A resveratrol nano-formulation for treating dry eye syndrome and its preparation method

Abstract

The present application belongs to the field of dry eye treatment, and relates to a resveratrol nano-preparation for treating dry eye and a preparation method thereof. The resveratrol nano-preparation comprises resveratrol and a composite carrier formed by interpenetration of sericin and glycyrrhizic acid, and the composite carrier encapsulates the resveratrol. The present application effectively solves the problem of poor curative effect of traditional resveratrol in the treatment of dry eye due to low bioavailability by encapsulating resveratrol with the composite carrier formed by interpenetration of sericin and glycyrrhizic acid. The composite carrier can encapsulate resveratrol to form a stable nanometer silk structure, and the nanometer silk structure has a hydrodynamic diameter of less than 10 nm, greatly improving the oral bioavailability of resveratrol and the distribution ability in the ocular tissue, and further significantly improving the drug efficacy of resveratrol in the oral treatment of dry eye.

Description

TECHNICAL FIELD

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

[0002] In recent years, the number of people suffering from dry eye in China has been increasing. This eye disease, which is mainly affected by multiple factors, is mainly manifested as eye dryness, foreign body sensation and visual fatigue. In severe cases, it may cause eye redness, obvious discomfort, swelling and burning sensation. 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, and is one of the important inducements and common clinical manifestations. Therefore, it is crucial to effectively control inflammation during treatment. At present, cyclosporine A, tacrolimus and glucocorticoids are the main drugs for treating dry eye. However, long-term use of these drugs may cause side effects, such as increased drug resistance, increased risk of cataract and increased intraocular pressure. Therefore, it is particularly important to explore new treatment strategies to cope with the challenges of dry eye.

[0003] Resveratrol (RES) is a natural polyphenolic compound widely found in grape skins, red wine, blueberries and other plants. It is known for its antioxidant, anti-inflammatory and neuroprotective effects, and has shown potential in treating dry eye. Resveratrol has anti-inflammatory and antioxidant effects, which can help improve dry eye symptoms. Its natural source can also reduce the risk of toxicity and side effects. Resveratrol has a specific effect on inflammation, which can effectively reduce eye inflammation symptoms, and has a neuroprotective effect, which can promote eye nerve repair. Overall, resveratrol has the advantages of high safety and good tolerance in treating dry eye.

[0004] Although resveratrol has shown potential in treating dry eye, its oral bioavailability is relatively low, which limits its therapeutic effect. Specifically, the oral bioavailability of resveratrol is less than 5%, which is due to its rapid metabolism and elimination in the body. This low bioavailability directly leads to insufficient intraocular concentration, thereby weakening its effect in treating dry eye.

[0005] Therefore, it is necessary to provide new technology to improve the oral bioavailability of resveratrol and thus improve its therapeutic effect on dry eye. SUMMARY

[0006] (1) Technical problem to be solved

[0007] In order to solve the problem of poor therapeutic effect of resveratrol in treating dry eye due to low bioavailability in the prior art, the present application provides a resveratrol nano-preparation for treating dry eye and a preparation method thereof.

[0008] (II) Technical Solution

[0009] To achieve the above object, the main technical scheme adopted by the present application comprises:

[0010] In a first aspect, the present application provides a resveratrol nano-preparation 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 application further provides a preparation method of the above-mentioned resveratrol nano-preparation, comprising the following steps:

[0013] S1: mixing a sericin aqueous solution with glycyrrhizic acid salt to obtain a composite carrier liquid;

[0014] S2: adding resveratrol into the composite carrier liquid, adjusting the pH of the system to alkaline, then performing ultrasonic treatment, followed by adjusting the pH of the system to neutral, and performing centrifugal treatment to obtain a supernatant;

[0015] S3: drying the supernatant to obtain the resveratrol nano-preparation.

[0016] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in steps S1 and S2, the mass ratio of resveratrol, sericin in the sericin aqueous solution, and glycyrrhizic acid salt is (4-6):(40-50):(40-50).

[0017] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in step S1, the glycyrrhizic acid salt is dipotassium glycyrrhizinate, disodium glycyrrhizinate, potassium glycyrrhizinate, or sodium glycyrrhizinate.

[0018] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in step S1, the sericin aqueous solution is mixed with the glycyrrhizic acid salt, and then stirring is performed, with a stirring time of 1-2h.

[0019] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in step S2, after adding resveratrol into the composite carrier liquid, the pH of the system is adjusted to 11.8-12.5 by a strong base.

[0020] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in step S2, the ultrasonic treatment is performed under ice bath conditions, with a treatment time of 3-5min, and the centrifugal treatment is performed for a time of 25-35min.

[0021] Preferably, in the preparation method of the resveratrol nano-preparation as described above, in step S2, the pH of the system is adjusted to neutral by hydrochloric acid.

[0022] Preferably, in step S3, the supernatant is subjected to freeze-drying treatment, and the freezing temperature is -40 to -50°C.

[0023] (III) Beneficial effects

[0024] The present application effectively solves the problem of poor curative effect of traditional resveratrol in the treatment of dry eye due to low bioavailability by encapsulating resveratrol in a composite carrier formed by interpenetration of sericin and glycyrrhizic acid. The composite carrier can encapsulate resveratrol to form a stable nanoscale silk structure, which has a hydrodynamic diameter of less than 10 nm, greatly improving the oral bioavailability of resveratrol and its distribution ability in eye tissues, and thus significantly improving the drug efficacy of resveratrol in the oral treatment of dry eye. In addition, glycyrrhizic acid can also improve the oral delivery efficiency and biocompatibility of the nanoscale preparation together with sericin.

[0025] The use of glycyrrhizic acid in combination with sericin can eliminate the isoelectric point of sericin, effectively prevent the degradation and rupture of sericin in the stomach environment, and ensure that resveratrol can be absorbed in a more complete and efficient form and targeted to eye tissues. Therefore, the resveratrol nanoscale preparation provided by the present application not only overcomes the problem of low bioavailability of resveratrol in the prior art, but also significantly enhances its therapeutic effect on dry eye, and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

[0029] Figure 3 B is a comparison chart of the stability 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 blood compatibility comparison diagram of resveratrol, dipotassium glycyrrhizate, sericin, sericin-glycyrrhizic acid complex and the product prepared in Example 1.

[0032] Figure 4 B is a color comparison diagram 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 A comparative diagram of oral compatibility of resveratrol, sericin-glycyrrhizic acid complex and the product prepared in Example 1;

[0035] Figure 6 Blood concentration detection graphs for resveratrol and the product prepared in Example 1;

[0036] Figure 7 According to Figure 6 The obtained pharmacokinetic data;

[0037] Figure 8 The distribution of resveratrol and the product prepared in Example 1 in the whole eye tissue is shown in the image.

[0038] Figure 9 A shows a comparison of fluorescent staining of corneas from different groups of mice;

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

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

[0041] Figure 9 D is a comparative graph of tear secretion statistics in different groups of mice;

[0042] Figure 9 E is Figure 9 A statistical chart of corneal thickness in section B;

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

[0044] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] This invention provides a resveratrol nanoformulation for treating dry eye syndrome, comprising resveratrol and a composite carrier formed by the interpenetration of sericin and glycyrrhizic acid, wherein the composite carrier is encapsulated on the surface of resveratrol.

[0046] This invention utilizes a composite carrier formed by the interpenetration of sericin and glycyrrhizic acid to encapsulate resveratrol, effectively addressing the problem of poor efficacy caused by low bioavailability in traditional resveratrol treatments for dry eye. This composite carrier encapsulates resveratrol, forming a stable nanofilament structure with a hydrodynamic diameter of less than 10 nm, significantly improving the oral bioavailability and distribution ability of resveratrol in ocular tissues, thereby significantly enhancing the efficacy of oral resveratrol in treating dry eye. Furthermore, glycyrrhizic acid, in conjunction with sericin, can further improve the oral delivery efficiency and biocompatibility of the nano-formulation.

[0047] The combined use of glycyrrhizic acid and sericin can eliminate the isoelectric point of sericin, effectively preventing its degradation and breakage in the gastric environment. This ensures that resveratrol can be absorbed in a more complete and efficient form and targeted to ocular tissues. Therefore, the resveratrol nano-formulation provided by this invention not only overcomes the problem of low bioavailability of resveratrol in existing technologies, but also significantly enhances its therapeutic effect on dry eye syndrome, showing promising application prospects.

[0048] This invention also provides a method for preparing the above-mentioned resveratrol nanoparticle formulation, comprising the following steps:

[0049] S1: Mix the sericin aqueous solution with glycyrrhizic acid and stir to obtain the composite carrier solution.

[0050] S2: Add resveratrol to the composite carrier solution, adjust the pH of the system to alkaline, then sonicate, adjust the pH of the system to neutral, and centrifuge to obtain the supernatant.

[0051] S3: The supernatant is dried to obtain resveratrol nano-formulation.

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

[0053] Preferably, in step S1 above, 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. These dipotassium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, or sodium glycyrrhizate are all salts of glycyrrhizic acid, possessing good solubility and amphiphilicity. After mixing the sericin aqueous solution with the glycyrrhizic acid salt, stirring is required for 1-2 hours at a speed of 800-1000 r / min.

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

[0055] In step S1 above, sericin and glycyrrhizate form an interpenetrating network structure through intermolecular hydrogen bonds, hydrophobic interactions, and electrostatic attraction. Specifically, the β-sheet domain of sericin can achieve steric hindrance complementarity with the triterpenoid saponin groups of glycyrrhizate, forming a dynamically cross-linked nanoscale network structure, providing anchoring sites for the subsequent encapsulation of resveratrol. This structure can directionally adsorb the phenolic hydroxyl groups of resveratrol through van der Waals forces, forming a "core-shell" nanoassembly. In addition, the carboxylic acid groups of glycyrrhizate and the amino groups of sericin can produce an acid-base neutralization effect, causing the isoelectric point of sericin to disappear, effectively avoiding recognition by the positively charged binding sites of pepsin.

[0056] Stirring for 1-2 hours ensures sufficient molecular diffusion, forming stable nano-assemblies.

[0057] In step S2 above, adjusting the pH of the system to 11.8-12.5 is one of the key preparation conditions of this invention. After adjusting the pH to 11.8-12.5, the alkaline environment can promote the unfolding of the α-helix of sericin, exposing the hydrophobic core and enhancing its π-π stacking interaction with resveratrol. Under this alkaline environment, the carboxylate group of glycyrrhizate is completely ionized, which can enhance the electrostatic attraction with resveratrol. This alkaline environment can also deprotonate the phenolic hydroxyl groups of resveratrol, further enhancing the π-π stacking with the Tyr residues of sericin.

[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 turn blackish-purple, the activity of resveratrol will decrease, and the biocompatibility will decline.

[0059] In step S2 above, the microjets generated by ultrasonic treatment can shear larger particles in the system, reducing the product particle size to <10nm. The ice bath condition can suppress the thermal effect and prevent sericin denaturation. The ultrasonic treatment time also has a significant impact on product performance. If the ultrasonic treatment time is less than 3 minutes, the encapsulation rate of resveratrol will be too low, resulting in incomplete encapsulation. If the ultrasonic treatment time is too long, it may cause sericin denaturation and partial damage to the nano-formulation.

[0060] In step S2 above, adjusting the pH to neutral allows for the protonation of the amino groups of sericin, forming a strong electrostatic cross-link with the carboxyl group of glycyrrhizic acid, locking the nanostructure, and reprotonating resveratrol, which is then encapsulated in a hydrophobic core. Additionally, adjusting the pH to neutral also allows glycyrrhizate to be converted to glycyrrhizic acid.

[0061] Furthermore, during the research process of this invention, to improve the bioavailability of resveratrol, we first attempted to use glycyrrhizic acid as a single carrier to encapsulate resveratrol. Testing revealed that glycyrrhizic acid exhibited good encapsulation efficiency and loading rate for resveratrol. However, the electrostatic repulsion of the carboxylate group of glycyrrhizic acid alone is weak, failing to resist ion-induced particle aggregation. Moreover, the binding of glycyrrhizic acid and resveratrol relies on dynamic hydrophobic interactions, lacking a rigid network support, resulting in an unstable product after 40 days of storage. To obtain a product with good encapsulation efficiency, loading rate, and stability, this invention further incorporates sericin to enhance stability. This sericin, along with glycyrrhizic acid and resveratrol, forms a composite carrier, improving drug delivery efficiency in vivo and reducing glycyrrhizic acid-induced side effects.

[0062] Furthermore, in the single glycyrrhizic acid encapsulation system, the resveratrol-glycyrrhizic acid complex has a larger average particle size. After introducing sericin, the hydrodynamic diameter of the composite encapsulation product was further reduced compared to the resveratrol-glycyrrhizic acid complex, and its morphology changed from the spherical shape of the resveratrol-glycyrrhizic acid complex to a uniform nanofilament structure.

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

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

[0065] 2. It has good blood compatibility and in vivo compatibility.

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

[0067] 4. In the pharmacokinetics of resveratrol, compared with resveratrol alone, R@SD showed a maximum increase of 6.82 times in the area under the curve, a maximum increase of 12.18 times in the maximum plasma concentration, and a delayed time to peak concentration.

[0068] 5. The distribution and concentration of resveratrol in the whole eye tissue of R@SD was significantly increased.

[0069] To further clarify the present invention and its technological advancements, the following description is provided in conjunction with specific embodiments and technical effects.

[0070] Example 1

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

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

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

[0074] S3: The supernatant was freeze-dried at -45℃ to obtain resveratrol nano-formulation.

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

[0076] Example 2

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

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

[0079] S2: Add resveratrol powder to the composite carrier solution, adjust the pH of the system to 11.8 with sodium hydroxide, and then sonicate in an ice bath for 4 minutes using a probe sonicator, keeping the switch on for 4 seconds and off for 4 seconds. Next, add hydrochloric acid to adjust the pH to 7, and then centrifuge at 8000×g for 25 minutes to obtain the supernatant.

[0080] S3: The supernatant was freeze-dried at -40℃ to obtain resveratrol nano-formulation.

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

[0082] Example 3

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

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

[0085] S2: Add resveratrol powder to the composite carrier solution, adjust the pH of the system to 12.5 with sodium hydroxide, and then sonicate in an ice bath using a probe sonicator for 5 min, keeping the switch on for 5 s and off for 5 s. Next, add hydrochloric acid to adjust the pH to 7, and then centrifuge at 8000×g for 35 min to obtain the supernatant.

[0086] S3: Freeze-dry the supernatant at -50℃ to obtain resveratrol nano-formulation.

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

[0088] Example 4

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

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

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

[0092] S3: The supernatant was freeze-dried at -45℃ to obtain resveratrol nano-formulation.

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

[0094] Example 5

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

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

[0097] S2: Add resveratrol powder to the composite carrier solution, adjust the pH of the system to 12.1 with sodium hydroxide, and then sonicate in an ice bath for 3.5 min using a probe sonicator, keeping the cycle on for 4 s and off for 3 s. Next, add hydrochloric acid to adjust the pH to 7, and then centrifuge at 8000×g for 32 min to obtain the supernatant.

[0098] S3: The supernatant was freeze-dried at -46℃ to obtain resveratrol nano-formulation.

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

[0100] Comparative Example 1

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

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

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

[0104] S3: The supernatant was freeze-dried at -45℃ to obtain glycyrrhizic acid nanocomposite loaded with resveratrol.

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

[0106] Comparative Examples 2-3

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

[0108] Comparative Example 4-5

[0109] Comparative Examples 4-5 provide a method for preparing resveratrol nanoparticles, which differs from Example 1 in that they are treated with a probe sonicator in an ice bath for 1 min and 8 min, respectively.

[0110] Figure 1 Transmission electron microscopy (TEM) images of sericin, dipotassium glycyrrhizate, 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 described in 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 resveratrol was not added.

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

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

[0113] In addition, it was observed that the transmission electron microscopy (TEM) images and scanning electron microscopy (SEM) images of the products prepared in Examples 2-5 were basically identical to the microstructure of the 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 As shown in A, glycyrrhizic acid and the glycyrrhizic acid-serice protein complex carrier have a good encapsulation effect 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 diagram of glycyrrhizic acid-encapsulated resveratrol and the product prepared in Example 1, through... Figure 3 As shown in B, the storage stability of resveratrol encapsulated with glycyrrhizic acid is poor. With the increase of storage time, the resveratrol retention rate decreases significantly, while R@SD has better storage stability.

[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 in Examples 2-5 are basically the same as those in Example 1.

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

[0119] Prepare 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. Add equal amounts of red blood cells to each of the above solutions, incubate for a period of time, and then centrifuge to precipitate the red blood cells. If the red blood cells are intact, they will sink directly to the bottom. If they are broken, it indicates that the drug is destructive to red blood cells. It is generally believed that the blood compatibility of the drug is better when the hemolysis percentage is below 5%.

[0120] pass Figure 4 A and Figure 4 As shown in C, except for the Triton and sodium hydroxide groups, the blood compatibility of all other groups was good. Furthermore, tests revealed that the R@SD samples in Examples 2-5 also exhibited good blood compatibility, with a hemolysis percentage of less than 5%.

[0121] Figure 5 This is a comparative diagram of the oral compatibility of resveratrol, the sericin-glycyrrhizic acid complex, and the product prepared in Example 1. PBS buffer was used as a control. The effects of resveratrol, the sericin-glycyrrhizic acid complex, and R@SD on liver function (AST and ALT) and kidney function (CREA and UREA) were detected. It can be seen that the effects of resveratrol, the sericin-glycyrrhizic acid complex, and R@SD on liver and kidney function are similar to those of the control group, indicating good oral compatibility. Similarly, R@SD from Examples 2-5 also showed good oral compatibility after testing.

[0122] Figure 6 The graph shows the blood concentration detection of resveratrol and the product prepared in Example 1. Figure 7 According to Figure 6 The obtained pharmacokinetic data. (Through...) Figure 6 as well as Figure 7 It can be seen that, compared with resveratrol alone, R@SD has a higher area under the curve (AUC) during the treatment period. (0-t) It increased by 6.82 times, with the maximum blood drug concentration C max It increased by 12.18 times, peak time T max The delay indicates that the product R@SD from Example 1 can significantly increase the blood concentration of RES. Furthermore, testing showed that R@SD from Examples 2-5 also significantly increased the blood concentration of RES.

[0123] Figure 8 This is a detection image of the distribution of resveratrol and the product prepared in Example 1 in the whole eye tissue, obtained through... Figure 8It can be seen that, compared with resveratrol alone, the concentration of R@SD distributed throughout the entire eye tissue was significantly improved. Furthermore, testing showed that R@SD in Examples 2-5 also increased the concentration distributed throughout the entire eye tissue.

[0124] Several healthy, female C57BL / 6 mice aged 6-8 weeks were selected, and mice with abnormal corneal fluorescein staining or significant differences in tear secretion between the two sides were excluded to ensure that the mice's eyes were normal. The mice were then randomly divided into 5 groups.

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

[0126] Drug treatment was initiated concurrently with the establishment of the dry eye model. Mice in groups 1 and 2 were orally administered an equal volume of PBS buffer. Mice in groups 3, 4, and 5 were orally administered equal volumes and concentrations of resveratrol, sericin-glycyrrhizic acid complex, and R@SD prepared in Example 1. The dosing frequency for all groups was once daily for 14 consecutive days via gavage, at a dose of 50 mg / kg / mouse and a volume of 100 μL / mouse.

[0127] On day 14 of drug administration, mice in each group were stained with 0.1 wt% sodium fluorescein solution. The sodium fluorescein solution was instilled into the mouse eyes and allowed to remain on the ocular surface for 10 seconds. Excess stain was then rinsed off with physiological saline, and the ocular surface was dried. The mice were then exposed to cobalt blue light under a slit lamp, and photographs were taken after adjusting the magnification. The staining pattern on the cornea of ​​each group of mice was recorded. Figure 9 A. Additionally, eyeballs from each group of mice were taken, fixed, embedded, and stained with hematoxylin and eosin (HE) to obtain... Figure 9 B.

[0128] The average tear secretion (unit: mm) of mice in each group was measured 14 days after drug administration using the phenol red cotton thread test. The procedure for the phenol red cotton thread test was as follows: A phenol red cotton thread was held with tweezers and placed at the outer canthus of the mouse's eye. After 60 seconds, it was removed, and the wetted length (mm) of the phenol red cotton thread was recorded for each group of mice at different time points. Figure 9 D.

[0129] pass Figure 9 According to AF, in a benzalkonium chloride-induced dry eye mouse model, R@SD can significantly reduce ocular surface damage, restore the normal structure of the cornea, and increase tear secretion in dry eye mice, and its therapeutic effect is significantly better than RES.

[0130] Furthermore, the encapsulation effect of the product prepared in Comparative Example 2 on resveratrol was significantly lower than that in Examples 1-5, while the product in Comparative Example 3 appeared blackish-purple, indicating a decrease in the activity and biocompatibility of resveratrol. The encapsulation rate of Comparative Example 4 was too low, resulting in incomplete encapsulation of resveratrol. The nano-formulation in Comparative Example 5 was damaged, but its encapsulation rate 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 nanoformulation of resveratrol for the treatment of dry eye, characterized in that, The complex carrier is formed by interpenetration of resveratrol, sericin and glycyrrhizic acid; The complex carrier encapsulates the resveratrol; The preparation method of the resveratrol nano-preparation comprises the following steps: S1: mixing a sericin aqueous solution with glycyrrhizic acid salt to obtain a complex carrier liquid; the glycyrrhizic acid salt is dipotassium glycyrrhizinate, disodium glycyrrhizinate, potassium glycyrrhizinate or sodium glycyrrhizinate; S2: adding resveratrol into the complex carrier liquid, adjusting the pH of the system to 11.8-12.5, then performing ultrasonic treatment under ice bath conditions for 3-5 min, then adjusting the pH of the system to neutral, and obtaining supernatant after centrifugal treatment; S3: drying the supernatant to obtain the resveratrol nano-preparation.

2. A method of preparing the nanoformulation of resveratrol as claimed in claim 1, characterized by, The preparation method of the resveratrol nano-preparation comprises the following steps: S1: mixing a sericin aqueous solution with glycyrrhizic acid salt to obtain a complex carrier liquid; the glycyrrhizic acid salt is dipotassium glycyrrhizinate, disodium glycyrrhizinate, potassium glycyrrhizinate or sodium glycyrrhizinate; S2: adding resveratrol into the complex carrier liquid, adjusting the pH of the system to 11.8-12.5, then performing ultrasonic treatment under ice bath conditions for 3-5 min, then adjusting the pH of the system to neutral, and obtaining supernatant after centrifugal treatment; S3: drying the supernatant to obtain the resveratrol nano-preparation.

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

4. The method of claim 2, wherein the resveratrol nanoformulation is prepared by, In step S1, the sericin aqueous solution is mixed with the glycyrrhizic acid salt, and then stirring is performed, and the stirring time is 1-2 h.

5. The method of claim 2, wherein the resveratrol nanoformulation is prepared by, In step S2, the centrifugal treatment time is 25-35 min.

6. The method of claim 2, wherein the resveratrol nanoformulation is prepared by, In step S2, the pH of the system is adjusted to neutral by hydrochloric acid.

7. The method of claim 2, wherein the resveratrol nanoformulation is prepared by, In step S3, the supernatant is subjected to freeze-drying treatment, and the freezing temperature is-40℃ to-50℃.

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