Resveratrol nano preparation for treating xerophthalmia and preparation method thereof

Abstract

The invention belongs to the field of xerophthalmia treatment, and relates to a resveratrol nano preparation for treating xerophthalmia and a preparation method thereof. The resveratrol nano preparation comprises resveratrol and a composite carrier formed by sericin and glycyrrhizic acid through interpenetration, and the resveratrol is encapsulated by the composite carrier. According to the invention, the composite carrier formed by interpenetration of sericin and glycyrrhizic acid is adopted to encapsulate resveratrol, so that the problem of poor curative effect caused by low bioavailability of traditional resveratrol in the process of treating xerophthalmia is effectively solved. The composite carrier can encapsulate the resveratrol to form a stable nano filamentous structure, the nano filamentous structure has a hydrodynamic diameter less than 10nm, the oral bioavailability of the resveratrol and the distribution capability of the resveratrol in eye tissues are greatly improved, and the efficacy of the oral resveratrol for treating dry eyes is further remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of dry eye treatment, and in particular to a resveratrol nano preparation for treating dry eye and a preparation method thereof. Background Art

[0002] In recent years, the number of patients with dry eye in my country has continued to increase. This eye disease, which is affected by multiple factors, is mainly manifested by dry eyes, foreign body sensation and visual fatigue. In severe cases, redness, obvious discomfort, swelling and burning sensation of the eyes may occur. If not treated in time, dry eye may even cause damage to vision. Inflammation plays a core role in the occurrence and development of dry eye. It is not only one of the important causes but also a common clinical manifestation. It is crucial to effectively control inflammation during the treatment process. At present, cyclosporine A, tacrolimus and glucocorticoids are the main drugs for the treatment of dry eye, but long-term use of these drugs may bring side effects, such as increased drug resistance, increased risk of cataracts and increased intraocular pressure. In view of this, exploring new treatment strategies is particularly important to meet the challenges of dry eye.

[0003] Resveratrol (RES) is a natural polyphenol compound that is widely found in plants such as grape skins, red wine, and blueberries. It is known for its antioxidant, anti-inflammatory, and neuroprotective effects, and shows potential in the treatment of dry eyes. Resveratrol has anti-inflammatory and antioxidant effects, which can help to comprehensively improve the symptoms of dry eyes. Its natural source can also reduce the risk of toxicity and side effects. Resveratrol has a significant specific effect on inflammation and can effectively reduce the symptoms of eye inflammation. It also has a neuroprotective effect and can promote ocular nerve repair. Overall, resveratrol has the advantages of high safety and good tolerability in the treatment of dry eyes.

[0004] While resveratrol has shown potential in treating dry eye, its relatively low oral bioavailability limits its therapeutic efficacy. Specifically, resveratrol's oral bioavailability is less than 5%, due to its rapid metabolism and elimination in the body. This low bioavailability directly leads to insufficient intraocular concentrations, thereby weakening its effectiveness in treating dry eye.

[0005] Therefore, new technologies need to be provided to improve the oral bioavailability of resveratrol, thereby enhancing its therapeutic effect on dry eye. Summary of the Invention

[0006] (1) Technical issues to be solved

[0007] In order to solve the problem of poor dry eye treatment effect caused by low bioavailability of resveratrol in the prior art, the present invention provides a resveratrol nanoformulation for treating dry eye and a preparation method thereof.

[0008] (2) Technical solution

[0009] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention provides a resveratrol nanoformulation for treating dry eye, comprising resveratrol and a composite carrier formed by interpenetration of sericin and glycyrrhizic acid;

[0011] The composite carrier encapsulates the resveratrol.

[0012] In a second aspect, the present invention further provides a method for preparing the above-mentioned resveratrol nanoformulation, comprising the following steps:

[0013] S1: mixing the sericin aqueous solution with glycyrrhizate to obtain a composite carrier solution;

[0014] S2: adding resveratrol to the composite carrier solution, adjusting the pH of the system to alkaline, then performing ultrasonic treatment, then adjusting the pH of the system to neutral, and performing centrifugation to obtain a supernatant;

[0015] S3: Drying the supernatant to obtain the resveratrol nanoformulation.

[0016] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S1 and step S2, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is (4-6):(40-50):(40-50).

[0017] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S1, the glycyrrhizic acid salt is dipotassium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate or sodium glycyrrhizate.

[0018] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S1, the sericin aqueous solution is mixed with glycyrrhizate, and then stirred for 1-2 hours.

[0019] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S2, after adding resveratrol to the composite carrier liquid, the pH of the system is adjusted to 11.8-12.5 by using a strong base.

[0020] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S2, ultrasonic treatment is performed under ice bath conditions, the ultrasonic treatment time is 3-5 minutes, and the centrifugal treatment time is 25-35 minutes.

[0021] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S2, the pH of the system is adjusted to neutral by hydrochloric acid.

[0022] In the above-mentioned method for preparing the resveratrol nanoformulation, preferably, in step S3, the supernatant is freeze-dried at a temperature of -40°C to -50°C.

[0023] (3) Beneficial effects

[0024] This invention uses a composite carrier formed by interpenetrating sericin and glycyrrhizic acid to encapsulate resveratrol, effectively addressing the poor efficacy of traditional resveratrol in treating dry eye due to its low bioavailability. This composite carrier encapsulates resveratrol, forming a stable nanofilamentous structure with a hydrodynamic diameter of less than 10 nm. This significantly enhances the oral bioavailability and distribution of resveratrol within ocular tissues, significantly improving the efficacy of resveratrol in treating dry eye. Furthermore, glycyrrhizic acid, in combination with sericin, enhances the oral delivery efficiency and biocompatibility of the nanoformulation.

[0025] The combination of glycyrrhizic acid and sericin can eliminate the isoelectric point of sericin, effectively preventing degradation and breakdown in the stomach environment. This ensures that resveratrol is absorbed in a more complete and efficient form and targeted to ocular tissue. Therefore, the resveratrol nanoformulation provided by the present invention not only overcomes the low bioavailability of resveratrol in existing technologies but also significantly enhances its therapeutic effect for dry eye, showing promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron micrographs of sericin, dipotassium glycyrrhizate, sericin-glycyrrhizic acid complex, sericin-encapsulated resveratrol, glycyrrhizic acid-encapsulated resveratrol, and the product prepared in Example 1;

[0027] Figure 2 Scanning electron micrographs of resveratrol, dipotassium glycyrrhizate, sericin, a sericin-glycyrrhizic acid complex, a physical mixture of resveratrol-dipotassium glycyrrhizate-sericin, and the product prepared in Example 1;

[0028] Figure 3 A is a comparison chart of the encapsulation capabilities of glycyrrhizic acid-encapsulated resveratrol, sericin-encapsulated resveratrol, and the product prepared in Example 1;

[0029] Figure 3 B is a stability comparison chart of glycyrrhizic acid-encapsulated resveratrol and the product prepared in Example 1;

[0030] Figure 3C is a size comparison diagram of sericin, dipotassium glycyrrhizate, sericin-glycyrrhizic acid complex, glycyrrhizic acid-encapsulated resveratrol, sericin-encapsulated resveratrol, and the product prepared in Example 1;

[0031] Figure 4 A is a comparison chart of the blood compatibility of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex, and the product prepared in Example 1;

[0032] Figure 4 B is a color comparison chart of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex, and the product prepared in Example 1;

[0033] Figure 4 C is a statistical graph of the blood compatibility of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex, and the product prepared in Example 1;

[0034] Figure 5 This is a comparison chart of the oral compatibility of resveratrol, sericin-glycyrrhizic acid complex, and the product prepared in Example 1;

[0035] Figure 6 This is a blood drug concentration test chart of resveratrol and the product prepared in Example 1;

[0036] Figure 7 Based on Figure 6 The pharmacokinetic data obtained;

[0037] Figure 8 This is a detection diagram of the distribution of resveratrol and the product prepared in Example 1 in the whole eye tissue;

[0038] Figure 9 A is a comparison of fluorescent staining of the cornea of ​​mice in different groups;

[0039] Figure 9 B is a comparison of HE staining of mouse eyeballs in different groups;

[0040] Figure 9 C is Figure 9 A's statistical chart;

[0041] Figure 9 D is a statistical comparison of tear secretion in mice of different groups;

[0042] Figure 9 E is Figure 9 Statistical graph of corneal thickness in B;

[0043] Figure 9 F is Figure 9 Statistical graph of corneal epithelial cell thickness in B. DETAILED DESCRIPTION

[0044] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] The present invention provides a resveratrol nano-preparation for treating dry eye, comprising resveratrol and a composite carrier formed by interpenetration of sericin and glycyrrhizic acid, wherein the composite carrier is encapsulated on the surface of the resveratrol.

[0046] This invention uses a composite carrier formed by interpenetrating sericin and glycyrrhizic acid to encapsulate resveratrol, effectively addressing the poor efficacy of traditional resveratrol in treating dry eye due to its low bioavailability. This composite carrier encapsulates resveratrol, forming a stable nanofilamentous structure with a hydrodynamic diameter of less than 10 nm. This significantly enhances the oral bioavailability and distribution of resveratrol within ocular tissues, significantly improving the efficacy of resveratrol in treating dry eye. Furthermore, glycyrrhizic acid, in combination with sericin, enhances the oral delivery efficiency and biocompatibility of the nanoformulation.

[0047] The combination of glycyrrhizic acid and sericin can eliminate the isoelectric point of sericin, effectively preventing degradation and breakdown in the stomach environment. This ensures that resveratrol is absorbed in a more complete and efficient form and targeted to ocular tissue. Therefore, the resveratrol nanoformulation provided by the present invention not only overcomes the low bioavailability of resveratrol in existing technologies but also significantly enhances its therapeutic effect for dry eye, showing promising application prospects.

[0048] The present invention also provides a method for preparing the above-mentioned resveratrol nanoformulation, comprising the following steps:

[0049] S1: Mixing the sericin aqueous solution and glycyrrhizic acid salt, and stirring to obtain a composite carrier liquid.

[0050] S2: adding resveratrol to the composite carrier solution, adjusting the pH of the system to alkaline, then performing ultrasonic treatment, then adjusting the pH of the system to neutral, and performing centrifugation to obtain a supernatant.

[0051] S3: Drying the supernatant to obtain the resveratrol nanoformulation.

[0052] Preferably, in step S1 and step S2, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is (4-6):(40-50):(40-50), more preferably 5:45:45.

[0053] Preferably, in step S1, the sericin can be dissolved in deionized water and stored overnight at 4°C to further complete hydration. The glycyrrhizic acid salt can be dipotassium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, or sodium glycyrrhizate. Dipotassium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, or sodium glycyrrhizate, all of which are salts of glycyrrhizic acid and have good solubility and amphiphilicity. After mixing the sericin aqueous solution and the glycyrrhizic acid salt, the mixture is stirred for 1-2 hours at a speed of 800-1000 rpm.

[0054] Preferably, in step S2, after adding resveratrol to the composite carrier liquid, the pH of the system is adjusted to 11.8-12.5 using a strong base / strong base solution, such as sodium hydroxide, potassium hydroxide, sodium hydroxide solution, or potassium hydroxide solution, and then ultrasonically treated under ice bath conditions, with each ultrasonic treatment lasting 3-5 seconds, and the total ultrasonic treatment time is 3-5 minutes. Next, the pH of the system is adjusted to 7 using hydrochloric acid, and then centrifuged at a centrifugal force of 8000×g for 25-35 minutes to remove the precipitate. The supernatant obtained after centrifugation is the resveratrol-loaded sericin-glycyrrhizic acid nanocomposite, and the supernatant is then freeze-dried at a freezing temperature of -40°C to -50°C to obtain a resveratrol nanoformulation.

[0055] In step S1, sericin and glycyrrhizate form an interpenetrating network structure through intermolecular hydrogen bonding, hydrophobic interactions, and electrostatic attraction. Specifically, the sericin's β-pleated sheet domains sterically complement the triterpenoid saponin groups of glycyrrhizate, forming a dynamically cross-linked nanoscale network structure that provides anchoring sites for the subsequent encapsulation of resveratrol. This structure enables targeted adsorption of resveratrol's phenolic hydroxyl groups via van der Waals forces, forming a "core-shell" nanoassembly. Furthermore, the carboxylic acid groups of glycyrrhizate and the amino groups of sericin produce an acid-base neutralization effect, eliminating the sericin's isoelectric point and effectively preventing recognition by the positively charged binding sites of pepsin.

[0056] Stirring for 1-2 hours can ensure sufficient diffusion of molecules and form stable nanoassemblies.

[0057] Adjusting the pH of the system to 11.8-12.5 in step S2 is a key preparation condition of the present invention. After the pH is adjusted to 11.8-12.5, the alkaline environment promotes the unfolding of the sericin's α-helix, exposing the hydrophobic core and enhancing its π-π stacking interaction with resveratrol. This alkaline environment completely ionizes the carboxylate groups of glycyrrhizate, strengthening the electrostatic attraction with resveratrol. This alkaline environment also deprotonates the phenolic hydroxyl groups of resveratrol, further enhancing π-π stacking with the sericin's Tyr residues.

[0058] If the pH of the system in step S2 is less than 11.8, the encapsulation effect of the composite carrier on resveratrol will be significantly reduced, resulting in insufficient bioavailability of the product. If the pH of the system in step S2 is greater than 12.5, the product may appear dark purple, the activity of resveratrol will be reduced, and the biocompatibility will be reduced.

[0059] In step S2, the microfluidics generated by ultrasonic treatment can shear larger particles in the system, reducing the product particle size to <10 nm. The ice bath condition can suppress thermal effects and prevent denaturation of sericin. The ultrasonic treatment time also significantly affects product performance. If the ultrasonic treatment time is less than 3 minutes, the resveratrol encapsulation efficiency will be too low, resulting in incomplete encapsulation. If the ultrasonic treatment time is too long, the sericin may denature and may also cause partial damage to the nanoformulation.

[0060] In step S2, adjusting the pH back to neutral allows the amino groups of sericin to protonate, forming strong electrostatic crosslinks with the carboxylate groups of glycyrrhizic acid, locking the nanostructure and allowing the resveratrol to be reprotonated and encapsulated within the hydrophobic core. Furthermore, adjusting the pH back to neutral allows the conversion of glycyrrhizate salt to glycyrrhizic acid.

[0061] Furthermore, during the research process of the present invention, to improve the bioavailability of resveratrol, an attempt was first made to encapsulate resveratrol with glycyrrhizic acid using glycyrrhizic acid salt as a single carrier. Testing revealed that glycyrrhizic acid exhibited good encapsulation and loading efficiency for resveratrol. However, the weak electrostatic repulsion of the carboxylate groups of glycyrrhizic acid alone could not resist ion-induced particle aggregation. Furthermore, the binding of glycyrrhizic acid to resveratrol relied on dynamic hydrophobic interactions and lacked a rigid network support, resulting in the product being unstable after 40 days of storage. To achieve a product with both good encapsulation efficiency, loading efficiency, and good stability, the present invention further added sericin to enhance stability. This composite carrier, combined with glycyrrhizic acid and resveratrol, enhanced drug delivery efficiency in the body and reduced the side effects caused by glycyrrhizic acid.

[0062] In addition, in the single glycyrrhizic acid encapsulation system, the average particle size of the resveratrol-glycyrrhizic acid complex is larger. After the introduction of sericin, the hydrodynamic diameter of the composite encapsulation product is further reduced compared with the resveratrol-glycyrrhizic acid complex, and its morphology is transformed from the spherical shape of the resveratrol-glycyrrhizic acid complex to a uniform nanofilamentous structure.

[0063] The resveratrol-loaded sericin-glycyrrhizic acid nanofilaments (R@SD) prepared by the present invention have the following advantages:

[0064] 1. It has higher encapsulation ability, high stability and smaller nano size.

[0065] 2. Good blood compatibility and body compatibility.

[0066] 3. It can significantly increase the blood concentration of resveratrol.

[0067] 4. In the pharmacokinetics of resveratrol, compared with single resveratrol, the area under the concentration-time curve of R@SD increased by up to 6.82 times, the maximum blood concentration increased by up to 12.18 times, and the peak time was delayed.

[0068] 5. The distribution concentration of resveratrol in the entire eye tissue of R@SD is greatly improved.

[0069] In order to further clarify the solution of the present invention and its technical advancement, the following description is made in conjunction with specific embodiments and technical effects.

[0070] Example 1

[0071] This embodiment provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0072] S1: Sericin protein (SER) was stirred in distilled water until completely dissolved and stored at 4°C overnight. A 45 mg / mL sericin aqueous solution was obtained by dilution. Dipotassium glycyrrhizinate (DG) was added to the sericin aqueous solution and stirred at 900 rpm for 1 hour to obtain a composite carrier solution.

[0073] S2: Add resveratrol powder to the composite carrier solution. Adjust the pH to 12 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 3 minutes, turning on for 3 seconds and then off for 3 seconds. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 30 minutes to obtain the supernatant.

[0074] S3: freeze-drying the supernatant at -45°C to obtain resveratrol nanoformulation.

[0075] In this embodiment, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is 5:45:45.

[0076] Example 2

[0077] This embodiment provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0078] S1: Sericin was stirred in distilled water until completely dissolved and stored at 4°C overnight. A sericin aqueous solution with a concentration of 75 mg / mL was obtained by dilution. Disodium glycyrrhizate was added to the sericin aqueous solution and stirred at 800 rpm for 2 hours to obtain a composite carrier solution.

[0079] S2: Add resveratrol powder to the composite carrier solution. Adjust the pH to 11.8 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 4 minutes, with a 4-second on-state and 4-second off-state. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 25 minutes to obtain the supernatant.

[0080] S3: freeze-drying the supernatant at -40°C to obtain resveratrol nanoformulation.

[0081] In this embodiment, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is 4:40:40.

[0082] Example 3

[0083] This embodiment provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0084] S1: Sericin was stirred in distilled water until completely dissolved and stored at 4°C overnight. A 60 mg / mL sericin aqueous solution was obtained by dilution. Potassium glycyrrhizate was added to the sericin aqueous solution and stirred at 1000 rpm for 1.5 hours to obtain a composite carrier solution.

[0085] S2: Add resveratrol powder to the composite carrier solution. Adjust the pH to 12.5 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 5 minutes, turning on for 5 seconds and then off for 5 seconds. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 35 minutes to obtain the supernatant.

[0086] S3: freeze-drying the supernatant at -50°C to obtain resveratrol nanoformulation.

[0087] In this embodiment, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is 6:50:50.

[0088] Example 4

[0089] This embodiment provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0090] S1: Sericin was stirred in distilled water until completely dissolved and stored at 4°C overnight. A 30 mg / mL sericin aqueous solution was obtained by dilution. Sodium glycyrrhizate was added to the sericin aqueous solution and stirred at 900 rpm for 1 hour to obtain a composite carrier solution.

[0091] S2: Add resveratrol powder to the composite carrier solution. Adjust the pH to 12 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 3 minutes, turning on for 3 seconds and then off for 3 seconds. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 30 minutes to obtain the supernatant.

[0092] S3: freeze-drying the supernatant at -45°C to obtain resveratrol nanoformulation.

[0093] In this embodiment, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is 5:45:45.

[0094] Example 5

[0095] This embodiment provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0096] S1: Sericin was stirred in distilled water until completely dissolved and stored at 4°C overnight. A 15 mg / mL sericin aqueous solution was obtained by dilution. Sodium glycyrrhizate was added to the sericin aqueous solution and stirred at 950 rpm for 1.6 hours to obtain a composite carrier solution.

[0097] S2: Add resveratrol powder to the composite carrier solution. Adjust the pH to 12.1 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 3.5 minutes, maintaining the on-state for 4 seconds and the off-state for 3 seconds. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 32 minutes to obtain the supernatant.

[0098] S3: freeze-drying the supernatant at -46°C to obtain resveratrol nanoformulation.

[0099] In this embodiment, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is 5:45:45.

[0100] Comparative Example 1

[0101] This comparative example provides a method for preparing a resveratrol nanoformulation, comprising the following steps:

[0102] S1: Dipotassium glycyrrhizate was stirred in distilled water until completely dissolved, and then diluted to obtain a dipotassium glycyrrhizate aqueous solution with a concentration of 45 mg / mL.

[0103] S2: Add resveratrol powder to a dipotassium glycyrrhizate aqueous solution. Adjust the pH to 12 with sodium hydroxide. Then, sonicate in an ice bath using a probe sonicator for 3 minutes, turning on for 3 seconds and then off for 3 seconds. Next, add hydrochloric acid to adjust the pH to 7. Centrifuge at 8000 × g for 30 minutes to obtain the supernatant.

[0104] S3: freeze-drying the supernatant at -45°C to obtain the resveratrol-loaded glycyrrhizic acid nanocomplex.

[0105] In this comparative example, the mass ratio of resveratrol to glycyrrhizate is 5:45.

[0106] Comparative Examples 2-3

[0107] Comparative Example 2-3 provides a method for preparing a resveratrol nanoformulation, which differs from Example 1 in that the pH of the system is adjusted to 11 and 13 respectively by sodium hydroxide.

[0108] Comparative Examples 4-5

[0109] Comparative Example 4-5 provides a method for preparing a resveratrol nanoformulation, which differs from Example 1 in that a probe ultrasonic machine is used for ice bath ultrasonic treatment for 1 min and 8 min, respectively.

[0110] Figure 1 Transmission electron micrographs of sericin, dipotassium glycyrrhizate, a sericin-glycyrrhizic acid complex, sericin-encapsulated resveratrol, glycyrrhizic acid-encapsulated resveratrol, and the product prepared in Example 1. Glycyrrhizic acid-encapsulated resveratrol was prepared using the method of Comparative Example 1. The preparation method of sericin-encapsulated resveratrol was similar to that of Comparative Example 1, except that dipotassium glycyrrhizate was replaced with sericin. The preparation method of the sericin-glycyrrhizic acid complex differed from that of Example 1 in that no resveratrol was added.

[0111] pass Figure 1 It can be seen that the resveratrol encapsulated by sericin, dipotassium glycyrrhizate, and glycyrrhizic acid is spherical, and the encapsulation effect of resveratrol encapsulated by sericin is poor. The sericin-glycyrrhizic acid complex and the product prepared in Example 1 are nanofilamentous, and the size of the resveratrol nanospheres in R@SD prepared in Example 1 is further reduced.

[0112] Figure 2 The scanning electron micrographs are of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex, resveratrol-dipotassium glycyrrhizate-sericin physical mixture and the product prepared in Example 1. Figure 2It can be seen that RES is a crystalline tubular structure, DG is a spherical structure, and SER is a lamellar structure. The three raw materials are relatively large in size, and the morphology presented by the physical mixture of the three raw materials is the superposition of the three. However, after being encapsulated by the SD composite carrier, the freeze-dried R@SD shows a micron-sized silkworm-like structure. Compared with the three raw materials, the morphology has changed significantly, and the size is significantly reduced compared with the raw materials.

[0113] In addition, it was observed that the transmission electron microscopy images and scanning electron microscopy images of the products prepared in Examples 2-5 were basically identical to the microscopic morphology of R@SD prepared in Example 1.

[0114] Figure 3 A is a comparison chart of the encapsulation capabilities of glycyrrhizic acid-encapsulated resveratrol, sericin-encapsulated resveratrol, and the product prepared in Example 1. Figure 3 A shows that glycyrrhizic acid and glycyrrhizic acid-sericin composite carriers have better encapsulation effects on resveratrol, while sericin has a poor encapsulation effect on resveratrol and has almost no encapsulation ability.

[0115] Figure 3 B is a stability comparison chart of glycyrrhizic acid encapsulated resveratrol and the product prepared in Example 1. Figure 3 B shows that the storage stability of resveratrol encapsulated by glycyrrhizic acid is poor. As the storage time increases, the retention rate of resveratrol decreases significantly. The storage stability of R@SD is better.

[0116] Figure 3 C is a size comparison diagram of sericin, dipotassium glycyrrhizate, sericin-glycyrrhizic acid complex, glycyrrhizic acid-encapsulated resveratrol, sericin-encapsulated resveratrol, and the product prepared in Example 1.

[0117] The encapsulation effect, storage stability and size of the products of Examples 2-5 are substantially the same as those of Example 1.

[0118] Figure 4 B is a color comparison chart of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex, and the product prepared in Example 1 itself. Using physiological saline, Triton, and sodium hydroxide as controls, it can be seen that R@SD itself has a certain color.

[0119] Sodium chloride solution, Triton solution, sodium hydroxide solution, 5 mg / mL resveratrol solution, dipotassium glycyrrhizate solution, sericin solution, sericin-glycyrrhizic acid complex solution, and R@SD solution were prepared. Equal amounts of red blood cells were added to each of these solutions. After incubation for a period of time, the mixture was centrifuged. Unbroken red blood cells will sink to the bottom. Broken red blood cells indicate that the drug is destructive to red blood cells. A hemolysis percentage below 5% is generally considered to indicate good drug hemocompatibility.

[0120] pass Figure 4 A and Figure 4 C shows that, except for the Triton and sodium hydroxide groups, the blood compatibility of all groups is good. In addition, after testing, the blood compatibility of R@SD of Examples 2-5 is also good, with the hemolysis percentage less than 5%.

[0121] Figure 5 This figure compares the oral compatibility of resveratrol, the sericin-glycyrrhizic acid complex, and the product prepared in Example 1. Using PBS buffer as a control, the effects of resveratrol, the sericin-glycyrrhizic acid complex, and R@SD on liver function (AST and ALT) and renal function (CREA and UREA) were tested. The effects of resveratrol, the sericin-glycyrrhizic acid complex, and R@SD on liver and kidney function were similar to those of the control group, indicating good oral compatibility. Similarly, testing showed that R@SD from Examples 2-5 also exhibited good oral compatibility.

[0122] Figure 6 This is a blood drug concentration test chart of resveratrol and the product prepared in Example 1. Figure 7 Based on Figure 6 Pharmacokinetic data were obtained. Figure 6 as well as Figure 7 It can be seen that compared with single resveratrol, the area under the drug-time curve (AUC) of R@SD is (0-t) Increased by 6.82 times, the maximum blood concentration C max Increased by 12.18 times, peak time T max The results show that the product R@SD of Example 1 can significantly increase the blood concentration of RES. In addition, the R@SD of Examples 2-5 can also significantly increase the blood concentration of RES.

[0123] Figure 8 This is a detection diagram of the distribution of resveratrol and the product prepared in Example 1 in the whole eyeball tissue. Figure 8It can be seen that, compared with resveratrol alone, the concentration of R@SD distributed throughout the eyeball tissues has also been significantly increased. In addition, after testing, the R@SD of Examples 2-5 can also increase the concentration of resveratrol distributed throughout the eyeball tissues.

[0124] Healthy, female C57BL / 6 mice aged 6-8 weeks were selected. Mice with corneal abnormalities, such as abnormal corneal sodium fluorescein staining and significant differences in tear secretion between the two eyes, were excluded to ensure normal eye function. The mice were then randomly divided into five groups.

[0125] An equal volume of PBS was instilled into both eyes of the first group of mice, serving as a normal control group. A 0.1% benzalkonium chloride solution was instilled into both eyes of the mice in the second, third, fourth, and fifth groups once daily for 14 consecutive days to induce a dry eye model.

[0126] The dry eye model was established while drug treatment was being performed. Mice in the first and second groups were orally administered an equal volume of PBS buffer. Mice in the third, fourth, and fifth groups were orally administered an equal volume and concentration of resveratrol, sericin-glycyrrhizic acid complex, and R@SD prepared in Example 1. Dosing frequency for each group was once daily for 14 consecutive days via oral gavage, at a dose of 50 mg / kg per mouse, in a volume of 100 μL per dose.

[0127] On the 14th day of administration, 0.1 wt% sodium fluorescein solution was used to stain the mice in each group. The sodium fluorescein solution was dripped into the mouse eyes and allowed to remain on the mouse ocular surface for 10 seconds. The excess dye was then rinsed off with normal saline. The mouse ocular surface was wiped dry. The eyeballs of each group of mice were exposed to cobalt blue light under a slit lamp. After adjusting the magnification, photos were taken to record the coloration of the cornea of ​​each group of mice. Figure 9 A. In addition, the eyeballs of mice in each group were fixed, embedded and stained with HE to obtain Figure 9 B.

[0128] The phenol red cotton thread test was used to measure the average tear secretion volume (unit: mm) of each group of mice 14 days after administration. The phenol red cotton thread test was performed as follows: the phenol red cotton thread was clamped with tweezers and placed on the outer canthus of the mouse's eye. After 60 seconds, the phenol red cotton thread wet length (mm) of each group of mice at different times was recorded to obtain the tear secretion volume (unit: mm). Figure 9 D.

[0129] pass Figure 9 AF showed that in the benzalkonium chloride-induced dry eye mouse model, R@SD could significantly reduce the ocular surface damage of dry eye mice, restore the normal structure of the cornea, and increase the amount of tear secretion. Its therapeutic effect was significantly better than RES.

[0130] Furthermore, the product prepared in Comparative Example 2 showed significantly lower resveratrol encapsulation efficiency compared to Examples 1-5, while the product in Comparative Example 3 exhibited a dark purple color, indicating decreased resveratrol activity and biocompatibility. The encapsulation efficiency in Comparative Example 4 was too low, indicating incomplete resveratrol encapsulation. The nanoformulation in Comparative Example 5 exhibited damage, but the encapsulation efficiency was still higher than that of Comparative Example 4.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A resveratrol nanoformulation for treating dry eye, characterized in that: It includes resveratrol, and a composite carrier formed by the interpenetration of sericin and glycyrrhizic acid; The composite carrier encapsulates the resveratrol.

2. A method for preparing the resveratrol nanoformulation according to claim 1, characterized in that: The steps include: S1: mixing the sericin aqueous solution with glycyrrhizate to obtain a composite carrier solution; S2: adding resveratrol to the composite carrier solution, adjusting the pH of the system to alkaline, then performing ultrasonic treatment, then adjusting the pH of the system to neutral, and performing centrifugation to obtain a supernatant; S3: Drying the supernatant to obtain the resveratrol nanoformulation.

3. The method for preparing the resveratrol nanoformulation according to claim 2, wherein: In step S1 and step S2, the mass ratio of resveratrol, sericin and glycyrrhizic acid salt in the sericin aqueous solution is (4-6):(40-50):(40-50).

4. The method for preparing the resveratrol nanoformulation according to claim 2, wherein: In step S1, the glycyrrhizic acid salt is dipotassium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate or sodium glycyrrhizate.

5. The method for preparing the resveratrol nanoformulation according to claim 2, characterized in that: In step S1, the sericin aqueous solution and glycyrrhizic acid salt are mixed and then stirred for 1-2 hours.

6. The method for preparing the resveratrol nanoformulation according to claim 2, characterized in that: In step S2, after resveratrol is added to the composite carrier solution, the pH of the system is adjusted to 11.8-12.5 using a strong base.

7. The method for preparing the resveratrol nanoformulation according to claim 2, characterized in that: In step S2, ultrasonic treatment is performed in an ice bath, the ultrasonic treatment time is 3-5 minutes, and the centrifugal treatment time is 25-35 minutes.

8. The method for preparing the resveratrol nanoformulation according to claim 2, characterized in that: In step S2, the pH of the system is adjusted to neutral by hydrochloric acid.

9. The method for preparing the resveratrol nanoformulation according to claim 2, wherein: In step S3, the supernatant is freeze-dried at a temperature of -40°C to -50°C.

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