polyphenol or a synthetic polyphenol obtained by processing; can be one component or a mixture of multiple components. Wherein the natural plant includes but is not limited to fruits and vegetables, chinese medicinal materials, etc., such as tea, apple, grape, orange, watermelon, spinach, broccoli, tomato, cucumber, aloe, ginseng, ginger, rehmannia root, gastrodia tuber, matrimony vine, corn, etc.; the synthetic polyphenol can be obtained by methods such as total synthesis, structure modification, polymerization reaction and the like. Still further, the polyphenol is selected from one or more of grape polyphenol, medlar polyphenol, corn polyphenol, tea polyphenol, cucumber polyphenol, epigallocatechin gallate, epigallocatechin, onion polyphenol, chlorogenic acid, gallic acid, tannin, quercetin, ferulic acid, curcumin, tannic acid, caffeic acid, quinic acid, catechin, agrimonine, sanguisorbain, rhodinin, geraniin, corilagin, kojic acid, oenotheracleum biennin, prairitin, punicin and cornusin.
Further wherein the lipid is derived from a natural plant, biological fluid, tissue and/or cell. Wherein the natural plants include but are not limited to fruits and vegetables, chinese medicinal materials and the like; wherein the biological fluids include, but are not limited to, milk, saliva, peritoneal fluid, blood, and urine; wherein the biological tissue includes but is not limited to tissue of visceral origin; wherein the cells include but are not limited to stem cells, tumor cells, immune cells, endothelial cells, and the like. Still further, the lipid is selected from one or more of grape lipid, tomato lipid, tumor cell lipid, ginseng lipid, aloe lipid, cucumber lipid, medlar lipid, and milk lipid.
A second object of the invention is to provide a process for the preparation of a protein/polypeptide loaded lipid composition comprising the steps of:
the polyphenol and the protein/polypeptide form a nano-composite through affinity action;
and then constructing a lipoid system carrying protein/polypeptide through the load effect of the lipoid.
The affinity is estimated to be that polyphenol is linked between proteins/polypeptides by means of a bridged bond in the above-mentioned nanocomplex, but the structure of the above-mentioned nanocomplex is not particularly limited.
Further, the polyphenol and the protein/polypeptide are mixed with each other in an aqueous solution, and the mixing process is not particularly limited. For example, the polyphenol and the protein/polypeptide may be added to water sequentially or simultaneously and mixed. For example, the mixture may be prepared by mixing a 3 to 50 weight percent aqueous solution of polyphenol with a 3 to 50 weight percent aqueous solution of protein/polypeptide. Preferably, the polyphenol solution is added dropwise to the protein/polypeptide solution and mixed.
Still further, the above mixing may be carried out under normal pressure or under pressure. The mixing temperature may be from-4 to 90 ℃ or preferably from 0 to 70 ℃ and more preferably from 15 to 30 ℃, but is not limited to these conditions.
Still further, the process for preparing the above-mentioned nanocomposite comprises a step of separating the above-mentioned nanocomposite from an aqueous solution and/or comprises a step of removing free polyphenol. The method for separating the polyphenol-protein/polypeptide complex may use any one of the methods known in the art. For example, pouring, centrifugation, filtration, evaporation, and the like.
Further, the step of loading the lipid with the nanocomposite comprises: and mixing the lipoid with the nano-composite solution, and carrying out physical and/or chemical treatment to obtain the lipoid composition loaded with the protein/polypeptide. The mixing order is not particularly limited. For example, the lipid and the nanocomposite may be mixed after each being prepared into an aqueous solution, or may be mixed by adding the lipid and the nanocomposite to water sequentially or simultaneously. Preferably, the lipid is dropped into the nanocomposite solution and mixed.
Still further, the physical and/or chemical treatment is a loading treatment method commonly used in the art, such as stirring, extrusion, heating, and the like. Preferably by extrusion.
Furthermore, the preparation method of the lipid body composition carrying the protein/polypeptide also comprises a preparation method of polyphenol. Such as polyphenol extraction and purification steps, and optionally, polyphenol synthesis.
Still further, the extraction and purification of polyphenols comprises:
extracting for one or more times by taking an ethanol solution as an extraction solvent under the conditions of proper material-liquid ratio, extraction temperature and extraction time; followed by purification with macroporous resin.
Still further, the process for synthesizing the polyphenol comprises:
weighing a proper amount of polyphenol, dissolving the polyphenol in a mixed solution of acetic acid, water and DMSO, adding acetaldehyde, reacting at a proper pH value, dialyzing a product, and freeze-drying the product.
Further, the preparation method of the protein/polypeptide-carrying lipid composition also comprises the extraction of the lipid component. For example, the juice (or biological fluid, tissue homogenate, cell culture supernatant) is centrifuged and precipitated to obtain a lipoid solution; optionally, lyophilization of the lipid solution is included.
Still further, the lipid component is extracted by:
centrifuging the juice (or biological fluid, tissue homogenate, and cell culture supernatant), collecting supernatant, adding PEG solution, mixing, incubating, and centrifuging for resuspension to obtain lipoid solution. Optionally, the lipid solution is stored lyophilized.
Still further, a method of preparing a protein/polypeptide loaded lipid composition comprising the steps of:
1. a preparation method of polyphenol (VTs) comprises the following steps:
a. extracting and purifying polyphenol in edible plants: taking 60-95% ethanol solution as an extraction solvent, wherein the material-liquid ratio is 1; then selecting the best type of macroporous resin (D101, AB-8, XAD-7 Hp) to purify polyphenol. The polyphenol content is expressed as the absorbance at 760 nm.
b. The method for synthesizing the polyphenol comprises the following steps: weighing a proper amount of polyphenol, dissolving the polyphenol in a mixed solution of acetic acid, water and DMSO, adding 20-60% of acetaldehyde, adjusting the pH to 2-5, reacting for 6-24 h under the protection of nitrogen, dialyzing a product, and freeze-drying.
2. And (3) extracting lipid bodies: squeezing edible plants to obtain juice, taking the juice (or biological body fluid, tissue homogenate and cell culture supernatant), centrifuging for 1h at 2000-5000 g, taking supernatant, adding 8% PEG6000 solution in the same volume, mixing uniformly, incubating at 4 ℃ for 6-24h, centrifuging for 1h at 5000-20000 g, taking the supernatant for precipitation, adding PBS for heavy suspension, and obtaining lipoid. Measuring particle size, lyophilizing, and storing at-80 deg.C.
3. Weighing appropriate amounts of polyphenol and protein/polypeptide obtained in the step 1, respectively dissolving in PBS buffer solution (pH 3-7.4), slowly dripping polyphenol solution into the protein/polypeptide solution, removing free polyphenol to obtain VPN compound, and measuring the particle size to be 30-200 nm.
4. Dripping the lipoid solution into the VPN compound solution, repeatedly extruding for 10-100 times by an extruder (double-layer 100nm polycarbonate film), and measuring the particle diameter to be 50-500 nm to obtain the lipoid composition VPN-Lip carrying the protein/polypeptide.
Compared with the prior art, the invention has the following advantages:
according to the invention, lipoid is selected to load protein/polypeptide, and natural properties of the lipoid are utilized, so that compared with a carrier synthesized artificially, the lipoid has the advantages of wide source, simple and convenient preparation, low cost and convenience for large-scale mass production; the biological stability and safety are better, and the cytotoxicity and immunogenicity are almost avoided; meanwhile, the electronegativity of the lipoid can shield the positive charge of the polypeptide, so that the biological safety is improved; and the protein/polypeptide has high encapsulation rate, high release effect and good utilization rate and efficacy.
Drawings
FIG. 1 is a graph showing the particle size distribution of the melittin-loaded lipid VPN-Lip prepared in example 1.
FIG. 2 is a VPN-Lip and VPN-EVs in vitro cumulative release profile.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific embodiments, but it is easily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are only for illustrating the present invention, and should not also limit the present invention described in detail in the claims.
Example 1:
a preparation method of melittin-loaded lipoid composition comprises the following steps:
1. extracting and purifying grape polyphenol: peeling grape skin and grape seed, freeze drying, and pulverizing. Accurately weighing 5.00g of grape skin and grape seed powder (the mass ratio of the grape skin to the grape seed powder is 5: 1). Extracting with 60% ethanol at a ratio of 1:10 at 50 deg.C for 50min for 2 times to obtain grape polyphenols crude extract; selecting XAD-7Hp type macroporous resin (BR, shanghai leaf Biotechnology limited) to purify polyphenol, wherein the purification conditions are as follows: the crude extract of grape polyphenol with the concentration of 4mg/mL is loaded for 7BV, washed with water for 5BV and eluted with 60 percent ethanol for 5BV.
2. Extraction of grape lipoid: removing seeds and peel of fructus Vitis Viniferae, and squeezing to obtain fruit juice. Taking grape juice, centrifuging for 1h at 4000g, taking supernatant, adding 8 percent PEG6000 solution in equal volume, mixing uniformly, incubating for 12h at 4 ℃, centrifuging for 1h at 10000g, collecting precipitate, and adding 10mL PBS for resuspension to obtain grape lipoid. Measuring the particle diameter (163.22 + -2.01) nm, lyophilizing, and storing at-80 deg.C.
3. Weighing appropriate amounts of the grape polyphenols and the melittin in the step 1, respectively dissolving in PBS buffer solution with pH7.4, slowly dripping 1mg/mL polyphenol solution into 1mg/mL melittin solution with the same volume, incubating at 25 ℃ for 30min, loading the compound solution into an ultrafiltration tube with molecular weight cutoff of 3kDa, ultrafiltering for 10min, removing free polyphenol to obtain VPN compound, and determining the particle size (46.47 +/-1.28) nm.
4. Dripping 5mL of grape lipoid into an equal volume VPN solution (the concentration of the melittin is 1 mg/mL), stirring for 30min at 25 ℃, repeatedly extruding the solution for 15 times through an extruder (a double-layer 100nm polycarbonate membrane), and measuring the particle size to be (236.13 +/-2.42) nm to obtain the melittin-loaded lipoid VPN-Lip.
5. And mixing the VPN-Lip with PBS (phosphate buffer solution), DMEM (DMEM) and 10% serum which are equal in volume, and incubating at 37 ℃ for 0, 2, 4, 8, 12, 24 and 48 hours, wherein the change of the particle size is not obviously different.
6. Blood is taken from rat orbit, blood cells are taken out by centrifugation, and the red blood cells are prepared into red blood cell suspension with the concentration of 2% by adding saline. Collecting four parts of erythrocyte suspension, adding melittin, grape polyphenols, VPN-Lip (melittin concentration is consistent), incubating at 37 deg.C for 1h, collecting erythrocyte suspension, centrifuging, collecting 100 μ L supernatant, and measuring absorbance at 570 nm. The results show that VPN-Lip greatly reduces the hemolytic activity of melittin.
TABLE 1 melittin-loaded lipoid VPN-Lip in vitro hemolysis Rate study
Example 2:
a preparation method of a lysozyme-loaded lipoid composition comprises the following steps:
1. and (3) poly tea polyphenol synthesis: weighing 1.0g of epigallocatechin gallate (EGCG) and dissolving in a mixed solution of 15mL of acetic acid, 45mL of water and 6mL of DMSO, adding 7.2mL of 40% acetaldehyde, adjusting the pH value to 2, reacting for 12h under the protection of nitrogen, dialyzing the product, and freeze-drying.
2. Extracting milk lipoid: centrifuging 2000g of milk for 1h, collecting supernatant, adding 8% of PEG4000 solution in equal volume, mixing, incubating at 6 deg.C for 12h, centrifuging 5000g of supernatant for 1h, collecting supernatant, precipitating, and adding PBS for resuspension to obtain lipoid. Measuring the particle size (146.51 + -1.97) nm, lyophilizing, and storing at-80 deg.C.
3. Weighing appropriate amount of poly tea polyphenol and lysozyme (chicken protein source, activity is more than or equal to 70 000units/mg, shanghai alading company) in the step 1, respectively dissolving the poly tea polyphenol and the lysozyme in HEPES buffer solution with pH7.4, slowly dropping polyphenol solution into lysozyme solution with the same volume and concentration of 1mg/mL, incubating for 30min at 25 ℃, removing free polyphenol through ultrafiltration to obtain a VPN compound, and determining the particle size (63.45 +/-2.16) nm.
4. Dripping 8mL of milk lipoid into an equal volume VPN solution (the concentration of the lysozyme is 1 mg/mL), stirring for 30min at 25 ℃, repeatedly extruding the solution for 20 times through an extruder (a double-layer 100nm polycarbonate film), and measuring the particle size to be (233.29 +/-3.61) nm to obtain the lipoid composition carrying the lysozyme.
5. The lysozyme-loaded lipoid composition is mixed with PBS, DMEM and 10% serum which are equal in volume, and incubated for 0, 2, 4, 8, 12, 24 and 48 hours at 37 ℃, and the particle size change is not significantly different.
6. Four parts of staphylococcus aureus suspension are planted into a 96-micropore plate, lysozyme, poly tea polyphenol, VPN and a lipoid body carrying the lysozyme (the concentration of the lysozyme is consistent) are respectively added, the mixture is uniformly mixed and then placed into an incubator at 37 ℃ for culture for 24 hours, and the OD value is measured by an enzyme-linked immunosorbent assay instrument at 600 nm. The results show that the lipid group carrying lysozyme has the strongest bacteriostatic effect.
Example 3:
a preparation method of a glutathione-loaded lipoid composition comprises the following steps:
1. and (3) extraction and purification of medlar polyphenol: crushing Ningxia wolfberry fruit and weighing wolfberry fruit powder in 5.00g. Taking 60% ethanol as an extraction solvent, wherein the ratio of material to liquid is 1:5, extracting for 30min at the extraction temperature of 60 ℃ for 2 times to obtain a medlar polyphenol crude extract; selecting AB-8 type macroporous resin (BR, shanghai leaf Biotech limited) for purifying polyphenol, wherein the purification conditions are as follows: and (3) loading 8BV of a 5mg/mL wolfberry polyphenol crude extract, washing with water for 6BV, and eluting with 80% ethanol for 5BV.
2. Extraction of tumor cell lipid bodies:
collecting SKOV3 cell culture solution supernatant, centrifuging for 1h at 5000g, collecting supernatant, adding PEG2000 solution at equal volume, mixing, incubating at 4 deg.C for 1h, centrifuging for 1h at 20000g, collecting supernatant, adding PBS, and resuspending to obtain lipoid. Measuring particle diameter (174.33 + -2.07) nm, lyophilizing, and storing at-80 deg.C.
3. Weighing appropriate amount of fructus Lycii polyphenol and glutathione (Shanghai Compound Dan Xiawa pharmaceutical industry Co., ltd.) in step 1, dissolving in PBS buffer solution with pH of 7.4, slowly dropping polyphenol solution into glutathione solution with the same volume concentration of 1mg/mL, incubating at 25 deg.C for 30min, ultrafiltering to remove free polyphenol to obtain VPN compound, and determining particle size (51.70 + -3.42) nm.
4. Dripping 5mL of tumor cell lipoid into equal volume VPN solution (glutathione concentration is 1 mg/mL), stirring at 25 deg.C for 30min, repeatedly extruding the solution for 20 times through an extruder (double-layer 100nm polycarbonate membrane), and measuring the particle diameter to be (189.34 + -2.36) nm to obtain the lipoid composition carrying glutathione.
Example 4:
a method for preparing a textured soy protein-loaded lipid composition, comprising the steps of:
1. extracting and purifying corn polyphenol: airing and crushing the corn. Accurately weighing 5.00g of corn powder. Taking 60% ethanol as an extraction solvent, wherein the ratio of material to liquid is 1:10, extracting for 40min at the extraction temperature of 55 ℃ for 2 times to obtain a corn polyphenol crude extract; selecting D101 type macroporous resin (BR, shanghai leaf Biotech limited) for purifying polyphenol, wherein the purification conditions are as follows: the corn polyphenol crude extract with the concentration of 5mg/mL is loaded for 7BV, washed with water for 4BV and eluted with 80 percent ethanol for 5BV.
2. Extracting tomato lipoid: juicing fructus Lycopersici Esculenti, taking out juice, centrifuging at 3000g for 1h, collecting supernatant, adding 8% PEG6000 solution at equal volume, mixing, incubating at 4 deg.C for 12h, centrifuging at 15000g for 1h, collecting supernatant, precipitating, adding PBS, and resuspending to obtain lipoid. Measuring the particle diameter (144.17 + -3.23) nm, lyophilizing, and storing at-80 deg.C.
3. Weighing appropriate amounts of the corn polyphenol and the soybean tissue protein (D302, anyang acquired Nature food, ltd.) in the step 1, respectively dissolving the corn polyphenol and the soybean tissue protein in PBS buffer solution with pH of 7.4, slowly dropping the polyphenol solution into the soybean protein solution with the same volume and the concentration of 1mg/mL, incubating for 30min at 25 ℃, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa for ultrafiltration of 10min, removing free polyphenol to obtain a VPN compound, and determining the particle size (74.17 +/-3.23) nm.
4. Dripping tomato lipoid 5mL into VPN solution (soybean protein concentration 1 mg/mL) with equal volume, stirring at 25 deg.C for 30min, repeatedly extruding the solution for 30 times through an extruder (double-layer 100nm polycarbonate film), and measuring the particle diameter to be (220.34 + -1.85) nm to obtain soybean protein-loaded lipoid composition.
Example 5:
extracting ginseng lipid: cutting Ginseng radix into pieces, squeezing, centrifuging at 4000g for 1h, collecting supernatant, adding PEG4000 solution at equal volume, mixing, incubating at 4 deg.C for 12h, centrifuging at 10000g for 1h, collecting the supernatant, precipitating, and adding PBS for resuspension to obtain lipoid. Measuring the particle diameter (157.34 + -3.10) nm, lyophilizing, and storing at-80 deg.C.
Taking a proper amount of tea polyphenol and epidermal growth factor EGF, respectively dissolving in PBS buffer solution with pH of 7.4, slowly dripping the polyphenol solution into equal volume of EGF solution with concentration of 1mg/mL, incubating at 25 ℃ for 30min, loading the compound solution into an ultrafiltration tube with molecular weight cutoff of 3kDa, ultrafiltering for 10min, removing free polyphenol to obtain VPN compound, and measuring the particle size (53.42 +/-2.25) nm.
Dropping 5mL of the ginseng lipoid into an equal volume of VPN solution (EGF concentration is 1 mg/mL), stirring at 25 ℃ for 30min, repeatedly extruding the solution through an extruder (double-layer 100nm polycarbonate membrane) for 20 times, and measuring the particle size to be (201.16 +/-3.05) nm to obtain the EGF-carrying lipoid. Mixing 80% of EGF-loaded lipid with 5% of propylene glycol, 10% of glycerol and 5% of Vc to obtain the essence liquid for repairing muscle base.
Example 6:
extracting aloe lipoid: juicing folium Aloe, centrifuging for 1h at 5000g, collecting supernatant, adding PEG6000 solution (8% by volume) at the same volume, mixing, incubating at 4 deg.C for 12h, centrifuging for 1h at 15000g, collecting the supernatant, precipitating, and adding PBS for resuspension to obtain lipoid. Measuring the particle diameter (125.31 + -2.04) nm, lyophilizing, and storing at-80 deg.C.
Taking a proper amount of tea polyphenol and fibroin, respectively dissolving in PBS buffer solution with the pH value of 7.4, slowly dripping polyphenol solution into the fibroin solution with the same volume and the concentration of 1mg/mL, incubating at 25 ℃ for 30min, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa, carrying out ultrafiltration for 10min, removing free polyphenol to obtain a VPN compound, and measuring the particle size (60.09 +/-2.11) nm.
Dripping 5mL of aloe lipoid into equal volume VPN solution (fibroin concentration 1 mg/mL), stirring at 25 deg.C for 30min, repeatedly extruding the solution for 20 times through an extruder (double-layer 100nm polycarbonate membrane), and measuring the particle diameter to be (196.42 + -2.21) nm to obtain fibroin-loaded lipoid. Mixing 80% fibroin-loaded lipoid with 5% propylene glycol, 10% glycerol, and 5% Vc to obtain whitening and moisturizing essence.
Example 7:
extracting and purifying cucumber polyphenol: drying and crushing cucumber slices, taking 5.00g of 50% ethanol as an extraction solvent, wherein the material-liquid ratio is 1:12, extracting for 60min at the extraction temperature of 55 ℃ for 2 times to obtain a cucumber polyphenol crude extract; selecting D101 type macroporous resin (BR, shanghai leaf Biotech limited) for purifying polyphenol, wherein the purification conditions are as follows: the cucumber polyphenol crude extract with the concentration of 5mg/mL is loaded with 7BV, washed with water with 4BV, and eluted with 80% ethanol with the concentration of 5BV.
Cucumber lipid extraction: juicing cucumber, taking out juice, centrifuging at 2000g for 1h, taking supernatant, adding 8% PEG6000 solution in equal volume, mixing uniformly, incubating at 4 deg.C for 12h, centrifuging at 12000g for 1h, taking the supernatant precipitate, adding PBS, and resuspending to obtain lipoid. Measuring the particle diameter (123.13 + -2.45) nm, lyophilizing, and storing at-80 deg.C.
Weighing a proper amount of the cucumber polyphenol and the collagen in the step 1, respectively dissolving the cucumber polyphenol and the collagen in PBS buffer solution with pH7.4, slowly dripping the polyphenol solution into the collagen solution with the same volume and the concentration of 1mg/mL, incubating for 30min at 25 ℃, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa, carrying out ultrafiltration for 10min, removing free polyphenol to obtain a VPN compound, and measuring the particle size (83.14 +/-2.03) nm.
Dripping 5mL of cucumber lipoid into VPN solution (collagen concentration is 1 mg/mL) with equal volume, stirring at 25 deg.C for 30min, repeatedly extruding the solution through an extruder (double-layer 100nm polycarbonate membrane) for 20 times, and measuring the particle size to be (202.32 + -3.18) nm to obtain the collagen-loaded lipoid. Mixing 90% of collagen-loaded lipoid and 10% of lavender essential oil in each facial mask to prepare the anti-aging facial mask.
Example 8:
and (3) extraction and purification of medlar polyphenol: crushing Ningxia wolfberry fruit and weighing wolfberry fruit powder in 5.00g. Taking 60% ethanol as an extraction solvent, wherein the ratio of material to liquid is 1:5, extracting for 30min at the extraction temperature of 60 ℃ for 2 times to obtain a medlar polyphenol crude extract; selecting AB-8 type macroporous resin (BR, shanghai leaf Biotech limited) for purifying polyphenol, wherein the purification conditions are as follows: 5mg/mL medlar polyphenol crude extract sample is loaded for 8BV, washed by water for 6BV, and eluted by 80 percent ethanol for 5BV.
Extracting lycium barbarum lipid: squeezing fructus Lycii, taking out juice, centrifuging at 3000g for 1h, collecting supernatant, adding 8% PEG6000 solution in equal volume, mixing, incubating at 4 deg.C for 12h, centrifuging at 8000g for 1h, collecting supernatant, precipitating, and adding PBS for resuspension to obtain lipoid. Measuring the particle size (154.26 + -2.17) nm, lyophilizing, and storing at-80 deg.C.
Weighing a proper amount of the medlar polyphenol and soybean tissue protein (D302, anyang acquired Nature food, ltd.) in the step 1, respectively dissolving the medlar polyphenol and the soybean tissue protein in PBS buffer solution with pH of 7.4, slowly dropping polyphenol solution into soybean protein solution with the same volume and the concentration of 1mg/mL, incubating for 30min at 25 ℃, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa, carrying out ultrafiltration for 10min, removing free polyphenol to obtain a VPN compound, and determining the particle size (86.35 +/-2.09) nm.
Dripping 5mL of the lycium barbarum lipoid into an equal volume VPN solution (the concentration of the soybean protein is 1 mg/mL), stirring for 30min at 25 ℃, repeatedly extruding the solution for 20 times through an extruder (a double-layer 100nm polycarbonate membrane), and measuring the particle size to be (210.24 +/-2.18) nm to obtain the soybean protein-loaded lipoid.
Comparative example 1:
a preparation method of a biological vesicle composition carrying melittin comprises the following steps:
1. extracting and purifying grape polyphenol: peeling grape skin and grape seed, freeze drying, and pulverizing. Accurately weighing 5.00g of grape skin and grape seed powder (the mass ratio of the grape skin to the grape seed powder is 5: 1). Extracting with 60% ethanol at a ratio of 1:10 at 50 deg.C for 50min for 2 times to obtain grape polyphenols crude extract; selecting XAD-7Hp type macroporous resin (BR, shanghai leaf Biotechnology limited) to purify polyphenol, wherein the purification conditions are as follows: the crude extract of grape polyphenol with the concentration of 4mg/mL is loaded with 7BV, washed with water for 5BV, and eluted with 60 percent ethanol for 5BV.
2. Extraction and purification of grape vesicles: removing seeds and skins of the grapes, and squeezing the pulp to obtain juice. The grape juice was removed and mixed with an equal volume of PBS (pH 7.4). Subpackaging the mixed solution of grape juice and PBS, centrifuging for 1h at 4000g, taking supernatant, and continuing centrifuging for 1h at 10000 g. The supernatant was poured into an ultracentrifuge tube and ultracentrifuged at 150000g for 2h. Taking the lower layer precipitate, adding 40mL PBS for suspension, ultrasonically dispersing uniformly, transferring the suspension into a sucrose density gradient (8, 30, 45 and 60%) solution, ultracentrifuging for 2h at 150000g, collecting sedimentation zones between 8/30% and 30/45% interfaces, adding PBS for centrifugation to wash out the sucrose, washing twice to obtain a grape vesicle solution, measuring the particle size (155.09 +/-3.03) nm, freeze-drying, and storing at-80 ℃ for later use.
3. Weighing appropriate amounts of the grape polyphenols and the melittin in the step 1, respectively dissolving in PBS buffer solution with pH7.4, slowly dripping 1mg/mL polyphenol solution into 1mg/mL melittin solution with the same volume, incubating at 25 ℃ for 30min, loading the compound solution into an ultrafiltration tube with molecular weight cutoff of 3kDa, ultrafiltering for 10min, removing free polyphenol to obtain VPN compound, and determining the particle size (46.47 +/-1.28) nm.
4. Dripping 5mL of grape vesicle into VPN solution (melittin concentration is 1 mg/mL) with the same volume, stirring at 25 deg.C for 30min, repeatedly extruding the solution through an extruder (double-layer 100nm polycarbonate membrane) for 15 times, and measuring the particle size to be (130.68 + -5.31) nm to obtain the melittin-loaded biological vesicle VPN-EVs.
Test example 1
Comparison of the liposome VPN-Lip loaded with melittin with the liposome/vesicle yield of the bio-vesicle VPN-EVs and the encapsulation efficiency of melittin. The results are shown in Table 2.
TABLE 2 comparison of melittin-loaded lipoids VPN-Lip with Bio-vesicle VPN-EVs
Test example 2
2mL of VPN-Lip solution or VPN-EVs solution is put into a dialysis bag with molecular weight cut-off of 25kDa, immersed in 18mL of PBS solution (pH 7.5), and put into a gas bath shaker at 37 +/-0.5 ℃ with the rotation speed of the shaker being 600rpm. 0.2mL of release medium was taken at 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48h, respectively, while 0.2mL of fresh medium was replenished. The content of melittin is detected by liquid phase, and is paralleled by 3 parts. The results show (FIG. 2) that the release amounts of VPN-Lip and VPN-EVs are gradually increased with time, the release of VPN-Lip is faster than that of VPN-EVs, the release is complete, and the melittin residue rate is low.
Test example 3
Kunming mice, male and female halves, depilating the back, exposing a test area of 4cm multiplied by 4cm, washing the skin with warm water, applying the collagen-loaded lipid prepared in example 7 every night, continuously applying for one month, removing newly grown mouse hair during the period, cutting skin tissues of the test area after 12h of final application, homogenizing, and determining the activity of superoxide dismutase SOD and the MDA content, wherein the test results are shown in the following table 3. The results show that the collagen-loaded lipid can improve the activity of SOD of the skin and reduce the content of MDA, which indicates that the collagen-loaded lipid has good anti-aging effect.
TABLE 3 anti-aging evaluation test for collagen-loaded lipids
Claims (5)
1. A protein/polypeptide loaded lipid body composition, which is a melittin loaded lipid body composition, prepared by a process comprising the steps of:
(1) Extracting and purifying grape polyphenol: peeling grape skin and grape seed, freeze drying, and pulverizing; accurately weighing 5.00g of grape skin and grape seed powder, wherein the mass ratio of the grape skin to the grape seed powder is 5: 1; extracting with 60% ethanol at a ratio of 1:10 at 50 deg.C for 50min for 2 times to obtain grape polyphenols crude extract; selecting XAD-7Hp type macroporous resin to purify polyphenol, wherein the purification conditions are as follows: loading 7BV of a 4mg/mL grape polyphenol crude extract, washing with water for 5BV, and eluting with 60% ethanol for 5BV;
(2) Extraction of grape lipoid: removing seeds and skins of the grapes, and squeezing pulp to obtain juice; taking grape juice, centrifuging for 1h at 4000g, taking supernatant, adding 8% PEG6000 solution in the same volume, mixing uniformly, incubating for 12h at 4 ℃, centrifuging for 1h at 10000g, collecting precipitate, and adding 10mL PBS for resuspension to obtain grape lipoid; determining particle size of 163.22 + -2.01 nm, lyophilizing, and storing at-80 deg.C;
(3) Weighing appropriate amounts of grape polyphenol and melittin in the step 1, respectively dissolving in PBS buffer solution with pH of 7.4, slowly dripping 1mg/mL polyphenol solution into 1mg/mL melittin solution with the same volume, incubating at 25 ℃ for 30min, loading the compound solution into an ultrafiltration tube with molecular weight cutoff of 3kDa, ultrafiltering for 10min, removing free polyphenol to obtain VPN compound, and determining the particle size to be 46.47 +/-1.28 nm;
(4) Dripping 5mL of grape lipoid into an equal volume VPN compound solution, wherein the concentration of the melittin in the VPN compound solution is 1mg/mL, stirring for 30min at 25 ℃, repeatedly extruding the solution for 15 times through an extruder with a double-layer 100nm polycarbonate membrane, and measuring the particle size to be 236.13 +/-2.42 nm to obtain the melittin-loaded lipoid VPN-Lip.
2. A protein/polypeptide loaded lipid composition, which is a collagen loaded lipid composition, prepared by a process comprising the steps of:
(1) Extracting and purifying cucumber polyphenol: drying and crushing cucumber slices, taking 5.00g of 50% ethanol as an extraction solvent, wherein the material-liquid ratio is 1:12, extracting for 60min at the extraction temperature of 55 ℃ for 2 times to obtain a cucumber polyphenol crude extract; selecting D101 type macroporous resin for purifying polyphenol, wherein the purification conditions are as follows: loading a cucumber polyphenol crude extract with the concentration of 5mg/mL for 7BV, washing with water for 4BV, and eluting with 80% ethanol for 5BV;
(2) Cucumber lipid extraction: juicing cucumber, taking out juice, centrifuging for 1h at 2000g, taking supernatant, adding 8% PEG6000 solution in the same volume, mixing uniformly, incubating at 4 ℃ for 12h, centrifuging at 12000g for 1h, taking the lower layer precipitate, adding PBS (phosphate buffer solution) for resuspension to obtain lipoid, measuring the particle size to be 123.13 +/-2.45 nm, freeze-drying, and storing at-80 ℃;
(3) Weighing a proper amount of cucumber polyphenol and collagen in the step (1), respectively dissolving the cucumber polyphenol and the collagen in PBS buffer solution with pH7.4, slowly dropping polyphenol solution into the collagen solution with the same volume and the concentration of 1mg/mL, incubating for 30min at 25 ℃, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa for ultrafiltration for 10min, removing free polyphenol to obtain a VPN compound, and determining the particle size to be 83.14 +/-2.03 nm;
(4) Dripping 5mL of cucumber lipoid into an equal volume VPN compound solution, wherein the concentration of the collagen in the VPN compound solution is 1mg/mL, stirring for 30min at 25 ℃, repeatedly extruding the solution for 20 times through an extruder with a double-layer 100nm polycarbonate membrane, and measuring the particle size to be 202.32 +/-3.18 nm to obtain the collagen-loaded lipoid.
3. A process for preparing the lipid composition of claim 1 comprising the steps of:
(1) Extracting and purifying grape polyphenol: peeling grape skin and grape seed, freeze drying, and pulverizing; accurately weighing 5.00g of grape skin and grape seed powder, wherein the mass ratio of the grape skin to the grape seed powder is 5: 1; extracting with 60% ethanol at a ratio of 1:10 at 50 deg.C for 50min for 2 times to obtain grape polyphenols crude extract; selecting XAD-7Hp type macroporous resin to purify polyphenol, wherein the purification conditions are as follows: loading 7BV of a 4mg/mL grape polyphenol crude extract, washing with water for 5BV, and eluting with 60% ethanol for 5BV;
(2) Extraction of grape lipid bodies: removing seeds and peel of fructus Vitis Viniferae, and squeezing to obtain fruit juice; taking grape juice, centrifuging for 1h at 4000g, taking supernatant, adding 8% PEG6000 solution in the same volume, mixing uniformly, incubating for 12h at 4 ℃, centrifuging for 1h at 10000g, collecting precipitate, and adding 10mL PBS for resuspension to obtain grape lipoid; determining particle size of 163.22 + -2.01 nm, lyophilizing, and storing at-80 deg.C;
(3) Weighing appropriate amounts of grape polyphenol and melittin in the step 1, respectively dissolving in PBS buffer solution with pH of 7.4, slowly dripping 1mg/mL polyphenol solution into 1mg/mL melittin solution with the same volume, incubating at 25 ℃ for 30min, loading the compound solution into an ultrafiltration tube with molecular weight cutoff of 3kDa, ultrafiltering for 10min, removing free polyphenol to obtain VPN compound, and determining the particle size to be 46.47 +/-1.28 nm;
(4) Dripping 5mL of grape lipoid into an equal volume of VPN compound solution, wherein the concentration of the melittin in the VPN compound solution is 1mg/mL, stirring for 30min at 25 ℃, repeatedly extruding the solution for 15 times through an extruder with a double-layer 100nm polycarbonate membrane, and measuring the particle size to be 236.13 +/-2.42 nm to obtain the melittin-loaded lipoid VPN-Lip.
4. A process for preparing the lipid composition of claim 2, comprising the steps of:
(1) Extracting and purifying cucumber polyphenol: drying and crushing cucumber slices, taking 5.00g of 50% ethanol as an extraction solvent, wherein the material-liquid ratio is 1:12, extracting for 60min at the extraction temperature of 55 ℃ for 2 times to obtain a cucumber polyphenol crude extract; selecting D101 type macroporous resin for purifying polyphenol, wherein the purification conditions are as follows: loading a cucumber polyphenol crude extract with the concentration of 5mg/mL for 7BV, washing with water for 4BV, and eluting with 80% ethanol for 5BV;
(2) Cucumber lipid extraction: juicing cucumber, taking out juice, centrifuging for 1h at 2000g, taking supernatant, adding 8% PEG6000 solution in the same volume, mixing uniformly, incubating at 4 ℃ for 12h, centrifuging at 12000g for 1h, taking the supernatant precipitate, adding PBS for resuspension to obtain lipoid, measuring the particle size to be 123.13 +/-2.45 nm, freeze-drying, and storing at-80 ℃;
(3) Weighing a proper amount of cucumber polyphenol and collagen in the step (1), respectively dissolving the cucumber polyphenol and the collagen in PBS buffer solution with pH7.4, slowly dropping polyphenol solution into the collagen solution with the same volume and the concentration of 1mg/mL, incubating for 30min at 25 ℃, putting the compound solution into an ultrafiltration tube with the molecular weight cutoff of 3kDa for ultrafiltration for 10min, removing free polyphenol to obtain a VPN compound, and determining the particle size to be 83.14 +/-2.03 nm;
(4) Dripping 5mL of cucumber lipoid into an equal volume of VPN compound solution, wherein the concentration of the collagen in the VPN compound solution is 1mg/mL, stirring for 30min at 25 ℃, repeatedly extruding the solution for 20 times through an extruder with a double-layer 100nm polycarbonate membrane, and measuring the particle size to be 202.32 +/-3.18 nm to obtain the collagen-loaded lipoid.
5. Use of a composition according to claim 1 or 2 for the preparation of a medicament, a food product, a nutraceutical product and a cosmetic product.
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