Resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel and preparation method thereof

Abstract

The invention discloses a resveratrol delivery system based on lactoferrin-polyglutamic acid composite nanogel and a preparation method thereof.The preparation method comprises the steps that a lactoferrin aqueous solution and a gamma-sodium polyglutamate aqueous solution are mixed to form a composite system, resveratrol is embedded with the composite system, and the resveratrol delivery system is obtained. And finally, adding calcium chloride into the resveratrol-loaded lactoferrin / polyglutamic acid mixed system, so that the calcium chloride and calcium ions are subjected to ionic crosslinking to form stable nanogel, and freeze-drying the nanogel to obtain the high-resveratrol nanogel powder. According to the invention, lactoferrin and polyglutamic acid are selected to be compounded to construct a resveratrol-loaded lactoferrin / polyglutamic acid nanogel system, a composite structure is formed by utilizing electrostatic interaction between protein and peptide polyacid, and then the composite structure is crosslinked with Ca < 2 + > to form a stable core-shell type three-dimensional nanogel structure. The water solubility, the drug loading capacity and the biological accessibility of the resveratrol are obviously improved.

Description

Technical Field

[0001] The present invention relates to the fields of food, nutritional health products and drug delivery, and in particular to a method for preparing a resveratrol nanogel delivery system based on lactoferrin and polyglutamic acid. Background Art

[0002] Resveratrol is a non-flavonoid polyphenol widely found in plants such as grapes, peanuts, and Japanese knotweed. It exhibits multiple physiological activities, including antioxidant, anti-inflammatory, anti-cancer, and lipid- and blood-sugar-lowering properties. However, resveratrol's poor water solubility, low bioavailability, and susceptibility to degradation and inactivation severely limit its application in food, medicine, and health supplements.

[0003] To improve the solubility, stability, and bioavailability of resveratrol, existing technologies mainly adopt the following strategies: (1) nanotechnology, such as using liposomes, solid lipid nanoparticles, nanocrystals, etc. to encapsulate resveratrol to improve dispersibility by reducing particle size; (2) inclusion technology, such as using cyclodextrin inclusion compounds or the cavity structure of metal-organic frameworks (MOFs) to encapsulate resveratrol molecules to improve their biocompatibility; (3) microemulsification technology, using surfactants to form a microemulsion system to enhance solubility; (4) chemical modification, introducing hydrophilic groups to improve solubility. However, the above methods have problems such as complex processes, insufficient carrier biocompatibility, or chemical modification that may affect activity.

[0004] In recent years, encapsulation and delivery systems based on natural proteins (such as whey protein, soy protein, and zein) have attracted attention due to their safety, biodegradability, and targeting potential. Proteins can bind to resveratrol through hydrophobic interactions and hydrogen bonds, forming a core-shell structure, which improves its loading rate and stability. For example, patent CN 118892197A discloses a method for preparing and using lactoferrin-based nanoparticles loaded with resveratrol. Encapsulating resveratrol with lactoferrin can effectively improve the dispersibility, stability, and antioxidant properties of resveratrol. Patent CN118844622A discloses a method for preparing and using whey protein-based nanoparticles loaded with resveratrol. Preparation of whey protein-based nanoparticles loaded with resveratrol by antisolvent precipitation can effectively improve the stability of resveratrol and increase its encapsulation rate and antioxidant properties. However, single-protein encapsulation systems have significant limitations. For example, the inherent structure of proteins limits their bioavailability, solubility, and dispersion stability. For example, the large particle size of resveratrol nanoparticles in lactoferrin white phoenix is not conducive to improving their dispersion stability. In addition, the stability of single proteins is easily affected by environmental pH, ionic strength, and digestive enzymes, resulting in sudden release or premature degradation, resulting in bioaccessibility that is lower than expected. Therefore, developing composite delivery systems to overcome the shortcomings of single proteins through structural optimization has become a key direction for improving the delivery efficiency of resveratrol.

[0005] Polyglutamic acid is a natural anionic polypeptide with good biocompatibility and biodegradability. It can form a stable structure through reactions such as hydrogen bonding and ionic crosslinking involving carboxyl groups between molecular chains. However, when the nanogel formed alone encapsulates hydrophobic drugs such as resveratrol, the particle size is prone to instability and the drug release is uneven due to the hydrophilic chain backbone repulsion effect. CN103751149A discloses a preparation method of γ-polyglutamic acid / gelatin nanoparticles loaded with drugs. By mixing the γ-polyglutamic acid aqueous solution loaded with drugs with the gelatin solution and freeze-drying, γ-polyglutamic acid / gelatin nanoparticles loaded with drugs are prepared. Although the biocompatibility and degradability of the drugs are improved, the particle size distribution range of the composite nanoparticles is large, the dispersion stability is poor, and the preparation process of the nanoparticles is relatively complex, requiring multiple pH adjustments. Although nanoparticles with a smaller average particle size may be obtained by adjusting experimental parameters, the dispersibility of the nanoparticles is still poor. Moreover, the method of first loading the drug and then complexing with the protein is not conducive to the formation of the gel structure between polyglutamic acid and the protein, resulting in poor drug encapsulation effect and poor bioaccessibility.

[0006] Lactoferrin is a natural cationic glycoprotein with a stable structure and certain hydrophobicity. When lactoferrin is used as a nanocarrier, it can provide a hydrophobic phase encapsulation environment and enhance the affinity for hydrophobic drugs. However, when it forms a colloid independently, it is prone to aggregation and precipitation, the particle size distribution is unstable, and the release controllability is poor.

[0007] Therefore, there is an urgent need to construct a new type of nanogel system that combines the functional advantages of proteins and polyglutamic acid to overcome the problems of poor solubility, low encapsulation efficiency, and poor controlled release performance existing in the existing systems in drug delivery, and to improve the structural stability, encapsulation rate, water solubility, bioaccessibility, etc. of the resveratrol delivery system. Summary of the Invention

[0008] Aiming at the above problems, the purpose of the present invention is to provide a preparation method of a resveratrol delivery system based on lactoferrin-sodium polyglutamate nanogel, so as to significantly improve the water solubility, drug loading amount, solubility, stability, bioaccessibility and other properties of resveratrol.

[0009] Aiming at the above purpose, the present invention first provides a preparation method of a resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel, including the following steps:

[0010] Step 1, prepare a lactoferrin solution and a γ-sodium polyglutamate solution respectively. After mixing and stirring evenly, a stable composite solution is formed;

[0011] Step 2: Dissolve resveratrol in 50% ethanol to form a resveratrol ethanol solution, and slowly drop the resveratrol ethanol solution into the composite solution obtained in Step 1, and stir at 30 °C for 20 - 30 min to obtain a lactoferrin - γ - polyglutamate sodium composite system loaded with resveratrol;

[0012] Step 3: Slowly drop a CaCl2 solution into the composite system obtained in Step 2 for ionic cross - linking to form a nanogel structure;

[0013] Step 4: Dialyze the nanogel obtained in Step 3, remove the free components, and then perform freeze - drying to obtain a resveratrol delivery system based on lactoferrin - polyglutamate nanogel.

[0014] In one embodiment of the present invention, the lactoferrin solution in Step 1 refers to a solution formed by dissolving lactoferrin in water, and the mass concentration of the lactoferrin solution is 1 - 2%.

[0015] In one embodiment of the present invention, the γ - polyglutamate sodium solution in Step 1 refers to a solution formed by dissolving γ - polyglutamate sodium in water, and the mass concentration of the γ - polyglutamate sodium solution is 0.5 - 1%.

[0016] In one embodiment of the present invention, the volume ratio of the lactoferrin solution to the γ - polyglutamate sodium solution in Step 1 is 1:1 - 2:1.

[0017] In one embodiment of the present invention, the mass concentration of the resveratrol ethanol solution in Step 2 is 4 - 8 mg / mL, and the ethanol concentration is 50% (v / v).

[0018] In one embodiment of the present invention, the concentration of resveratrol in the composite system in Step 2 is 0.4 - 0.8 mg / mL.

[0019] In one embodiment of the present invention, the mass concentration of the CaCl2 solution in Step 3 is 5 - 10% (w / v), the addition rate is controlled within 0.3 - 0.5 mL / min, and after adding the CaCl2 solution, the mass concentration of CaCl2 in the mixed system is 1.5% - 2.0%.

[0020] In one embodiment of the present invention, during the ionic cross - linking in Step 3, the pH is 6.8 - 7.2, the temperature is 30 - 35 °C, continuous stirring is carried out for the cross - linking reaction, and the cross - linking reaction time is 20 - 40 min.

[0021] In one embodiment of the present invention, the cut - off molecular weight of the dialysis bag used during dialysis in Step 4 is 3500 Da, and the dialysis is carried out by refrigerating at 4 °C for 12 - 24 h.

[0022] In one embodiment of the present invention, the conditions for freeze-drying in step four are to freeze at -80°C for 8 hours first, and then conduct freeze-drying under vacuum conditions for 24 to 48 hours.

[0023] The present invention also discloses a resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel prepared by the above method.

[0024] The present invention also discloses the application of the above resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel in food, medicine and health products.

[0025] Beneficial effects:

[0026] (1) The present invention provides a preparation method for a lactoferrin-sodium polyglutamate nanogel resveratrol delivery system with high solubility, high drug loading capacity and high bioaccessibility. The whole process is completed under normal temperature conditions, without the need for ultrasonic or high-pressure equipment. The technical solution is simple, the operation is safe, the carrier is green, and it is easy to promote.

[0027] (2) The present invention selects the combination of lactoferrin and polyglutamic acid to construct a lactoferrin / polyglutamic acid nanogel system loaded with resveratrol, and uses the electrostatic interaction between the protein and the peptide polyacid to form a composite structure. The composite structure is cross-linked with Ca 2 + to form a stable "core-shell type three-dimensional nanogel structure", which significantly improves the water solubility, drug loading capacity and bioaccessibility of resveratrol.

[0028] (3) In the present invention, lactoferrin and polyglutamic acid are first combined, and then the complex of the protein and polyglutamic acid is used to load the drug, which significantly reduces the particle size of the nanoparticles, improves their stability and encapsulation effect. Then, the nanoparticles are cross-linked with calcium ions, and the formed dense network structure can further improve their stability and biocompatibility.

[0029] (4) The encapsulation efficiency of the nanogel prepared by the method of the present invention for resveratrol can reach more than 90%. Compared with unencapsulated resveratrol, the solubility improvement multiple is as high as 15.4 times. The average particle size of the nanogel is 80 - 250 nm, and the PDI is less than 0.3, indicating that its distribution is concentrated, the morphology is uniform, and it has good colloidal stability. It shows high bioaccessibility in in vitro simulated digestion experiments. Specific embodiments

[0030] The following further elaborates on the present invention in detail in combination with specific embodiments, so that those skilled in the art can implement the present invention with reference to the description in the specification.

[0031] Example 1

[0032] A preparation method of a resveratrol delivery system based on lactoferrin - polyglutamic acid nanogel, comprising the following steps:

[0033] Step 1: Prepare 50 mL of 1% lactoferrin aqueous solution and 1% sodium polyglutamate solution respectively, mix them in a volume ratio of 1:1, control the temperature at 30 °C, and stir magnetically for 40 min to form a uniform mixture.

[0034] Step 2: Dissolve resveratrol in 50% ethanol to prepare a resveratrol solution with a concentration of 6 mg / mL, and slowly add it dropwise to the mixture in Step 1 to form a mixed solution. Control the final concentration of resveratrol to be 0.6 mg / mL, and stir and react for 30 min.

[0035] Step 3: Slowly add 10% (w / v) CaCl2 solution to the mixed solution in Step 2 at a rate of 0.3 mL / min until the final Ca 2 + concentration reaches 2%, maintain the pH at 7.0, the temperature at 35 °C, and stir and react for 40 min to form a nanogel solution.

[0036] Step 4: Load the nanogel solution formed in Step 3 into a 3500 Da dialysis bag and dialyze at 4 °C for 20 h to remove unloaded drugs and impurities.

[0037] Step 5: Pre - freeze the dialyzed product at - 80 °C for 8 h, and then dry it in a vacuum freeze - dryer for 48 h to obtain resveratrol nanogel powder.

[0038] Example 2

[0039] The difference between Example 2 and Example 1 is only that in Step 1, the mass ratio of lactoferrin to sodium polyglutamate is 2:1. Step 1 is specifically as follows:

[0040] Prepare 50 mL of 1% lactoferrin aqueous solution and 0.5% sodium polyglutamate solution respectively, mix them in a ratio of 1:1, control the temperature at 30 °C, and stir magnetically for 40 min to form a uniform mixture.

[0041] Example 3

[0042] The difference between Example 3 and Example 1 is that in Step 2, the mass concentration of the resveratrol solution is 5 mg / mL, and the final concentration of resveratrol in the mixed solution is 0.5 mg / mL.

[0043] Comparative Example 1

[0044] The difference between Comparative Example 1 and Example 1 is that in Step 1, lactoferrin is not contained.

[0045] Step 1 is specifically as follows: Prepare 100 mL of 1% (w / v) sodium polyglutamate solution, and stir magnetically until completely dissolved.

[0046] Comparative Example 2

[0047] The difference between Comparative Example 2 and Example 1 is that in Step 1, γ-polyglutamic acid sodium is not contained, and the solution in Step 1 is 100 mL of lactoferrin with a mass concentration of 1%.

[0048] Comparative Example 3

[0049] The difference between Comparative Example 3 and Example 1 is that 1% of whey protein is used to replace lactoferrin in Example 1.

[0050] Comparative Example 4

[0051] The difference between Comparative Example 4 and Example 1 is that Step 3 is omitted.

[0052] Comparative Example 5

[0053] A preparation method of a resveratrol delivery system based on gelatin-polyglutamic acid nanogel, comprising the following steps:

[0054] Step 1: Prepare 100 mL of 0.1% γ-polyglutamic acid sodium solution.

[0055] Step 2: Dissolve resveratrol with 50% ethanol to prepare a mother liquor with a concentration of 6 mg / mL. Take 10 mL and add it to 90 mL of γ-polyglutamic acid sodium solution and mix evenly. Adjust the pH value to 8.0 with 1 mol / L sodium hydroxide to obtain a γ-polyglutamic acid aqueous solution containing resveratrol.

[0056] Step 3: Prepare 0.4% gelatin solution and adjust the pH value to 3.0 with 1 mol / L hydrochloric acid.

[0057] Step 4: Drop 5 mL of the γ-polyglutamic acid sodium solution of resveratrol into 5 mL of gelatin solution, control the dropping rate at 2.0 mL / h, and continue to control the stirring rate at 300 r / min for stirring for 30 min after the dropping is completed to obtain a y-polyglutamic acid / gelatin nanoparticle solution loaded with resveratrol.

[0058] Step 5: Load the nanoparticle solution formed in Step 4 into a 3500 Da dialysis bag and dialyze at 4°C for 20 h to remove unencapsulated drugs and impurities.

[0059] Step 6: Pre-freeze the product after dialysis at -80°C for 8 h, and then dry it in a vacuum freeze dryer for 48 h to obtain resveratrol nanoparticle powder.

[0060] Comparison of the effects of examples and comparative examples:

[0061] 1. Comparison of resveratrol drug loading and encapsulation efficiency

[0062] The free resveratrol content before dialysis and the total added resveratrol content in the nanogel solution were detected by HPLC. The encapsulation efficiency and drug loading were calculated according to the following formulas:

[0063] Encapsulation efficiency (EE, %) = (Ct - Cf) / Ct * 100%

[0064] Where: Ct is the total added resveratrol content, and Cf is the free resveratrol content.

[0065] After disrupting the resveratrol delivery system powder with acetonitrile and removing impurities by centrifugation, the resveratrol content was determined by HPLC. The formula for calculating the drug loading is as follows:

[0066] Drug loading (mg / g) = resveratrol content / mass of freeze-dried powder.

[0067] Table 1 Encapsulation efficiency and drug loading of resveratrol in Examples 1 - 3 and Comparative Examples 1 - 5

[0068] Serial number Sample name Entrapment efficiency (%) Drug loading (mg / g) 1 Example 1 85.3 17.2 2 Example 2 82.7 16.7 3 Example 3 90.1 15.3 4 Comparative example 1 40.8 7.5 5 Comparative example 2 47.3 9.3 6 Comparative example 3 61.5 12.4 7 Comparative example 4 47.3 8.2 8 Comparative example 5 67.6 9.6

[0069] Table 1 shows the encapsulation efficiency and drug loading of resveratrol in Examples 1 - 3 and Comparative Examples 1 - 5. From the results in Table 1, it can be seen that a decrease in the concentration or addition amount of sodium polyglutamate will lead to a decrease in the encapsulation efficiency of resveratrol, thereby reducing the drug loading of the delivery system. Reducing the concentration of resveratrol helps to improve the encapsulation efficiency of the drug, but at the same time, the drug loading of the delivery system relatively decreases. Generally speaking, the encapsulation efficiency of Examples 1 - 3 is greater than 80%, and the drug loading is greater than 15 mg / g.

[0070] In Comparative Example 1 and Comparative Example 2, lactoferrin and sodium polyglutamate were omitted respectively. The encapsulation effect of a single sodium polyglutamate or lactoferrin on the drug was poor, resulting in a significant decrease in drug loading. In Comparative Example 3, lactoferrin was replaced with whey protein, and the encapsulation effect of the drug also decreased significantly, indicating that the type of protein has a great influence on the encapsulation effect of resveratrol, and the combination of lactoferrin and sodium polyglutamate has the best effect. In addition, in the present invention, the cross-linking of calcium ions helps to form nanogels, which can better encapsulate drugs and improve the drug loading.

[0071] 2. Comparison of the improvement effect on the water solubility of resveratrol

[0072] 10 g of nanogel freeze-dried powder was dissolved in 100 mL of water, disrupted with acetonitrile, and impurities were removed by centrifugation. Then the resveratrol concentration was determined by HPLC. The solubility of resveratrol was calculated in μg of resveratrol per mL of solution. Based on the solubility of free resveratrol being about 35 μg / mL, the solubility enhancement factor was calculated according to the following formula:

[0073] Enhancement factor = solubility of resveratrol / 35.

[0074] Table 2 Solubility and enhancement of delivery systems of Examples 1 to 3 and Comparative Examples 1 to 5

[0075] Serial number Sample name Solubility (μg / mL) Enhancement factor 1 Resveratrol 35 0 2 Example 1 510 14.6 3 Example 2 485 13.9 4 Example 3 540 15.4 5 Comparative example 1 110 3.1 6 Comparative example 2 123 3.5 7 Comparative example 3 282 8.1 8 Comparative example 4 135 3.9 9 Comparative example 5 432 12.3

[0076] Table 2 compares the solubility and solubility enhancement of the delivery systems of Examples 1-3 and Comparative Examples 1-5. Combined with the data in Table 2, it can be seen that reducing the amount of sodium polyglutamate added leads to a slight decrease in solubility. Furthermore, the Examples significantly enhance the water solubility of resveratrol. However, due to structural instability or lack of encapsulation, the solubility enhancement effect of the Comparative Examples is limited. This demonstrates that the nanogel significantly improves the hydrophilicity of resveratrol.

[0077] 3. Comparison of Resveratrol Bioaccessibility

[0078] Resveratrol nanogels were prepared into aqueous solutions at the same resveratrol concentration and incubated in simulated gastric fluid for 2 hours and then in simulated small intestinal fluid for 4 hours using an in vitro simulated digestion method. The digestion fluid was placed in a 3500Da dialysis bag and dialyzed at 4°C for 20 hours. The dialysate was broken with acetonitrile and centrifuged to remove impurities, and the resveratrol concentration was determined by HPLC. The bioaccessibility of resveratrol was calculated according to the following formula:

[0079] Bioaccessibility (%) = (resveratrol content before digestion - resveratrol content in digestion dialysate) / resveratrol content before digestion × 100%

[0080] Table 3 Bioaccessibility of the delivery systems of Examples 1 to 3 and Comparative Examples 1 to 5

[0081] Serial number Sample name Bioaccessibility (%) 1 Resveratrol 16.5 2 Example 1 75.2 3 Example 2 71.4 4 Example 3 78.1 5 Comparative example 1 28.3 6 Comparative example 2 30.5 7 Comparative example 3 45.2 8 Comparative example 4 29.8 9 Comparative example 5 56.2

[0082] Table 3 shows the bioaccessibility of the delivery systems of Examples 1-3 and Comparative Examples 1-5. As can be seen from the table, drug encapsulation helps improve drug bioaccessibility. However, the lack of lactoferrin or sodium polyglutamate, or the absence of ionic crosslinking, can lead to structural instability or poor encapsulation of the delivery system, resulting in poor bioaccessibility. While whey protein or gelatin combined with sodium polyglutamate can also form a certain amount of drug encapsulation, the encapsulation effect is poor, and the bioaccessibility thereof falls far short of the standards of the present invention.

[0083] 4. Comparison of nanogel particle size

[0084] The average particle size, particle size distribution and polydispersity index of the nanogel solution were determined by dynamic light scattering.

[0085] Table 4 Gel particle size of the delivery system of Examples 1 to 3 and Comparative Examples 1 to 5

[0086]

[0087]

[0088] Table 4 gives the data of the gel particle size and dispersion coefficient of the delivery systems of Examples 1 to 3 and Comparative Examples 1 to 5. It can be seen from Table 4 that the particle size of the examples is smaller and the polydispersity index is low (<0.25), indicating a uniform structure; while the particle size of the comparative examples is larger and the distribution is wider, and the polydispersity index increases significantly, indicating structural instability or aggregation.

[0089] The examples provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A preparation method of a resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel, characterized in that, It includes the following steps: Step 1: Prepare a lactoferrin solution and a γ-polyglutamic acid sodium solution respectively. After mixing and stirring evenly, a stable composite solution is formed. Step 2: Dissolve resveratrol in 50% ethanol to form a resveratrol ethanol solution. Slowly add the resveratrol ethanol solution to the composite solution in Step 1, and stir at 30°C for 20 - 30 min to obtain a lactoferrin–γ-polyglutamic acid sodium composite system loaded with resveratrol. Step 3: Slowly add a CaCl2 solution to the composite system in Step 2 for ionic cross-linking to form a nanogel structure. Step 4: Dialyze the nanogel obtained in Step 3 to remove free components, and then freeze-dry to obtain a resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel.

2. The preparation method according to claim 1, wherein, In Step 1, the mass concentration of the lactoferrin solution is 1 - 2%, and the mass concentration of the γ-polyglutamic acid sodium solution is 0.5 - 1%.

3. The preparation method according to claim 1, characterized in that, In Step 1, the volume ratio of the lactoferrin solution to the γ-polyglutamic acid sodium solution is 1:1 - 2:

1.

4. The preparation method according to claim 1, wherein In Step 2, the mass concentration of the resveratrol ethanol solution is 4 - 8 mg / mL, and the concentration of resveratrol in the composite system is 0.4 - 0.8 mg / mL.

5. The preparation method according to claim 1, wherein, In Step 3, the mass concentration of the CaCl2 solution is 5 - 10%, the addition rate is controlled within 0.3 - 0.5 mL / min. After adding the CaCl2 solution, the mass concentration of CaCl2 in the mixed system is 1.5% - 2.0%.

6. The preparation method according to claim 1, characterized in that, When performing the ionic cross-linking in Step 3, the pH is 6.8 - 7.2, the temperature is 30 - 35°C, and continuous stirring is carried out for the cross-linking reaction. The cross-linking reaction time is 20 - 40 min.

7. The preparation method according to claim 1, characterized in that, When dialyzing in Step 4, the cut-off molecular weight of the dialysis bag used is 3500 Da, and the dialysis is carried out at 4°C for cold dialysis for 12 - 24 h.

8. The preparation method according to claim 1, characterized in that, The conditions for freeze-drying in Step 4 are to freeze at -80°C for 8 h first, and then freeze-dry under vacuum conditions for 24 - 48 h.

9. A resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel prepared by the method according to any one of claims 1 - 8.

10. Use of the resveratrol delivery system based on lactoferrin-polyglutamic acid nanogel according to claim 9 in food, medicine and health products.

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