A method for preparing a functional lutein complex and application for relieving dry eye

Abstract

The application discloses a functional lutein compound preparation method and application for relieving dry eye syndrome and belongs to the technical field of medicines. The application provides a lutein compound, wherein lutein is primarily embedded by phycocyanin, trihydroxybenzoic acid is grafted onto yeast beta glucan through esterification, and modified yeast beta glucan and chitosan are further self-assembled through electrostatic interaction to form a compound with improved hydrophobic nutrients; the application improves the stability of the physical and chemical properties of lutein, can realize gastric acid protection of lutein, improves the concentration of lutein in blood and eyeballs, relieves the dry eye syndrome of mice, the embedding protection mode of the method constructs a functional characteristic nano-carrier system and fully improves the absorption and utilization of natural nutrients.

Description

Technical Field

[0001] This invention relates to a method for preparing a functional lutein complex and its application in relieving dry eye syndrome, belonging to the field of pharmaceutical technology. Background Technology

[0002] Lutein is a carotenoid that is fat-soluble and insoluble in water. It is a natural active nutrient widely found in fruits, vegetables, flowers, and algae, and has color-developing properties. Since the human body cannot synthesize lutein, it must be obtained through diet or supplementation. Lutein contains abundant conjugated double bonds and hydroxyl groups, giving it excellent free radical scavenging capabilities. Lutein possesses various biological activities such as antioxidant, anti-inflammatory, anti-lipid peroxidation, anti-aging, eye protection, and prevention of age-related macular degeneration. In the human body, lutein is mainly distributed in the macula and the entire retina, playing a vital role in the development and maintenance of normal function of the visual system.

[0003] However, lutein's low water solubility leads to poor dispersion in polar solvents, severely limiting its absorption and utilization. Furthermore, its strong antioxidant properties make it easily oxidized and decomposed in vivo. Therefore, improving the solubility, dispersibility, and stability of lutein, and enhancing its intestinal absorption and utilization, is crucial.

[0004] Currently, the protection, delivery, and controlled release of nutritional functional factors are mainly achieved by constructing delivery systems. Common types of delivery systems include emulsions, complexes, Pickering emulsions, liposomes, solid lipid complexes, and hydrogels. In existing technologies, lutein-encapsulated complexes have low stability in the gastrointestinal environment, releasing lutein before intestinal absorption, resulting in a large amount of lutein failing to enter the bloodstream and exert its function.

[0005] There is an urgent need for a complex that can target and release lutein to improve the bioavailability of lutein in the intestine and serum, so that the activity of the natural active substance can be fully exerted. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art by utilizing the electrostatic layer-by-layer self-assembly between phycocyanin, chitosan, and yeast β-glucan to protect lutein from the extreme environment of gastric acid, promote the concentrated accumulation and release of lutein in the intestine, and enhance the absorption and utilization of lutein; and by using yeast β-glucan to specifically target the resident immune cells on Pell's plaques in the intestine to enhance the absorption and utilization of lutein in the intestine, thereby enhancing the eye-protecting function of lutein.

[0007] This invention provides a method for preparing a functional lutein complex, comprising the following steps: S1. Dissolve tricarboxyphenylboronic acid in a 90-98% dimethyl sulfoxide solution to achieve a final concentration of 10-30 mg / mL, and stir until fully dissolved; then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at a concentration of 20-50 mg / mL and 4-dimethylaminopyridine at a concentration of 20-30 mg / mL, and stir for 3-5 hours to obtain an activated tricarboxyphenylboronic acid solution; S2. Dissolve yeast β-glucan thoroughly in dimethyl sulfoxide solution to achieve a final concentration of 2-5 mg / mL; then add it dropwise to the tricarboxyphenylboronic acid activation solution described in step S1, and gradually heat to 40-60°C. Stir the reaction at 40-60°C for 30-50 h; after the reaction is complete, dialyze for 2-3 days, and then freeze-dry to obtain the yeast β-glucan-tricarboxyphenylboronic acid complex. S3. Dissolve phycocyanin fully in deionized water to achieve a final concentration of 2-4 mg / mL; then add a lutein ethanol solution with a concentration of 5-20 mg / mL, and shear and break it to obtain a sheared solution of phycocyanin and lutein. S4. Preparation of lutein complex: A yeast β-glucan-tricarboxyphenylboronic acid complex solution with a final concentration of 10-30 mg / mL and a chitosan solution with a final concentration of 2-4 mg / mL were added to the shearing solution of phycocyanin and lutein in step S3 for layer-by-layer self-assembly. The reaction was stirred for 12-24 h. After the reaction was completed, the mixture was freeze-dried to obtain the lutein complex.

[0008] In one embodiment, the dialysis in step S2 uses a dialysis bag with a capacity of 100-1000 Da.

[0009] In one embodiment, the shearing conditions in step S3 are 5000~8000 rpm for 4~6 min.

[0010] In one embodiment, in step S4, the chitosan solution has a pH of 4.5-5.5 and a concentration of 0.03%-0.2% w / v; the yeast β-glucan-tricarboxyphenylboronic acid concentration is 2-4 mg / mL.

[0011] In one embodiment, the concentration of glacial acetic acid in step S4 is 0.02~0.05% w / v, and the pH is 4.0~6.0.

[0012] In one embodiment, the stirring speed in step S4 is 600~800 rpm.

[0013] The present invention also provides a functional lutein complex prepared by the above method, comprising the following components: phycocyanin content of 50-60% w / w, tricarboxyphenylboronic acid-yeast β-glucan content of 10-20% w / w, chitosan content of 10-15% w / w, and lutein content of 5-25% w / w.

[0014] In one embodiment, the lutein complex comprises the following components: phycocyanin content of 55% w / w, tricarboxyphenylboronic acid-yeast β-glucan complex content of 13.8% w / w, chitosan content of 12.5% ​​w / w, and lutein content of 18.7% w / w.

[0015] The present invention also provides a method for improving the bioavailability of lutein, wherein a lutein complex is prepared by using lutein as the core material and phycocyanin, tricarboxyphenylboronic acid-yeast β-glucan complex and chitosan as the wall material.

[0016] The present invention also provides the use of functional lutein complexes in the preparation of pharmaceuticals for relieving dry eye syndrome.

[0017] In one embodiment, the pharmaceutical product further includes a drug carrier and / or excipients.

[0018] Beneficial effects: This invention utilizes phycocyanin for primary encapsulation of lutein, and further constructs a functional complex that enhances lutein bioavailability through layer-by-layer self-assembly of the tricarboxyphenylboronic acid-yeast β-glucan complex and chitosan via electrostatic interactions. This invention protects lutein from the extreme acidic environment of the stomach, increases lutein concentration in the blood (by 2.5 times), and compared to free lutein, the lutein complex significantly alleviates dry eye symptoms in mice. This encapsulation method constructs a functional nano-carrier system, significantly improving the absorption and utilization of natural nutrients. Attached Figure Description

[0019] Figure 1 The image shows a TEM transmission electron microscope (TEM) image (×40K) of the lutein complex in Example 1. Figure 2 This is a TEM transmission electron microscope (TEM) particle size distribution diagram of the lutein complex in Example 1; Figure 3 The encapsulation efficiency of the lutein complex in Comparative Example 1, Comparative Example 2 and Example 1; Figure 4 The loading rate of the lutein complex in Comparative Example 1, Comparative Example 2 and Example 1; Figure 5 The UV stability of the lutein complexes in Comparative Example 1, Comparative Example 2 and Example 1; Figure 6The heating stability of the lutein complexes in Comparative Example 1, Comparative Example 2 and Example 1; Figure 7 This is a laser confocal image taken after FITC-labeled lutein complex cells were incubated with RAW264.7 cells for 8 hours in Example 1. Figure 8 Laser confocal images of RAW264.7 cells after 8 hours of incubation with FITC-labeled lutein complex in Example 1 following inhibitor pretreatment. Figure 9 This is a cell viability graph of corneal epithelial cells after incubation for 24 hours with different concentrations of lutein complex in Example 1. Figure 10 This is a section of mouse liver stained with hematoxylin and eosin after 56 days of gavage administration of lutein complex in Example 1; Figure 11 Hematoxylin and eosin stained sections of liver from healthy mice; Figure 12 The release rate of the lutein complex in simulated gastric and intestinal fluids in Example 1; Figure 13 The graph shows the lutein content of lutein and lutein complex in rat serum at different time points in Example 1. Figure 14 This is a graph showing the lutein content of lutein and lutein complex in rat eyes at different time points in Example 1; Figure 15 The image shows the corneal fluorescence scores of mice with dry eye at different time points in Example 1, including lutein, commercial lutein, and lutein complex. Figure 16 The image shows the corneal rose score of mice treated with lutein, commercial lutein and lutein complex at different time points in Example 1. Figure 17 The graph shows the tear secretion of mice with dry eye at different time points in Example 1, including lutein, commercial lutein and lutein complex. Figure 18 The image shows the tear film breakup time of mice treated with lutein, commercial lutein, and lutein complex at different time points in Example 1.

[0020] Appendix Figure 9 In 15~18, the symbols a, b, c and d indicate significance, P<0.05. Detailed Implementation

[0021] The present invention will be further described below through specific embodiments. The following examples illustrate the method for determining lutein content: (1) Spectrophotometry for lutein content: Preparation of standard curves: Lutein standard (98%, Chengdu Pusi Biotechnology Co., Ltd.) was diluted to 0.5, 1, 1.5, 2, 2.5, and 3 μg / mL standard solutions. The absorbance at 450 nm was measured by UV spectrophotometer, and standard curves were plotted.

[0022] (2) Detection of lutein content by high performance liquid chromatography (HPLC): Lutein standard (98%, Chengdu Pusi Biotechnology Co., Ltd.) was diluted to standard solutions of 0.2, 0.4, 0.78, 1.56, 3.13, and 6.75 μg / mL, and a standard curve was plotted by HPLC. The centralized mobile phases were (A) dichloromethane:methanol:acetonitrile:water (5:85:45:4.5, v / v) and (B) dichloromethane:methanol:acetonitrile:water (22:28:45:4.5, v / v), with linear gradient elution (0-8 min, 0% A; 8-20 min, 100% A). HPLC analysis was performed at 450 nm using a C18 column (4 μm, 4.6 × 150 mm) at a flow rate of 1 mL / min.

[0023] Example 1: Preparation of Lutein Complex A lutein complex with improved bioavailability and relief of dry eye syndrome comprises the following components: phycocyanin content of 55% w / w, chitosan content of 12.5% ​​w / w, tricarboxyphenylboronic acid-yeast β-glucan content of 13.8% w / w, and lutein content of 18.7% w / w.

[0024] The method for preparing the lutein complex with improved bioavailability and relief of dry eye syndrome includes the following steps: S1. Preparation of carboxyl-activated tricarboxyphenylboronic acid solution: Weigh 400 mg of tricarboxyphenylboronic acid and dissolve it in 20 ml of 98% dimethyl sulfoxide buffer solution. Stir thoroughly at 800 rpm to obtain a tricarboxyphenylboronic acid solution. Then add 400 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 400 mg of N-hydroxysuccinimide to the tricarboxyphenylboronic acid solution and stir thoroughly at 800 rpm for 5 h. S2. Preparation of yeast β-glucan-tricarboxyphenylboronic acid complex: Yeast β-glucan was fully dissolved in dimethyl sulfoxide solution to a final concentration of 2 mg / mL; the obtained yeast β-glucan solution was then added dropwise to the tricarboxyphenylboronic acid activation solution described in step S1, and the mixture was gradually heated to 55°C and stirred at 55°C for 40 h; after the reaction was completed, the mixture was dialyzed using a 500 Da dialysis bag for 3 days, and then freeze-dried to obtain the yeast β-glucan-tricarboxyphenylboronic acid complex; S3. Preparation of phycocyanin and lutein shearing solution: Phycocyanin is fully dissolved in deionized water to make the final concentration of phycocyanin 2 mg / mL; then lutein ethanol solution is added to make the final concentration of lutein 20 mg / mL, and the mixture is sheared and broken under ice bath conditions at 4℃, with shearing conditions of 8000 rpm for 4~6 min to obtain phycocyanin and lutein shearing solution. S4. Preparation of lutein complex with improved bioavailability and relief of dry eye syndrome: The yeast β-glucan-tricarboxyphenylboronic acid complex described in step S2 was dissolved in deionized water to obtain a yeast β-glucan-tricarboxyphenylboronic acid complex solution with a final concentration of 2 mg / mL; chitosan was dissolved in glacial acetic acid solution to obtain a chitosan solution with a final concentration of 2 mg / mL; then 10 ml of yeast β-glucan-tricarboxyphenylboronic acid complex solution and 10 ml of chitosan solution were added to 40 ml of the phycocyanin and lutein shearing solution described in step S3 for layer-by-layer self-assembly, and the reaction was stirred at 800 rpm for 24 h. After the reaction was completed, the mixture was freeze-dried to obtain the lutein complex with improved bioavailability and relief of dry eye syndrome.

[0025] In step S4, the concentration of glacial acetic acid is 0.02% w / v, and the pH is 5.0. The chitosan solution has a pH of 4.5-5.5 and a concentration of 0.03%-0.2% w / v.

[0026] Comparative Example 1 A method for preparing a lutein complex with improved bioavailability and relief of dry eye syndrome, the specific implementation method is as described in Example 1, the difference being that the lutein content is different, and step S3 is as follows: S3. Dissolve phycocyanin fully in deionized water to achieve a final concentration of 2 mg / mL; then add lutein ethanol solution to achieve a final concentration of 10 mg / mL. Shear and break the phycocyanin under ice bath conditions at 4°C and 8000 rpm to obtain a sheared solution of phycocyanin and lutein.

[0027] Comparative Example 2 A method for preparing a lutein complex with improved bioavailability and relief of dry eye syndrome, the specific implementation method is as described in Example 1, the difference being that the lutein content is different, and step S3 is as follows: S3. Dissolve phycocyanin fully in deionized water to achieve a final concentration of 2 mg / mL; then add lutein ethanol solution to achieve a final concentration of 5 mg / mL. Shear and break the phycocyanin under ice bath conditions at 4°C and 8000 rpm to obtain a sheared solution of phycocyanin and lutein.

[0028] Example 2: Characterization of the properties of the lutein complex 1. Particle size analysis The "lutein complex for improving bioavailability and relieving dry eye syndrome" described in step S4 of Example 1 was subjected to TEM scanning electron microscopy imaging. (See attached image.) Figure 1 As shown in the TEM scanning electron microscope image, the lutein complex has a nearly spherical morphology. (See attached image.) Figure 2 As shown, particle size analysis of the TEM images of the lutein complex was performed using ImageJ software, and the results showed that the size of the lutein complex was concentrated at 82.78 nm.

[0029] 2. Encapsulation rate and load factor The lutein complex described in Comparative Examples 1, 2, and 1 ("enhanced bioavailability and relief of dry eye") was dissolved in 1 mL of deionized water, mixed with 3 mL of n-hexane, vortexed for 1 min, and then centrifuged at 6000 rpm for 5 min. The absorbance of the supernatant at 450 nm was measured. The lutein content in the supernatant was determined using a pre-established standard curve, and the encapsulation efficiency and loading rate were calculated. (See attached...) Figure 3 As shown, the lutein encapsulation efficiencies of the lutein complexes in Comparative Examples 1, 2, and 1 were 94.85%, 94.12%, and 95.87%, respectively, indicating that the complex prepared in the steps of Example 1 had the best encapsulation effect. The lutein loading rates of the lutein complexes were 3.26 μg / mg, 6.21 μg / mg, and 11.98 μg / mg, respectively, indicating that the complex prepared in the steps of Example 1 had the best loading effect.

[0030] 3. Stability Lutein was dissolved in anhydrous ethanol. The "lutein complex for improving bioavailability and relieving dry eye" described in Comparative Examples 1, 2, and 1 was dissolved in deionized water to ensure that the final lutein concentrations of the four solutions were the same. The samples were then subjected to UV irradiation for 1, 2, 3, 4, and 5 hours, or heated in a 60°C water bath for 30, 60, 90, 120, 150, and 180 minutes. Lutein was extracted from the samples using n-hexane, and the lutein content in the solution was determined using a pre-established standard curve, and the lutein retention rate was calculated. (See attached...) Figure 5 As shown, after 5 hours of UV irradiation, the lutein retention rates of lutein alone, Comparative Example 1, Comparative Example 2, and the lutein complex in Example 1 were 62.45%, 71.87%, 74.57%, and 76.87%, respectively. This indicates that the lutein complex prepared in Example 1 has better UV stability. (See attached image.) Figure 6As shown, after 14 days of storage at room temperature, the lutein retention rates of lutein alone, Comparative Example 1, Comparative Example 2, and the lutein complex in Example 1 were 48.52%, 55.62%, 60.74%, and 66.17%, respectively. This indicates that the lutein complex prepared in Example 1 has better thermal stability.

[0031] 4. Cell-targeting experiments Yeast β-glucan can specifically target resident immune cells on Pell's plaques in the intestine via the dectin-1 receptor. RAW264.7 cells are a type of resident immune cell. To observe the targeting effect of the "lutein complex for improving bioavailability and alleviating dry eye" described in Example 1 on the dectin-1 receptor on RAW264.7 cells, the lutein complex was labeled with FITC. A control group, an experimental group, and an inhibition group were set up. RAW264.7 cells (1×10⁻⁶) were used. 6 Cells were cultured in DMEM medium (Gibco, Thermo Fisher Scientific) in cell culture dishes for 24 hours. The inhibitor group was pretreated for 3 hours with medium containing 1 mg / mL laminarin, a dectin-1 receptor inhibitor, which specifically inhibits dectin-1. The control and experimental groups did not receive this treatment. After treatment, 1 mg / mL lutein complex was added to both the experimental and inhibitor groups and incubated for 8 hours. The cell culture medium was then removed, and the targeting results were observed by flow cytometry.

[0032] As attached Figure 7 As shown, after 8 hours of incubation with the lutein complex, its average fluorescence intensity was 502, indicating that the lutein complex could aggregate on RAW264.7 cells. Conversely, as shown in the attached figure... Figure 8 The results showed that RAW264.7 cells pretreated with the dectin-1 receptor inhibitor laminarin exhibited an average fluorescence intensity of only 10⁸ over 8 hours. This indicates that the lutein complex prepared in Example 1 can specifically target resident immune cells—RAW264.7 cells—via the dectin-1 receptor.

[0033] 5. Biosafety Corneal epithelial cells (HCECs) were loaded at a rate of 1×10 6Cells were inoculated into 96-well plates and cultured for 24 hours. The lutein complex described in Example 1, which enhances bioavailability and alleviates dry eye, was dissolved in DMEM medium (Gibco, Thermo Fisher Scientific) to final concentrations of 1.57, 3.13, 6.25, 12.5, 25, and 50 μg / mL. After 24 h of incubation, 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetraazole bromide (MTT) reagent was added, and incubation continued for another 4 h. Finally, 150 μL of DMSO was added to the cells, and the mixture was shaken for 10 min. Cell viability was assessed using a microplate reader. (See attached image.) Figure 9 As shown, after incubating corneal epithelial cells with different concentrations of the lutein complex in Example 1 for 24 hours, the cell viability remained above 80%, indicating that the lutein complex in Example 1 did not have significant cytotoxicity.

[0034] The "lutein complex for improving bioavailability and alleviating dry eye syndrome" described in Example 1 was administered by gavage to male BALB / c mice (6 weeks old, weighing 24±2g) for 56 consecutive days. Afterward, the mice and healthy mice were simultaneously sacrificed, and their livers were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. (See attached...) Figure 10 and 11 As shown, liver slices of the lutein complex from long-term gavage in Example 1 were not significantly different from liver slices from healthy mice, indicating the biocompatibility of the lutein complex.

[0035] Example 4: Release characteristics of lutein complex in the gastrointestinal tract Take 10 mg of the lutein complex with improved bioavailability and dry eye relief described in step S4 of Example 1, dissolve the complex sample in 5 mL of simulated saliva (Shanghai Yuanye Biotechnology Co., Ltd.), and stir at 100 r / min for 5 min on a shaking table at 37 ℃. Add another 20 mL of simulated gastric juice (Shanghai Yuanye Biotechnology Co., Ltd.) to the reaction system, and continue incubation at 100 rpm for 2 h at 37 ℃. Finally, add 50 mL of simulated intestinal juice (Shanghai Yuanye Biotechnology Co., Ltd.) to the mixed solution, and shake at 100 r / min for 5 h. Every 30 minutes, take 4 mL of supernatant from the mixed solution, detect its lutein concentration, and replenish with an equal volume of simulated intestinal juice.

[0036] As attached Figure 12The figure shows the release of lutein from the lutein complex in simulated gastric and intestinal fluids. The results indicate that after 5 minutes of simulated saliva digestion, only 5.46% of the lutein from the lutein complex was released. After 2 hours of simulated gastric digestion, the relative cumulative release rate of lutein reached 22.03%. With prolonged digestion time in simulated intestinal fluids, the relative cumulative release rate of lutein increased slowly. After 5 hours of digestion in simulated intestinal fluids, the release rate of lutein was 41.83%, indicating that 58.17% of the lutein nanoparticles could still be transported by resident immune cells. The delayed release of the lutein complex in simulated gastric and intestinal fluids is due to the resistance of yeast β-glucan in the carrier to gastric acid degradation and chitosan adhesion in the intestine.

[0037] Example 5: Lutein Complex Improves the Bioavailability of Lutein in Blood and Eyes After a 24-hour fast, SD rats were divided into two groups (n=3 per group) and administered lutein and a lutein complex (10 mg / kg lutein) as described in step S4 of Example 1 to improve bioavailability and alleviate dry eye symptoms, respectively, via gavage. Blood samples were collected at different time points into centrifuge tubes containing the anticoagulant ETDA. Similarly, after gavage at different time points, the SD rats were sacrificed, and ocular tissue was harvested and stored at -80°C. The plasma was centrifuged at 10,000 rpm for 10 min at 4°C to obtain the supernatant plasma. Physiological saline was then added to the ocular tissue, and the tissue was homogenized in an ice bath. The supernatant plasma and ocular homogenate were added to a chloroform:methanol mixture of 2:1 (v / v), vortexed for 1 min, then hexane was added, and the mixture was centrifuged for 10 min. The organic layer was dried under nitrogen and resuspended in dichloromethane. The lutein content in rat serum and ocular tissue was determined using high-performance liquid chromatography (HPLC).

[0038] As attached Figure 13 The figure shows the concentrations of lutein and lutein complex in rat serum at different time points. The highest concentration increased from 428.33 ng / mL to 1072.33 ng / mL, a 2.5-fold increase. (See attached figure.) Figure 14 The figure shows the lutein concentrations of lutein and its complex in rat eyes at different time points. The highest concentration increased from 251.8 ng / mL to 469.4 ng / mL, an increase of 1.86 times. These results indicate that the lutein complex significantly increased the lutein concentrations in blood and eyes, thereby improving the bioavailability of lutein in both, and providing support for the use of the lutein complex in treating dry eye.

[0039] Example 6: Lutein Complex Improves Dry Eye Symptoms Male BALB / c mice (6 weeks old, weighing 24±2g) were randomly divided into 5 groups of 5 mice each. The mice in each group received one of the following treatments: no gavage (positive control group), gavage with 0.9% saline (control group), lutein (98%, Chengdu Pusi Biotechnology Co., Ltd.), commercial lutein (Laiyi brand lutein chewable tablets, lutein dose 50 mg / kg / day, powdered and administered by gavage), and the lutein complex described in step S4 of Example 1 (lutein dose 50 mg / kg / day) for improving bioavailability and alleviating dry eye syndrome. After 14 days of oral administration, except for the control group, all other groups were given 0.2% benzalkonium chloride eye drops to establish a dry eye model. Successful establishment of the DED model was determined by tear volume measurement, corneal fluorescein staining, and Bengal rose red staining score. After model establishment, tear volume, tear film breakup time, and slit-lamp imaging were assessed on days 0, 7, 14, 21, and 28. In simple terms, tear volume was measured using a tear film test strip placed at the outer canthus of the lower eyelid, and its length was measured with a ruler to determine the tear volume. 1 μL of 0.125% sodium fluorescein was instilled into the conjunctival sac of mice, and after three manual blinks, observation was performed using a slit lamp. The tear film breakup time was recorded from the blink until the first black lesion appeared. 1% sodium fluorescein and Bengal rose red were instilled into the conjunctival sac of mice, and imaging was performed using a slit lamp. Each cornea was divided into four quadrants, and a scoring system was calculated using a four-point scale: 0 points (missing); 1. <30 punctate staining points; 2. >30 punctate staining points; 3. Large-area diffuse staining; 4. Positive fluorescein patch staining. Scoring was performed by three double-blind evaluators.

[0040] Appendix Figure 15 The results of corneal fluorescence scoring in mice show that, after modeling, all groups except the control group maintained obvious fluorescent spots in the cornea, with fluorescence scores all above 10, indicating severe corneal damage. With increasing intervention time, the production of corneal fluorescent spots decreased in the lutein, commercially available lutein products, and lutein complex groups. Notably, the lutein complex showed a stronger intervention effect than commercially available lutein and free lutein. After 28 days, the fluorescence score of the positive control group was 12.33±1.15, while the fluorescence scores of the lutein complex, commercially available lutein, and free lutein were 3±1, 5.33±0.58, and 9±2, respectively, indicating that the lutein complex had a better alleviating effect on corneal damage. Compared with the positive control group, the fluorescence intensity of the lutein complex group decreased by 4.11 times, and the corneal damage in mice recovered to a level comparable to that of the control group.

[0041] Corneal rose staining can reflect the tear film breakage condition. (Attached) Figure 16The results of the mouse corneal rose score are shown in the figure. The results indicate that after 28 days of intervention, the fluorescence score of the positive control group was 12.67±0.58, the rose score of the lutein complex group was 2.67±1.15, the commercial lutein group and the free lutein group were 5.33±0.58 and 10.67±1.15, respectively, suggesting that the lutein complex has a better effect on tear film relief than the commercial lutein group.

[0042] Appendix Figure 17 The figure shows the results of tear secretion in mice. After 28 days of intervention, the tear secretion volumes in the lutein group, the commercial lutein group, and the lutein complex group were 2.33±0.3 mm, 2.97±0.18 mm, and 4.57±0.14 mm, respectively. Compared with the positive control group, the tear secretion volume in the lutein complex group increased by 3.26 times, and compared with the commercial lutein group, the tear secretion volume in the lutein complex group increased by 1.54 times, recovering to a level comparable to the control group. This indicates that the lutein complex has a better effect on relieving tear dryness.

[0043] Appendix Figure 18 The image shows the results of tear film breakup time in mice, which can be used as an indicator of tear film stability. The results showed that after 28 days of oral administration, the positive control group had the shortest tear film breakup time (1.53 ± 0.21 seconds), while the lutein group, commercial lutein group, and lutein complex group had times of 2.04 ± 0.16 seconds, 3.07 ± 0.15 seconds, and 3.52 ± 0.18 seconds, respectively. This indicates that the lutein complex group was more effective than the lutein group and the commercial lutein group in restoring tear film stability. These results demonstrate that the lutein complex significantly alleviated benzalkonium chloride-induced dry eye symptoms in mice. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a functional lutein complex, characterized in that, Includes the following steps: S1. Preparation of carboxyl-activated tricarboxyphenylboronic acid solution: Prepare a tricarboxyphenylboronic acid solution with a final concentration of 10-30 mg / mL, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a final concentration of 20-50 mg / mL and N-hydroxysuccinimide with a final concentration of 20-30 mg / mL to the tricarboxyphenylboronic acid solution, stir and react for 3-5 h to obtain the carboxyl-activated tricarboxyphenylboronic acid solution; S2. Preparation of yeast β-glucan-tricarboxyphenylboronic acid complex: Prepare a yeast β-glucan solution with a final concentration of 2-5 mg / mL, then add the yeast β-glucan solution dropwise to the carboxyl activation solution of tricarboxyphenylboronic acid described in step S1, heat to 40-60℃, and stir for 30-50 h; after the reaction is completed, dialyze for 2-3 days and dry to obtain yeast β-glucan-tricarboxyphenylboronic acid complex; S3. Preparation of phycocyanin and lutein shearing solution: Prepare a phycocyanin solution with a final concentration of 2~4 mg / mL, then add a lutein solution with a final concentration of 5~20 mg / mL, and shear to obtain phycocyanin and lutein shearing solution. S4. Preparation of lutein complex: A yeast β-glucan-tricarboxyphenylboronic acid complex solution with a final concentration of 1-3 mg / mL and a chitosan solution with a final concentration of 2-4 mg / mL were added to the shearing solution of phycocyanin and lutein in step S3 for layer-by-layer self-assembly. The reaction was stirred for 12-24 h. After the reaction was completed, the mixture was freeze-dried to obtain the lutein complex.

2. The method according to claim 1, characterized in that, The dialysis described in step S2 uses a dialysis bag with a capacity of 100~1000 Da.

3. The method according to claim 2, characterized in that, The shearing conditions described in step S3 are 5000~8000 rpm and 4~6 min.

4. The method according to claim 3, characterized in that, In step S4, the chitosan solution has a pH of 4.5 to 5.5 and a concentration of 0.03% to 0.2% w / v.

5. The lutein complex prepared by any one of the methods described in claims 1 to 4.

6. The lutein complex according to claim 5, characterized in that, The lutein complex contains 50-60% w / w phycocyanin, 10-20% w / w tricarboxyphenylboronic acid-yeast β-glucan, 10-15% w / w chitosan, and 5-25% w / w lutein.

7. The use of the lutein complex of claim 5 or 6 in the preparation of a medicament for relieving dry eye syndrome.

8. The application according to claim 7, characterized in that, The drug also includes a drug carrier.

9. The application according to claim 8, characterized in that, The drug also includes excipients.

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