Oral preparation for relieving xerophthalmia

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an oral preparation for relieving xerophthalmia, a preparation method of the oral preparation comprises the following steps: S1, dispersing soybean phospholipid in MCT to obtain a composite embedding medium, dispersing astaxanthin, vitamin E and a food-grade emulsifier in the composite embedding medium, and performing high-pressure homogenization at 120-150MPa to obtain astaxanthin nanoemulsion; s2, performing wet granulation and enteric coating on the chitosan modified omega-3 fatty acid nanoparticle powder, peony seed oil and microcrystalline cellulose to obtain omega-3 fatty acid pellets; the preparation method of the chitosan modified omega-3 fatty acid nanoparticle powder comprises the following steps: dissolving omega-3 fatty acid and soybean lecithin in an oil phase solvent, and carrying out ultrasonic dispersion to obtain an oil phase; dissolving chitosan in an acetic acid aqueous solution, and adding a cross-linking agent STPP to obtain a water phase; and injecting the oil phase into the water phase, stirring, carrying out high-pressure homogenization at 120-150MPa, standing, centrifuging, collecting nanoparticles, adding a freeze-drying protective agent, and freeze-drying, thereby obtaining the product. And S3, filling soft capsules with the astaxanthin nanoemulsion and the omega-3 fatty acid pellets.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to an oral preparation for relieving dry eye disease. Background Art

[0002] Dry eye is a chronic ocular surface disease caused by multiple factors. It is caused by abnormalities in the quality, quantity, and dynamics of tears, resulting in unstable tear film or imbalance of the ocular surface microenvironment. It may be accompanied by ocular surface inflammatory reactions, tissue damage, and nerve abnormalities, causing various discomfort symptoms and / or visual function disorders in the eyes. Its pathological mechanism is closely related to oxidative stress, decreased tear film stability, and inflammatory reactions. In patients with dry eye disease, meibomian gland dysfunction (MGD) leads to an increased rate of tear evaporation (such as insufficient lipid secretion or compositional changes), and there are ocular surface inflammation and oxidative stress. For example, pro-inflammatory factor cascades, IL-1β, IL-6, and TNF-α activate the NF-κB pathway, inducing apoptosis of corneal epithelial cells. The accumulation of reactive oxygen species (ROS) leads to oxidative damage to the cornea.

[0003] Studies have shown that the lipid layer in tears is mainly secreted by the meibomian glands. Omega-3 fatty acids can regulate meibomian gland function, improve the quality of the tear film, and conduct anti-inflammatory regulation, such as inhibiting the COX-2 / PGE2 pathway and reducing the level of IL-17. Omega-3 fatty acids also promote the normalization of meibomian gland secretion. As the strongest natural antioxidant, astaxanthin can play a role in transmembrane antioxidant, quench corneal mitochondrial ROS, scavenge free radicals, and inhibit inflammatory factors (such as IL6 and TNFα). Astaxanthin can also inhibit the nuclear translocation of NF-κB, form a double blockade with Ω-3 fatty acids, reduce the expression of MMP-9, and reduce corneal epithelial erosion. Ω-3 regulates the systemic inflammatory background, while astaxanthin focuses on local oxidative damage.

[0004] However, when combining astaxanthin and Ω-3 fatty acids into an oral preparation product, the following problems exist: low absorption and bioavailability, among which Ω-3 fatty acids rely on bile emulsification and have a low absorption rate under the action of gastric acid. Astaxanthin has very poor stability, is sensitive to light and heat, and is easily oxidized and inactivated in advance during the processing, production, and storage processes. In addition, the Ω-3 fatty acids in the oral preparation have poor targeting and low local concentration, making it necessary for patients to take frequent oral doses. It is difficult for patients to adhere to the oral preparation taken 2-3 times a day for a long time, and the compliance is poor.

[0005] In view of the above problems, it is necessary to optimize the formulation or delivery technology to improve the therapeutic effect of the drug. Summary of the Invention

[0006] (1) Technical Problems to be Solved

[0007] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides an oral preparation for relieving dry eye syndrome. By encapsulating natural astaxanthin to form a nanoemulsion, preparing coated pellets with chitosan-modified Ω-3 fatty acids and auxiliary materials, and filling the nanoemulsion and coated pellets into soft capsules, the stability and bioavailability of astaxanthin and Ω-3 fatty acids are greatly improved. This not only reduces the frequency of gavage but also shows a significant improvement effect on dry eye syndrome.

[0008] (II) Technical Solution

[0009] In the first aspect, the present invention provides a preparation method of an oral preparation for relieving dry eye syndrome, which includes:

[0010] S1. Prepare astaxanthin nanoemulsion

[0011] Disperse soy lecithin in medium-chain triglyceride (MCT) to obtain a composite embedding agent. Disperse astaxanthin, vitamin E, and food-grade emulsifier in the composite embedding agent, and perform high-pressure homogenization treatment 3-6 times at 120-150 MPa (high-pressure homogenization is used to promote emulsification and control the particle size of the nanoemulsion) to obtain astaxanthin nanoemulsion;

[0012] S2. Prepare Ω-3 fatty acid pellets

[0013] Prepare wet granules from the powder of chitosan-modified Ω-3 fatty acid nanoparticles, peony seed oil, and microcrystalline cellulose, and perform fluidized bed coating with an enteric coating material to obtain Ω-3 fatty acid pellets;

[0014] The preparation method of the powder of chitosan-modified Ω-3 fatty acid nanoparticles is as follows: Dissolve Ω-3 fatty acid and soy lecithin in an oil-phase solvent, and perform ultrasonic dispersion to obtain an oil phase; dissolve chitosan in an acetic acid aqueous solution, add a sodium tripolyphosphate crosslinking agent to obtain an aqueous phase; inject the oil phase into the aqueous phase, stir to form a primary emulsion, perform high-pressure homogenization treatment 3-6 times at 120-150 MPa (high-pressure homogenization is used to promote crosslinking and control the particle size of the nanoparticles) to form nanoparticles, centrifuge, collect the nanoparticles, add a freeze-drying protectant, and freeze-dry to obtain;

[0015] S3. Fill the astaxanthin nanoemulsion and Ω-3 fatty acid pellets into soft capsules.

[0016] According to a preferred embodiment of the present invention, in S1, the astaxanthin is astaxanthin derived from Haematococcus pluvialis; the food-grade emulsifier is Tween 80.

[0017] According to a preferred embodiment of the present invention, in S1, the temperature of the high-pressure homogenization treatment is 25±2°C to avoid isomerization of astaxanthin caused by high temperature.

[0018] According to a preferred embodiment of the present invention, in S1, the mass ratio of astaxanthin, soy lecithin, medium-chain triglyceride MCT, vitamin E, and Tween 80 is 15-20:15-20:400-600:1-2:2-5.

[0019] According to a preferred embodiment of the present invention, in S1, first disperse soy lecithin in MCT, stir at 40 °C for 30-40 min, cool to 25±2 °C, then add astaxanthin, vitamin E, and Tween 80, and circulate 4 times under 135 MPa using a high-pressure homogenizer. The particle size of the astaxanthin nanoemulsion is controlled to be 80-120 nm, and PDI < 0.2.

[0020] According to a preferred embodiment of the present invention, in S2, the Ω-3 fatty acid is sourced from deep-sea fish oil or algal oil, where EPA:DHA = 2:1.

[0021] According to a preferred embodiment of the present invention, in S2, the mass ratio of chitosan-modified Ω-3 fatty acid nanoparticle powder, peony seed oil, and microcrystalline cellulose MCC is 25-35:5-7:60-70; when wet granulating, use HPMC with a mass concentration of 5%±1% as the binder, and the dosage of the binder is 30-35% of the total mass of microcrystalline cellulose MCC and peony seed oil.

[0022] Preferably, wet granulation is carried out by fluidized bed granulation, with an inlet air temperature of 40 °C, a material temperature ≤ 35 °C, and an end moisture content ≤ 3%.

[0023] According to a preferred embodiment of the present invention, in S2, the enteric coating material is L100-55 or Eudragit L30D-55, and the enteric coating material is 5-10% of the mass of the granules obtained by wet granulation. Preferably, the parameters of fluidized bed coating are: spraying rate is 2.5-4 mL / min, atomization pressure is 1.2 bar, inlet air temperature is 42-58 °C, and outlet air temperature is 32-34 °C.

[0024] According to a preferred embodiment of the present invention, in S2, when preparing chitosan-modified Ω-3 fatty acid nanoparticle powder, use chitosan with a degree of deacetylation of 85-95%. Among them, the oil phase solvent is food-grade dichloromethane; among them, the mass ratio of Ω-3 fatty acid, soy lecithin, chitosan, and sodium tripolyphosphate is 15-25:8-12:4-6:0.5-1. Among them, chitosan is dissolved in about 1% acetic acid aqueous solution (pH = 4.5-5.5 is acceptable) to obtain a chitosan solution of 5-30 g / L. Chitosans with different degrees of deacetylation and molecular weights have different solubilities in acetic acid aqueous solution. Preferably, prepare a chitosan solution with a concentration 20-30% lower than the saturation concentration.

[0025] According to a preferred embodiment of the present invention, in S2, colostrum is subjected to 4 times of high-pressure homogenization treatment at 135 MPa to form nanoparticles. The nanoparticles are collected and a freeze-drying protectant is added. The freeze-drying protectant is mannitol or trehalose, and the addition amount is 5-8% of the mass of the freeze-dried nanoparticles.

[0026] According to a preferred embodiment of the present invention, in S3, astaxanthin nanoemulsion and Ω-3 fatty acid pellets are mixed. After adding 0.4-0.8% of xanthan gum for thickening, soft capsules are filled.

[0027] In a second aspect, the present invention provides an oral preparation for relieving dry eye, which is prepared by the method of any one of the above embodiments; preferably, each soft capsule contains 6-12 mg of astaxanthin, 300-600 mg of EPA, 150-300 mg of DHA, 150-300 mg of peony seed oil, 0.4-0.6 mg of vitamin E, and 200-350 mg of medium-chain triglyceride MCT.

[0028] (III) Beneficial effects

[0029] The present invention adopts the high-pressure homogenization method to disperse astaxanthin into nanoemulsion droplets of about 80-120 nm, reduce the light scattering effect, and reduce the light sensitivity of astaxanthin; uses a composite embedding agent (soybean phospholipid + MCT): the phospholipid bilayer wraps astaxanthin to isolate oxygen and moisture; MCT is used as a fat-soluble matrix to inhibit the isomerization of astaxanthin, so that the retention rate of astaxanthin is ≥95%. When embedding astaxanthin, vitamin E is added to extend the oxidation half-life of astaxanthin. The nano-sized emulsion droplets are beneficial for absorption through the endocytosis of intestinal M cells, avoiding the dependence on bile emulsification, and effectively improving the bioavailability; among them, MCT is an oil carrier, and MCT can be quickly metabolized into monoglyceride, and can also promote the lymphatic transport of astaxanthin.

[0030] In order to solve the problems of Ω-3 gastric acid degradation and low absorption rate, the present invention uses chitosan to modify Ω-3 nanoparticles. Chitosan has cationic properties and can electrostatically adsorb with the negative charge of intestinal mucus, effectively extending the retention time to 8-12 h, increasing the absorption rate, and reducing the frequency of taking medicine (it is only necessary to administer once a day, and the compliance of administration is good). Using sodium polyphosphate (TPP) as a cross-linking agent, a pH-sensitive gel can be formed, which remains intact in gastric juice (pH 1.2) and quickly disintegrates in intestinal juice (pH 6.8), greatly improving the release efficiency in intestinal juice. The Ω-3 fatty acid pellets are coated with enteric coating materials and dissolve at pH≥5.5, accurately delivered to the duodenum, effectively reducing the damage of gastric acid to astaxanthin and Ω-3 fatty acids.

[0031] The present invention prepares an oral preparation for relieving dry eye. The chitosan-modified Ω-3 fatty acid nanoparticles can be absorbed through Peyer's patches to increase the local concentration of Ω-3, and lymphatic targeting can enhance the drug concentration at the lesion site. At the same time, peony seed oil rich in α-linolenic acid (ALA, with a content of over 40%) is used to synergistically enhance the effect. It is rich in α-linolenic acid (ALA), which can be continuously converted into EPA / DHA in the body, reducing the demand for exogenous Ω-3 and playing a role in continuously supplying and replenishing an appropriate amount of Ω-3. The α-linolenic acid in peony seed oil slows down the aging of retinal cells through antioxidant effects and has a certain preventive effect on adult vision degradation and age-related macular degeneration.

[0032] Among them, astaxanthin is a nanoemulsion encapsulated by enteric coating, and its release rate is relatively fast; the Ω-3 fatty acid pellets include an outer enteric coating and an inner chitosan aggregation, and its release rate is slow (the sustained release of Ω-3 pellets can reach 12 h). The synergistic effect of rapid release and sustained release fills the technical gap of long-acting sustained release of oral preparations for dry eye. The astaxanthin nanoemulsion and Ω-3 fatty acid pellets are filled into soft capsules (HPMC material) to isolate oxygen and ultraviolet rays, effectively improving the stability of astaxanthin and Ω-3 fatty acids during storage, transportation, sales, etc.

[0033] The present invention can reduce the administration frequency from the currently common 3 times / day to 1 time / day. MCT and peony seed oil (peony seed oil has a unique fragrance) can also mask the fishy smell of astaxanthin or DHA; peony seed oil is rich in unsaturated fatty acids, especially α-linolenic acid (ALA), which helps to maintain cardiovascular health, and its natural antioxidant properties may also help to protect sensitive components from oxidative damage. Compared with long-chain fatty acids, MCT is also easier to digest, reducing the risk of gastrointestinal discomfort caused by the intake of certain supplements. Both chitosan and peony seed oil are of natural origin, reducing immunogenicity. Chitosan can select different degrees of deacetylation (such as 85%-95%) to obtain nanoparticles with different crosslinking densities, and can customize different release curves of Ω-3 in the body.

[0034] In summary, the solution of the present invention systematically solves the key technical bottlenecks of oral preparations for dry eye through multi-dimensional innovations in nanotechnology, targeted modification, and pharmaceutical engineering. Description of the Drawings

[0035] Figure 1 Comparison of the retention rates of astaxanthin in the Ω-3 / astaxanthin soft capsules of Examples 1-4 and Comparative Examples 1-2 after being placed at high temperature in the laboratory for 48 h (a) and under light conditions for 48 h (b).

[0036] Figure 2 Comparison of the concentrations of Ω-3 in the plasma and lymph fluid collected after 24 h of oral administration of the Ω-3 / astaxanthin soft capsules of Examples 1-4 and Comparative Examples 1-2 to SD rats.

[0037] Figure 3 Comparison of the concentrations of astaxanthin in plasma and lymph fluid collected 24 h after intragastric administration of Ω-3 / astaxanthin soft capsules in Examples 1-4 and Comparative Examples 1-2 to SD rats.

[0038] Figure 4 Levels of MDA (malondialdehyde) and SOD (superoxide dismutase) in tears 1 week after intragastric administration of Ω-3 / astaxanthin soft capsules in Example 1 and Comparative Examples 1-2 to SD rats.

[0039] Figure 5 mRNA expression levels of IL-1β, TNF-α, and MMP-9 in corneal tissues of rats 1 week after intragastric administration of Ω-3 / astaxanthin soft capsules in Example 1 and Comparative Examples 1-2 to SD rats. Detailed implementation manners

[0040] For better explaining the present invention for easy understanding, the present invention will be described in detail below in conjunction with the accompanying drawings through specific implementation manners.

[0041] Example 1

[0042] This example provides a preparation method of an oral preparation for relieving dry eye, which includes:

[0043] (1) 20 g of soybean phospholipids are dispersed in 500 g of MCT (medium-chain triglycerides), stirred at 40 °C for 30 min to obtain a composite embedding agent; after cooling to 25 °C, 15 g of astaxanthin, 1.0 g of vitamin E, and 5 g of Tween 80 are dispersed in the composite embedding agent, and subjected to 4 times of high-pressure homogenization treatment at 135 MPa, and the internal temperature is kept not exceeding 26 °C during the homogenization process to obtain astaxanthin nanoemulsion. After detection, the particle size of the nanoemulsion is 80-120 nm, and PDI < 0.2. Among them, the astaxanthin is astaxanthin derived from Haematococcus pluvialis, and the content is 90%.

[0044] (2) 21 g of Ω-3 fatty acids (EPA:DHA = 2:1) and 10 g of soybean phospholipids are dissolved in food-grade dichloromethane, and ultrasonically dispersed for 20 min to obtain an oil phase. 5.5 g of chitosan (degree of deacetylation ≥ 90%) is dissolved in an acetic acid aqueous solution with pH = 5 to obtain a 7.5 g / L chitosan solution, and 0.9 g of sodium tripolyphosphate is added, stirred evenly to obtain an aqueous phase. The oil phase is injected into the aqueous phase (the volume of the aqueous phase is 4 times that of the oil phase), stirred to form a primary emulsion, and the primary emulsion is subjected to 4 times of high-pressure homogenization treatment at 135 MPa to form nanoparticles, centrifuged at 8000 rpm for 10 min, the nanoparticles are collected, 5% mannitol is added, and freeze-dried to obtain chitosan-modified Ω-3 fatty acid nanoparticle powder with a particle size of 50-200 nm.

[0045] (3) Mix the chitosan-modified Ω-3 fatty acid nanoparticle powder prepared in step (2), peony seed oil, and microcrystalline cellulose in a mass ratio of 35:6:60, and perform fluidized bed wet granulation using an aqueous HPMC solution with a mass concentration of 5% as the binder. During the granulation process, the inlet air temperature is 40 °C, and the material temperature ≤ 35 °C. After granulation, pellets with a water content ≤ 3% are obtained. Then, use L100-55 enteric coating material to coat the pellets. After the coating is completed, Ω-3 fatty acid pellets are obtained.

[0046] The coating solution formulation (per 100 g) contains: 12% L100-55 enteric coating material, 1.5% triethyl citrate (TEC), 3.0% talc powder, 83.5% (isopropanol: water v / v = 3:7). The weight gain after coating is 8.7%. The parameters for fluidized bed coating are: spraying rate is 3 mL / min, inlet air temperature is 44 °C, atomization pressure is 1.2 bar, and outlet air temperature is 33 °C.

[0047] (4) Mix the astaxanthin nanoemulsion in step (1) and the Ω-3 fatty acid pellets obtained in step (3), add 0.5% xanthan gum after mixing, thicken, and pour into soft capsules made of HPMC material to obtain the finished soft capsule product. Each soft capsule contains 8 mg of astaxanthin, 400 mg of EPA, 200 mg of DHA, 186 mg of peony seed oil, 0.53 mg of vitamin E, and 266 mg of medium-chain triglyceride MCT.

[0048] Example 2

[0049] The difference between this example and Example 1 is that step (1) is as follows: Take 15 g of soy lecithin and disperse it in 450 g of MCT (medium-chain triglyceride), stir at 40 °C for 30 min to obtain a composite embedding agent; after cooling to 25 °C, disperse 15 g of astaxanthin, 0.8 g of vitamin E, and 4 g of Tween 80 in this composite embedding agent, and perform 4 times of high-pressure homogenization treatment at 135 MPa. During the homogenization process, keep the internal temperature not exceeding 26 °C to obtain astaxanthin nanoemulsion. After testing, the particle size of the nanoemulsion is 80 - 120 nm, and PDI < 0.2. Among them, the astaxanthin is astaxanthin from Haematococcus pluvialis, with a content of 90%. Other steps and conditions refer to Example 1. Finally, each soft capsule prepared in this example contains 8.5 mg of astaxanthin, 425 mg of EPA, 213 mg of DHA, 198 mg of peony seed oil, 0.45 mg of vitamin E, and 255 mg of MCT.

[0050] Example 3

[0051] The difference between this example and Example 1 is that step (2) is as follows:

[0052] Dissolve 24 g of Ω-3 fatty acids (EPA:DHA = 2:1) and 12 g of soybean phospholipids in food-grade dichloromethane, and ultrasonically disperse for 20 min to obtain an oil phase. Dissolve 6 g of chitosan (degree of deacetylation ≥ 85%) in an acetic acid aqueous solution with pH = 5, add 1 g of sodium tripolyphosphate, and stir evenly to obtain an aqueous phase. Inject the oil phase into the aqueous phase (the volume of the aqueous phase is 4 times that of the oil phase), stir to form a primary emulsion, perform 4 times of high-pressure homogenization treatment on the primary emulsion at 135 MPa to form nanoparticles, centrifuge at 8000 rpm for 10 min, collect the nanoparticles, add 5% mannitol, and freeze-dry to obtain chitosan-modified Ω-3 fatty acid nanoparticle powder with a particle size of 50 - 200 nm. Other steps and conditions refer to Example 1.

[0053] Finally, each soft capsule prepared in this example contains 8.2 mg of astaxanthin, 455 mg of EPA, 228 mg of DHA, 211 mg of peony seed oil, 0.54 mg of vitamin E, and 274 mg of MCT.

[0054] Example 4

[0055] The difference between this example and Example 1 is that step (3) is as follows:

[0056] Mix the chitosan-modified Ω-3 fatty acid nanoparticle powder, peony seed oil, and microcrystalline cellulose according to a mass ratio of 30:5:65, and perform fluidized bed wet granulation with an aqueous solution of HPMC with a mass concentration of 5% as the binder. During the granulation process, the inlet air temperature is 40 °C, and the material temperature ≤ 35 °C. After granulation, obtain granulated materials with a moisture content ≤ 3%. Then, use L100-55 enteric coating material to coat the granulated materials. The coating solution formulation refers to Example 1. After coating, the weight gain is 9.1% to obtain Ω-3 fatty acid pellets. Other steps and conditions refer to Example 1.

[0057] Finally, each soft capsule prepared in this example contains 8.1 mg of astaxanthin, 400 mg of EPA, 200 mg of DHA, 183 mg of peony seed oil, 0.54 mg of vitamin E, and 271 mg of medium-chain triglyceride MCT.

[0058] Comparative Example 1

[0059] The main difference between this comparative example and Example 1 is that it does not include the astaxanthin embedding step in step (1). Instead, 15 g of astaxanthin and 1.0 g of vitamin E are directly dispersed in 520 g of corn germ oil, and then mixed with the Ω-3 fatty acid pellets obtained in step (3). After mixing, 0.5% xanthan gum is added and stirred to thicken, and then filled into soft capsules made of HPMC material to obtain the finished soft capsule product. The content of astaxanthin, EPA, DHA, and peony seed oil in each soft capsule is prepared according to the same specifications as in Example 1, with the error controlled within ±5%.

[0060] Comparative Example 2

[0061] The main difference between this comparative example and Example 1 is that it does not include the modification step of chitosan. The Ω-3 fatty acids (EPA:DHA = 2:1), soy lecithin, peony seed oil, and astaxanthin nanoemulsion are directly mixed, 0.5% xanthan gum is added and stirred to thicken, and then filled into soft capsules made of HPMC material to obtain the finished soft capsule product. The content of astaxanthin, EPA, DHA, peony seed oil, vitamin E, and MCT in each soft capsule is prepared according to the same specifications as in Example 1, with the error controlled within ±5%.

[0062] The stability, bioavailability, ROS scavenging, anti-inflammatory and other effects of the soft capsules of the above examples were measured, and the measurement methods and results are as follows. It should be noted that for the convenience of the experimental operation of gavage for rats, the soft capsules in each example and comparative example were miniaturized to prepare mini soft capsules with a diameter of <2 mm (suitable for experimental rats), and the content of a single mini soft capsule is 1 / 10 of the content in Examples 1-4 and Comparative Examples 1-2.

[0063] 1. Stability test

[0064] Take 3 soft capsules each from Examples 1-4 and Comparative Examples 1-2 and place them in the dark and sealed at 40 °C for 48 h, and test the retention rate of astaxanthin compared to the initial value after placement. Take the average of 3 tests. Take 3 soft capsules each from Examples 1-4 and Comparative Examples 1-2 and place them in the light (ultraviolet + visible light) at 4500 lux and sealed at 25 °C for 48 h, and test the retention rate of astaxanthin compared to the initial value after placement. Take the average of 3 tests. The test results are as Figure 1 shown. After the soft capsules of Examples 1-4 were placed at high temperature and under light for 48 h, the astaxanthin contained in them still had a high retention rate. In Comparative Example 1, the astaxanthin dispersed in corn germ oil had a lower retention rate. In Comparative Example 2, the same astaxanthin embedding treatment as in Example 1 was used, and the astaxanthin retention rate was close to that of Example 1.

[0065] 2. Select 18 male SD rats with similar body weights and activity levels from the purchased male SD rats. Their body weights are between 460 g and 480 g, and they are randomly divided into 6 groups of 3 rats each. Each group of rats is orally administered 2 micro-soft capsules prepared according to Examples 1-4 and Comparative Examples 1-2 per day per rat for 2 consecutive days. The administration time is from 8 to 9 in the morning, and the rats are fed normal rat feed and drinking water. 24 hours after the end of the second administration, the plasma and axillary lymph fluid of each group of rats are extracted, and the relative concentrations of astaxanthin and Ω-3 in the plasma and lymph fluid are detected (with the average plasma concentration after administering the micro-soft capsules prepared according to Example 1 as the reference).

[0066] Sampling method for axillary lymph fluid: Ensure that the rat is in an appropriate anesthetic state, shave or cut short the hair in the axillary area, and clean the skin with a disinfectant; locate the lymph nodes and find the position of the axillary lymph nodes by palpation; fine needle aspiration (FNA), use a sterile fine needle connected to a syringe. Carefully insert the needle into the lymph node and slowly pull the syringe piston to aspirate a small amount of lymph fluid.

[0067] Sampling method for plasma: Collect blood from the tail vein. Gently heat the rat's tail (for example, using a warm water bag) to dilate the blood vessels in the tail. After disinfecting the tail, gently insert a fine needle along the direction of the tail vein and slowly aspirate the required amount of blood.

[0068] The test results are as Figures 2-3 shown. The figure shows that after administering the soft capsules twice continuously and 24 hours after the end of the last administration, the detected distribution concentration of Ω-3 in the plasma is greater than that in the lymph fluid, while the distribution concentration of astaxanthin in the lymph fluid is greater than that in the plasma. As Figure 2 shown, for the SD rats administered with the soft capsules of Examples 1-4, the distribution concentrations of Ω-3 in their plasma and lymph fluid are significantly higher than those of Comparative Example 2, but there is no obvious difference from Comparative Example 1. As Figure 3 shown, for the SD rats administered with the soft capsules of Examples 1-4, the distribution concentrations of astaxanthin in their plasma and lymph fluid are significantly higher than those of Comparative Example 1, but there is no obvious difference from Comparative Example 2.

[0069] 3. Establish a dry eye model for SD rats. After the model is successfully established, continuously administer the SD rats for one week according to the above method. 24 hours after the end of the last administration, sample the tear fluid of each group of rats, and use ELISA to detect the relative levels of MDA (malondialdehyde) and SOD (superoxide dismutase) in the tear fluid (with healthy blank control rats as the reference).

[0070] Dry eye model establishment method: Use 0.1% benzalkonium chloride solution, instill 1 drop into each eye every day for 14 days, and the model is successfully established.

[0071] Administration groups (3 rats in each group, taking the average value):

[0072] Healthy blank control group: Each rat was intragastrically administered an equal amount of normal saline each time.

[0073] Model control group: After successful modeling, each rat was intragastrically administered an equal amount of normal saline each time.

[0074] Experimental group: After successful modeling, each rat was intragastrically administered 2 micro-soft capsules prepared according to Example 1 and Comparative Examples 1-2 per day per rat, and the administration was continued for 7 consecutive days.

[0075] The rats in each of the above groups were fed normal rat feed and drinking water.

[0076] Capillary method was used for sampling rat tears: A micro-capillary or a specially designed tear collection capillary was used. The operation steps were as follows: Gently pull the eyelid of the rat to keep the eye open, gently place the capillary at the corner of the eye, and try to avoid touching the surface of the eyeball to avoid discomfort or injury. Wait for the capillary to absorb tears naturally. This process may take several minutes to collect a sufficient sample volume.

[0077] The test results are as Figure 4 shown. Compared with healthy rats, the MDA level of model rats increased significantly, while the SOD level decreased significantly. The MDA in the tears of model rats was about 2 times that of healthy rats, and the SOD level in the tears of model rats was about 40% of that of healthy rats. After intragastric administration of the micro-soft capsules (with the inclusion content of 1 / 10) prepared according to the methods of Example 1 and Comparative Examples 1-2, the degree of decrease in MDA in the tears of the rats in the group corresponding to Example 1 was greater than that in the group corresponding to Comparative Examples 1-2, and the degree of increase in SOD was greater than that in the group corresponding to Comparative Examples 1-2. This shows that the soft capsule prepared in Example 1 of the present invention can improve dry eye faster and more effectively and reduce the degree of oxidative stress.

[0078] 4. Intragastrically administer the soft capsules continuously for 1 week according to the above method, take the corneal tissues of the rats in each group, and analyze the mRNA expression levels of IL-1β, TNF-α, and MMP-9 in the corneal tissues by qPCR.

[0079] Steps for taking the cornea: Place the anesthetized rat on a stable platform, and a foam pad or other soft material can be used to support the body to keep the head stable. Gently pull open the upper and lower eyelids to expose the eyeball. A special eyelid retractor can be used to keep the eye open. Use micro-scissors to carefully cut the conjunctiva and the surrounding fascia tissue to better approach the cornea. Use micro-scissors or a corneal trephine to make a precise cut around the corneal edge. Gently lift the cut corneal edge and use forceps or a curette to completely remove the cornea. Once the cornea is removed, immediately place it in a pre-cooled preservation solution (such as PBS buffer) for standby.

[0080] The test results are as Figure 5As shown. Compared with healthy rats, the expression level of IL-1β in the corneal tissue of model rats is about 4 times that of healthy rats, TNF-α is about 5 times that of healthy rats, and MMP-9 is about 7 times that of healthy rats. In the rat dry eye model, the levels of IL-1β, TNF-α and MMP-9 in the corneal tissue will increase significantly, reflecting the inflammatory state of the ocular surface and potential tissue damage. After continuous administration of the micro soft capsule for 1 week, in the rats of the corresponding group of Example 1, the relative expression levels of IL-1β, TNF-α and MMP-9 in the corneal tissue decreased significantly. The relevant indicators of the rats in the corresponding groups of Comparative Examples 1-2 also decreased, but the degree of decrease was less than that of the corresponding group of Example 1. This shows that the soft capsule of the present invention has a positive therapeutic effect on dry eye, especially in reducing inflammation, protecting the ocular surface and promoting the recovery of the eye to health.

[0081] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements, or in the case where the technical features in the above embodiments do not conflict with each other, can be combined in the manner described in the embodiments, and these modifications, replacements or combinations do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an oral preparation for relieving dry eye, characterized in that: The steps include: S1. Preparation of astaxanthin nanoemulsion Dispersing soybean lecithin in medium chain triglyceride (MCT) to obtain a composite embedding agent, dispersing astaxanthin, vitamin E, and a food-grade emulsifier in the composite embedding agent, and performing high pressure homogenization treatment at 120-150 MPa for 3-6 times to obtain an astaxanthin nanoemulsion; S2. Preparation of omega-3 fatty acid pellets Chitosan-modified omega-3 fatty acid nanoparticle powder, peony seed oil and microcrystalline cellulose are wet-granulated, and fluidized bed coating is performed, and the coating material is an enteric coating material to obtain omega-3 fatty acid micropellets; The preparation method of chitosan-modified omega-3 fatty acid nanoparticle powder is as follows: dissolving omega-3 fatty acid and soybean lecithin in an oil phase solvent, and performing ultrasonic dispersion to obtain an oil phase; dissolving chitosan in an acetic acid aqueous solution, and adding a sodium tripolyphosphate crosslinking agent to obtain an aqueous phase; injecting the oil phase into the aqueous phase, and stirring to form colostrum; performing high-pressure homogenization treatment on the colostrum at 120-150 MPa for 3-6 times to form nanoparticles; collecting the nanoparticles by centrifugation; adding a freeze-drying protective agent, and freeze-drying to obtain the powder; S3, filling the astaxanthin nanoemulsion and omega-3 fatty acid microcapsules into soft capsules.

2. The preparation method according to claim 1, characterized in that: In S1, the temperature of the high pressure homogenization treatment is 25±2°C.

3. The preparation method according to claim 1, characterized in that: In S1, the mass ratio of astaxanthin, soybean lecithin, medium chain triglyceride MCT, vitamin E, and Tween 80 is 15-20:15-20:400-600:1-2:2-5.

4. The preparation method according to claim 2 or 3, characterized in that: In S1, soybean lecithin was first dispersed in MCT, stirred at 40°C for 30-40 min, cooled to 25±2°C, and then astaxanthin, vitamin E and Tween 80 were added. A high-pressure homogenizer was used to circulate 4 times at 135 MPa. The particle size of astaxanthin nanoemulsion was controlled to 80-120 nm, and PDI was <0.

2.

5. The preparation method according to claim 1, characterized in that: In S2, the omega-3 fatty acids are derived from deep-sea fish oil or algae oil, wherein EPA:DHA=2:

1.

6. The preparation method according to claim 1, characterized in that: In S2, the mass ratio of chitosan-modified omega-3 fatty acid nanoparticle powder, peony seed oil and microcrystalline cellulose MCC is 25-35:5-7:60-70; during wet granulation, HPMC with a mass concentration of 5%±1% is used as a binder.

7. The preparation method according to claim 1, characterized in that: In S2, the moisture content of the granules produced by wet granulation is ≤3%; the enteric coating material is L100-55 or Eudragit L30D-55, the enteric coating material is 5-10% of the mass of the granules obtained by wet granulation.

8. The preparation method according to claim 1, characterized in that: In S2, when preparing chitosan-modified Ω-3 fatty acid nanoparticle powder, chitosan with a deacetylation degree of 85-95% is used, wherein the oil phase solvent is food-grade dichloromethane; wherein the mass ratio of Ω-3 fatty acid, soybean lecithin, chitosan, and sodium tripolyphosphate is 15-25:8-12:4-6:0.5-1.

9. The preparation method according to claim 1, characterized in that: In S3, astaxanthin nanoemulsion and omega-3 fatty acid pellets are mixed, 0.4-0.8% xanthan gum is added for thickening, and then soft capsules are filled.

10. An oral preparation for relieving dry eye, characterized in that: The method is prepared by any one of claims 1 to 9.

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