Application of a small molecule active peptide in the preparation of products for preventing and treating dry eyes

Abstract

The present invention provides an application of a small molecule active peptide in the preparation of a product for preventing and treating dry eyes, relates to the field of biomedicine, and specifically relates to an application of a tilapia skin peptide in improving dry eyes. The tilapia skin peptide of the present invention has been proven by in vitro and in vivo experiments to effectively improve dry eyes with little side effects. At the same time, the tilapia skin peptide of the present invention is derived from tilapia skin, has a wide source of raw materials, has a low preparation cost, improves the utilization rate of tilapia skin, is energy-saving and environmentally friendly, and provides a new direction for the fine processing of tilapia skin.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a small molecule active peptide in improving dry eyes. Background Art

[0002] Dry eye disease (DED), also known as corneal syndrome, is a chronic ophthalmic disease induced by multiple factors. Patients' eyes are often accompanied by symptoms such as foreign body sensation, burning sensation, dryness, photophobia, etc., which leads to a decline in the quality of life of patients and even psychological problems such as depression. Dry eye is one of the most common diseases in ophthalmology clinics. In addition, according to the "Chinese Dry Eye Expert Consensus (2020)", the incidence of dry eye in my country is about 21% to 30%, that is, one in every five people in my country suffers from dry eye. Dry eye has seriously endangered human life and health.

[0003] At present, there is no ideal drug that can completely cure dry eye at home and abroad. The current clinical drugs for treating dry eye have disadvantages such as causing ocular surface pain, osteoporosis, only temporarily relieving dry eye symptoms, and stopping the drug may cause the disease to rebound. In addition, although some clinical first-line drugs for treating dry eye, such as cyclosporine, have a fast effect, the high price of the drug also brings huge economic pressure to patients. Therefore, it is urgent to develop a drug that is highly effective, has few side effects and is low-cost for the prevention and treatment of dry eye.

[0004] Tilapia skin peptides, as natural food-borne bioactive substances, have multiple biological activities such as anti-inflammatory, antioxidant, anti-hypertensive, antibacterial, hypoglycemic, anti-skin photoaging, promoting osteoblast proliferation and differentiation, promoting wound healing, and improving ovarian function. At the same time, as a food-borne bioactive substance, it has the characteristics of safety and good absorption, so it is favored by scientific researchers. At present, the tilapia processing model in my country is relatively extensive, and a large amount of tilapia skin is directly discarded or processed into low-value feed, causing serious environmental pollution and great waste of resources. So far, there has been no report on the improvement of dry eyes by tilapia skin peptides. Summary of the invention

[0005] The purpose of the present invention is to provide a small molecule active peptide with high efficiency, low side effects and low cost and the function of preventing and treating dry eyes.

[0006] To achieve the above object, the technical solution adopted by the present invention is: use of a small molecule active peptide in the preparation of a product for preventing and treating dry eyes, wherein the small molecule active peptide is tilapia skin peptide.

[0007] In one aspect, the present invention provides an application of a small molecule active peptide in the preparation of a product for preventing and treating dry eyes, wherein the small molecule active peptide is a tilapia skin peptide.

[0008] Tilapia skin peptides are derived from tilapia skin, and tilapia is widely available and easy to breed, so the yield of tilapia skin is also high. At the same time, tilapia, as a food-borne bioactive substance, has the characteristics of safety and harmlessness. Further, the present invention has been verified through in vivo and in vitro experiments that tilapia skin peptides have the effect of preventing and treating dry eyes, which expands the scope of use of tilapia skin peptides and improves the economic value and utilization rate of tilapia skin peptides, reducing the waste of resources caused by extensive processing of tilapia skin.

[0009] As a preferred embodiment of the use of the small molecule active peptide of the present invention in the preparation of a product for preventing and treating dry eyes, the molecular weight of the small molecule active peptide is less than 6.00 kDa.

[0010] The present invention obtains a tilapia skin peptide with a molecular weight less than 6.00 kDa through a specific preparation method, and verifies through in vivo and in vitro experiments that the tilapia skin peptide has a good effect of improving dry eyes.

[0011] As a preferred embodiment of the use of the small molecule active peptide of the present invention in the preparation of a product for preventing and treating dry eyes, the molecular weight of the small molecule active peptide is 0.40 kDa to 2.00 kDa.

[0012] After the tilapia skin peptide of the present invention is separated by dextran gel G-25, a total of 5 components are obtained, which are named TSP-Ⅰ, TSP-Ⅱ, TSP-Ⅲ, TSP-Ⅳ and TSP-Ⅴ respectively. Through the in vitro anti-dry eye activity of the above components, it is found that the anti-dry eye active components of tilapia skin peptide are mainly concentrated in TSP-Ⅱ and TSP-Ⅲ. According to LC / MS / MS analysis, the molecular weight of TSP-Ⅱ is distributed between 0.61kDa and 1.93kDa, most of which are located between 1.00kDa and 1.50kDa. The molecular weight distribution of TSP-Ⅲ is mainly concentrated between 0.50kDa and 1.00kDa, among which the largest molecular weight is 1.52kDa and the smallest molecular weight is 0.44kDa. Therefore, tilapia skin peptides in the molecular weight range of 0.40kDa to 2.00kDa have better effects in preventing and treating dry eyes.

[0013] As a preferred embodiment of the application of the small molecule active peptide of the present invention in the preparation of a product for preventing and treating dry eyes, the preparation method of the tilapia skin peptide comprises the following steps:

[0014] (a) Sample pretreatment: Tilapia skin is descaled, defatted, and dried to obtain tilapia skin dry powder;

[0015] (b) the tilapia skin dry powder is dissolved, enzymatically hydrolyzed, centrifuged, and the supernatant is taken;

[0016] (c) ultrafiltration the supernatant obtained in step (b) using an ultrafiltration membrane.

[0017] The preparation method has low preparation cost, simple operation, high tilapia skin peptide extraction efficiency, and can realize quantitative production.

[0018] As a preferred embodiment of the use of the small molecule active peptide of the present invention in the preparation of products for preventing and treating dry eyes, the products for preventing and treating dry eyes include eye injections, eye drops, eye tablets, eye gels, eye ointments, eye sprays or eye patches.

[0019] On the other hand, the present invention provides a preparation for preventing and treating dry eyes, wherein the preparation for preventing and treating dry eyes comprises the above-mentioned small molecule active peptide.

[0020] As a preferred embodiment of the preparation for preventing and treating dry eyes of the present invention, the preparation for preventing and treating dry eyes is eye drops; in the eye drops, the concentration of the small molecule active peptide is 1 to 2000 μg / mL.

[0021] The eye drops prepared by the invention have the effect of preventing and treating dry eyes when the concentration of the small molecule active peptide is 1-2000 μg / mL.

[0022] As a preferred embodiment of the preparation for preventing and treating dry eyes of the present invention, the concentration of the small molecule active peptide in the eye drops is 250 to 1000 μg / mL.

[0023] The present invention has been proved through experiments that when the concentration of the small molecule active peptide in the eye drops is 250-1000 μg / mL, the comprehensive effect of preventing and treating dry eyes is better. Among them, when the concentration of the small molecule active peptide in the eye drops is 1000 μg / mL, the effect of preventing and treating dry eyes is the best.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The anti-dry eye active peptide prepared by the present invention is derived from tilapia skin, is a food-derived peptide, is safe and non-toxic, and improves the utilization rate of tilapia skin resources;

[0026] (2) The anti-dry eye active peptide prepared by the present invention is extracted from tilapia skin, the raw material source is wide and the price is low, and it is easy to expand the production on a large scale;

[0027] (3) The anti-dry eye active peptide prepared by the present invention has good anti-dry eye activity, and has important social value and guiding significance in the development of medicines, functional foods and other products for preventing and treating dry eyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the MTT result of the NaCl-induced human corneal epithelial cell dry eye model described in Test Example 1 (n=4, ): Compared with the blank group, # p<0.05,## p<0.01.

[0029] Figure 2 The MTT result of the cytotoxicity of the tilapia skin anti-dry eye active peptide described in Test Example 1 to human corneal epithelial cells (n=5, ): Compared with the blank group, # p<0.05, ## p<0.01.

[0030] Figure 3 This is the MTT result of the anti-dry eye activity of the tilapia skin anti-dry eye active peptide described in Test Example 1 (n=4, ): Compared with the blank group, # p<0.05, ## p<0.01, compared with the model group, * p<0.05, ** p<0.01.

[0031] Figure 4 This is the tear secretion result of mice described in Test Example 2 (n=16, ): Compared with the blank group, # p<0.05, ## p<0.01, compared with the model group, * p<0.05, ** p<0.01.

[0032] Figure 5 This is the scoring result of the mouse tear fern crystals in Test Example 2 (n=17, Scale bar = 50 μm): Compared with the blank group, # p<0.05, ## p<0.01, compared with the model group, * p<0.05, ** p<0.01.

[0033] Figure 6 The results of mouse corneal tissue morphology staining and corneal epithelial thickness change in test example 2 (n = 9, Scale bar = 50 μm): Compared with the blank group, # p<0.05, ## p<0.01, compared with the model group, * p<0.05, ** p<0.01.

[0034] Figure 7 The results of mouse conjunctival tissue morphology staining and goblet cell counting in Test Example 2 (n=7, Scale bar = 50 μm), where compared with the blank group, # p<0.05, ## p<0.01, compared with the model group, * p<0.05, ** p<0.01.

[0035] Figure 8 This is the test result diagram of test case 3, where: Figure 8 A is the gel filtration chromatography result of TSP in Test Example 3, Figure 8 B is the in vitro anti-dry eye activity test result of TSP in Test Example 3 (n=4, ).

[0036] Fig. 9 The in vivo anti-dry eye activity test diagram of TSP in Test Example 3 (n=14, ).

[0037] Fig.10 LC / MS / MS analysis of TSP in Test Example 3, where Fig.10 A and Fig.10 B is the LC / MS / MS analysis of TSP-Ⅱ; Fig.10 C and Fig.10 D is the LC / MS / MS analysis chart of TSP-Ⅲ. DETAILED DESCRIPTION

[0038] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope and implementation methods of the present invention are not limited thereto.

[0039] Example 1

[0040] An embodiment of the use of the tilapia skin peptide of the present invention in the preparation of a product for preventing and treating dry eyes.

[0041] The preparation method of tilapia skin peptide comprises the following steps:

[0042] (I) Sample pretreatment. First, descale the tilapia skin, rinse it with running water and drain the water. Use a tissue crusher to mince the tilapia skin, homogenize it for 3 minutes each time, and continue 4 times. Add 4 times the volume of isopropanol to the homogenate and defat it for 4 hours, changing the liquid 4 times during the period. The entire degreasing process is carried out on a magnetic stirrer, setting the stirring speed to 100 rpm and the stirring temperature to 55°C. After defatting, centrifuge it in a high-speed centrifuge (centrifugation parameters: 12000rpm, 10min), collect the filter residue, and dry it at 50°C in an electric constant temperature oven to obtain tilapia skin dry powder.

[0043] (Ⅱ) Enzymolysis. Add 5 times the volume of ultrapure water to soak the tilapia skin dry powder, add 3% neutral protease by weight of the fish skin dry powder, maintain the solution system pH at 7.0±0.2, and perform enzymolysis at 50±5℃ for 2h. After 2h, add 0.3% alkaline protease by weight of the fish skin dry powder, maintain the solution system pH at 9.0±0.2, and perform enzymolysis at 50±5℃ for 2h. All enzymolysis processes were stirred on a magnetic stirrer for 1min every 15min. After the enzymolysis is completed, heat the enzymolysis solution to 100℃ for 10min to interrupt the enzymolysis process. Centrifuge the enzymolysis solution, collect the filtrate and store it at -80℃ for later use.

[0044] (III) Ultrafiltration. After the above filtrate is thawed, ultrapure water is added in a ratio of 1:5, and the solution is poured into the liquid inlet cylinder of the pellicon tangential flow ultrafiltration system. The inlet pressure is adjusted to 30psi, the reflux pressure is 10psi, and a 10kDa ultrafiltration membrane is used for ultrafiltration at 4°C. The ultrafiltrate is collected and concentrated using a rotary evaporator with the water bath temperature set to 55°C. The concentrate is freeze-dried using a Labconco freeze dryer at -50°C and 0.20mBar operating parameters to obtain tilapia skin peptides (TSP) powder. Test Example 1 In vitro anti-dry eye activity test of tilapia skin peptides.

[0045] (1) Culture of human corneal epithelial cells.

[0046] Human corneal epithelial cells were cultured in MEM (modified eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Human corneal epithelial cells were cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium was replaced every two days, and human corneal epithelial cells were passaged at a ratio of 1:2 when they grew to the logarithmic phase.

[0047] (2) Construction of dry eye cell model.

[0048] The internationally accepted dry eye cell model was used (PARK B, et al. Nutrients, 2018, 10(10)1550). Human corneal epithelial cells in the logarithmic growth phase were obtained and cultured at 2×10 4The cells were inoculated into a 96-well plate at a density of 100 mM / mL. The cells were randomly divided into a blank group, NaCl experimental group 1 (containing 20 mM NaCl), NaCl experimental group 2 (containing 40 mM NaCl), NaCl experimental group 3 (containing 60 mM NaCl), NaCl experimental group 4 (containing 80 mM NaCl) and NaCl experimental group 5 (containing 100 mM NaCl). Five replicate wells were set up in each group. After human corneal epithelial cells were cultured in the well plate for 24 hours, the blank group was cultured with 100 μL maintenance solution (MEM culture medium containing 1% fetal bovine serum), and the remaining experimental groups were cultured with 100 μL maintenance solution containing 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM NaCl, respectively. After 12 hours of culture, the relative viability of cells in each group was detected by MTT method.

[0049] Cell relative viability test method (MTT detection method): ① After the incubation time is over, the supernatant in the well plate is discarded, and the human corneal epithelial cells are washed with PBS (phosphate buffered saline); ② 100 μL of 0.5 mg / mL MTT solution is added, and the supernatant in the well plate is discarded after continuing to incubate in a cell culture incubator for 4 hours; ③ 150 μL of dimethyl sulfoxide is added, and the shaker is shaken at room temperature for 3 minutes. The OD value of each group is detected at a wavelength of 490 nm using a spectrophotometer, and the relative viability of each group of cells is calculated according to the following formula (I).

[0050] Relative cell viability (%) = (OD 实验组 / OD 空白组 )×100%Formula (I)

[0051] (3) Detection of TSP cytotoxicity.

[0052] Human corneal epithelial cells in logarithmic growth phase were collected and plated at 2×10 4 The cells were randomly divided into a blank group, TSP experimental group 1 (containing 250μg / mL TSP), TSP experimental group 2 (containing 500μg / mL TSP), TSP experimental group 3 (containing 1000μg / mL TSP) and TSP experimental group 4 (containing 2000μg / mL TSP). Five replicate wells were set up in each group. After human corneal epithelial cells were cultured in the well plate for 24 hours, the blank group was cultured with 100μL maintenance solution (MEM culture medium containing 1% fetal bovine serum), and the other experimental groups were cultured with 100μL maintenance solution containing 250μg / mL, 500μg / mL, 1000μg / mL and 2000μg / mL TSP, respectively. After 12 hours of culture, the relative viability of cells in each group was detected by the above-mentioned MTT method.

[0053] (4) Detection of the anti-dry eye activity of tilapia skin peptides.

[0054] Human corneal epithelial cells in logarithmic growth phase were collected and plated at 2×10 4 The cells were randomly divided into a blank group, a model group (containing 100 mM NaCl), a TSP treatment group 1 (containing 250 μg / mL TSP) and a TSP treatment group 2 (containing 500 μg / mL TSP). Five replicate wells were set up for each group. After the human corneal epithelial cells were cultured in the well plate for 24 hours, the blank group and the model group were cultured with 100 μL maintenance medium (MEM medium containing 1% fetal bovine serum) for 2 hours, and the remaining groups were cultured with 100 μL maintenance medium containing 250 μg / mL and 500 μg / mL TSP, respectively. After the culture was completed, the liquid in the wells was discarded, the blank group was cultured with fresh maintenance solution (MEM medium containing 1% fetal bovine serum), the model group was cultured with maintenance solution containing 100mM NaCl, and the remaining groups were added with maintenance solution solutions with final concentrations of 100mM NaCl+250μg / mL TSP and 100mM NaCl+500μg / mL TSP, respectively, and the volume of solution added to each well of each group was 200μL. After 12h of treatment, the relative cell viability was detected according to the aforementioned MTT method, and the relative cell viability reflected the anti-dry eye activity of tilapia skin peptide.

[0055] (5) Data processing

[0056] One-way ANOVA was performed between the groups using GraphPadPrism 8.0, followed by multiple comparisons using the Dunnett method. All data are presented as mean ± standard error ( ). Compared with the blank group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01.

[0057] (6) Experimental conclusions.

[0058] The above experimental results showed that compared with the blank group, after 12h treatment of human corneal epithelial cells with 100mM NaCl, the cell viability decreased to 70.47% (p<0.01, Figure 1 ), which is the ideal concentration for constructing a dry eye cell model, and subsequent related experiments were conducted based on this concentration. When the tilapia skin peptide concentration is less than 500 μg / mL, it has no cytotoxicity to human corneal epithelial cells, which indicates that tilapia skin peptide can continue to be used in subsequent anti-dry eye research ( Figure 2 Compared with the model group, the relative activity of human corneal epithelial cells significantly increased after being treated with 250μg / mL and 500μg / mL tilapia skin peptide, respectively, to 81.27% and 75.32% (p<0.05, p<0.01, Figure 3), which indicates that tilapia skin peptide has anti-dry eye effect in dry eye cell model. Test Example 2 In vivo anti-dry eye activity test of tilapia skin peptide.

[0059] (1) Rearing of model organisms.

[0060] This experiment used 7- to 8-week-old C57BL / 6 male mice, each weighing approximately 20-23 g. The animal experiment protocol was approved by the Animal Ethics Committee of Guangdong Ocean University and was conducted in accordance with the requirements of the "Guidelines for the Care and Use of Laboratory Animals of Guangdong Ocean University".

[0061] (2) Animal grouping and drug intervention.

[0062] In this experiment, a dry eye animal model was constructed according to the dry eye animal modeling method recommended by the Chinese Association of Traditional Chinese Medicine Experimental Pharmacology Committee (Chinese Association of Traditional Chinese Medicine Experimental Pharmacology Committee. Chinese Journal of Experimental Traditional Chinese Medicine, 2018, 24 (19): 6-9). The mice were randomly divided into a blank group, a model group, a TSP treatment group and a positive drug group, with 9 mice in each group (n = 18 eyes / group). The blank group mice were instilled with 5 μL of normal saline per eye as a control, the model group mice were instilled with 5 μL of 0.3% benzalkonium chloride (BAC) per eye to induce dry eye-like damage on the mouse ocular surface, the TSP treatment group mice were instilled with 5 μL of 0.3% BAC and 0.1% tilapia skin peptide prepared in Example 1 per eye, and the positive drug group mice were instilled with 5 μL of 0.3% BAC and commercially available sodium hyaluronate eye drops (sodium hyaluronate concentration was 0.1%) per eye. All eye drop procedures were performed twice a day, at 9:00 am and 19:00 pm, for 14 days. After the experiment, the mice were tested for tear secretion and tear fern crystals on the 15th and 16th days. On the 17th day, the mice were euthanized, and the intact eyeballs and their accessory organs (including cornea, conjunctiva, and upper and lower eyelids) were removed and paraffin sections were made. Paraffin sections were used for hematoxylin-eosin staining (H&E) and periodic acid-schiff staining (PAS).

[0063] (3) Measurement of tear secretion.

[0064] Tear secretion was measured at 9:00 am on the 15th day. The specific operation was as follows: In a clean laboratory with good lighting and air humidity controlled at 55% to 60%, 10 mg / kg of sodium pentobarbital was intraperitoneally injected into the mice. After they were fully anesthetized, the lower eyelid of the mice was gently pulled down with sterile ophthalmic forceps, and the tear detection phenol red cotton thread was gently placed into the 1 / 3 of the conjunctival sac. After 30 seconds, the cotton thread was removed and the length of the cotton thread that turned red was measured to evaluate the tear secretion.

[0065] (4) Tear fern crystal scoring.

[0066] The tear fern crystal determination was started at 9:00 am on the 16th day. The specific operation was as follows: in a clean laboratory with good light and air humidity controlled at 55% to 60%, 3 μL of washing solution (normal saline) was taken to wash the mouse eye surface three times (to prevent the loss of washing solution and damage to the eye surface, avoid contact with the eye surface tissue during the washing process and the action should be gentle and slow), the washing solution was gently sucked away and applied on a clean glass slide, and after drying at room temperature for 1 hour, the tear fern crystal was observed under a Leica microscope.

[0067] The standard scoring of tear ferning is as follows:

[0068] Level 1: Uniform, dense fern-like crystals can be seen throughout the entire observation range, with small intervals between crystal branches;

[0069] Level 2: The number of crystal branches decreases, the shape becomes smaller, and the gaps between branches increase;

[0070] Level 3: The crystal branches are significantly reduced, the gaps between the branches are significantly increased, and it is difficult for the branches to form new and complete crystals;

[0071] Level 4: No fern-like crystals are observed or the number of crystals is very small in the entire observation range.

[0072] (5) Morphological changes of corneal epithelial cells.

[0073] In this experiment, paraffin sections were stained with H&E, the mouse corneal epithelial area was photographed using a Leica microscope and its imaging system, and the corneal epithelial thickness was measured using Image J software.

[0074] (6) Conjunctival pathological examination.

[0075] In this experiment, paraffin sections were stained with PAS, the mouse corneal epithelial area was photographed with a Leica microscope and its imaging system, and the number of conjunctival goblet cells was measured using Image pro plus 8.0 software.

[0076] (7) Data processing

[0077] One-way ANOVA was performed between the groups using GraphPadPrism 8.0, followed by multiple comparisons using the Dunnett method. All data are presented as mean ± standard error ( ). Compared with the blank group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01.

[0078] (8) Experimental conclusions.

[0079] Tear secretion results Figure 4 As shown, compared with the blank group, the tear secretion of mice in the model group decreased significantly, indicating that the 0.3% BAC-induced dry eye model was successful; compared with the model group, the tear secretion of mice in the TSP treatment group and the positive drug group increased significantly, indicating that tilapia skin peptide can improve the tear secretion function of BAC-induced dry eye mice and that tilapia skin peptide is equivalent to the positive drug in improving the tear secretion function of mice.

[0080] The results of the fern crystallization experiment are as follows Figure 5 As shown: From the perspective of crystal morphology and number, compared with the blank group, the crystal morphology of the model group was incomplete in the entire observation field, and only a few incomplete crystals could be observed; compared with the model group, the crystal morphology of the TSP treatment group and the positive drug group was relatively complete, and the number of crystals increased significantly. From the results of data statistical analysis, the fern-like crystal rating of the model group was significantly higher than that of the blank group, and the fern-like crystal rating of the TSP treatment group and the positive drug group was significantly lower than that of the model group. This shows that tilapia skin peptide can restore the fern-like crystal morphology of tears of BAC-induced dry eye mice, and tilapia skin peptide is equivalent to the positive drug in improving the fern-like crystal morphology of mouse tears.

[0081] The results of morphological changes of corneal epithelial cells are as follows Figure 6 As shown in the figure, the blank group had smooth corneal epithelial cell margins, and the corneal epithelial cells were distributed in 4 to 6 layers; compared with the blank group, the model group had detached and missing corneal epithelial cells, and the entire corneal epithelial cell layer became thinner; compared with the model group, after treatment with tilapia skin peptide and positive drugs, the thickness of the corneal epithelial cell layer increased and the margins were smoother. At the same time, the effect of the TSP treatment group was not much different from that of the positive drug.

[0082] The morphological changes of conjunctival goblet cells are as follows Figure 7 As shown, the blank group had more complete conjunctival goblet cells, while the model group had almost no conjunctival goblet cells. The conjunctival goblet cells in the tilapia skin peptide treatment group and the positive drug group were significantly more than those in the model group, and the tilapia skin peptide was as effective as the positive drug in preventing the loss of conjunctival goblet cells in mice.

[0083] The tilapia skin peptide of the present invention can significantly restore the cell viability of corneal epithelial cells induced by 100mM NaCl, significantly improve the tear secretion function of dry eye mice induced by 0.3% BAC, and restore the integrity of corneal epithelial cells and the number of conjunctival goblet cells. Tilapia skin peptide showed similar potential to positive drugs in in vivo experiments.

[0084] Test Example 3 Isolation and purification of tilapia skin anti-dry eye active peptides.

[0085] (1) Sephdex G-25 chromatography and anti-dry eye activity testing of each component.

[0086] TSP dry powder was prepared into a 50 mg / mL aqueous solution, filtered through a 0.45 μm filter membrane, and the filtrate was loaded onto a Sephdex G-25 chromatography column, using sterile ultrapure water as the mobile phase, the elution rate was 0.45 mL / min, and each chromatographic peak component was collected at a monitoring wavelength of 280 nm. The in vitro anti-dry eye activity of the separated components was detected according to the method in Test Example 1, and the concentration of each component was 250 μg / mL.

[0087] The in vivo anti-dry eye activity of TSP-Ⅱ and TSP-Ⅲ was further tested according to the method in Test Example 2, and the tear secretion was used as an indicator to reflect the in vivo anti-dry eye activity of TSP-Ⅱ and TSP-Ⅲ. The concentrations of TSP-Ⅱ and TSP-Ⅲ used in the in vivo experiment were both 500 μg / mL.

[0088] (2) LC / MS / MS analysis.

[0089] The TSP-Ⅱ and TSP-Ⅲ obtained above were subjected to LC / MS / MS analysis to obtain their molecular weights and other relevant information.

[0090] (3) Data processing

[0091] One-way ANOVA was performed between the groups using GraphPadPrism 8.0, followed by multiple comparisons using the Dunnett method. All data are presented as mean ± standard error ( ). Compared with the blank group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01.

[0092] (4) Experimental conclusions.

[0093] The gel filtration chromatography results of TSP are as follows Figure 8 As shown in A, after TSP was separated by dextran gel G-25, a total of 5 components were obtained, named TSP-Ⅰ, TSP-Ⅱ, TSP-Ⅲ, TSP-Ⅳ and TSP-Ⅴ. The in vitro anti-dry eye activity of each component was further studied, and the results were shown in Figure 8 As shown in Figure B, the anti-dry eye active components of TSP are mainly concentrated in TSP-Ⅱ and TSP-Ⅲ, among which TSP-Ⅱ is the component with the strongest anti-dry eye activity.

[0094] The anti-dry eye activity of TSP-Ⅱ and TSP-Ⅲ was tested in vivo. Fig. 9 As shown, 500 μg / mL of TSP-Ⅱ and TSP-Ⅲ can significantly restore the tear secretion of dry eye mice, and their ability to improve the tear secretion function of mice is equivalent to that of positive drugs.

[0095] TSP-Ⅱ and TSP-Ⅲ were analyzed by LC / MS / MS. Fig.10 The molecular weight of TSP-Ⅱ is distributed between 0.61kDa and 1.93kDa, mainly concentrated between 0.50kDa and 1.50kDa ( Fig.10 A and B). The molecular weight distribution of TSP-Ⅲ is mainly concentrated between 0.50kDa and 1.00kDa, with the largest molecular weight being 1.52kDa and the smallest being 0.44kDa ( Fig.10 C and D). In summary, tilapia skin peptides within the molecular weight range of 0.44 kDa to 2.00 kDa have better effects in preventing and treating dry eyes.

[0096] In summary, the tilapia skin peptide described in the present invention was separated by a G-25 gel column to obtain five components. In vitro experiments have shown that TSP-Ⅱ and TSP-Ⅲ can significantly restore the cell viability of human corneal epithelial cells at a dose of 250 μg / mL; animal experiments have shown that TSP-Ⅱ and TSP-Ⅲ can significantly reduce the dry eye symptoms of dry eye mice at a dose of 500 μg / mL.

[0097] Example 2

[0098] An embodiment of the dry eye prevention and treatment product of the present invention is an eye drop. The eye drop is composed of the tilapia skin peptide prepared in Example 1, a phosphate buffer solution with a pH of 7.2 to 7.4, and water for injection. The effective concentration of the tilapia skin peptide is 2000 μg / mL, and the preparation method of the phosphate buffer solution is as follows: weigh 0.10 g of KCl, 0.10 g of KH2PO4, 4.00 g of NaCl and 1.45 g of Na2HPO4·12H2O2 and dissolve them in water for injection, adjust the solution pH to 7.2 to 7.4 with 1 M HCl or 1 M NaOH, and make up to 500 mL.

[0099] Example 3

[0100] An embodiment of the dry eye prevention and treatment product of the present invention is different from Embodiment 2 only in that in this embodiment, the effective concentration of the tilapia skin peptide is 1000 μg / mL.

[0101] Example 4

[0102] An embodiment of the dry eye prevention and treatment product of the present invention is different from Embodiment 2 only in that in this embodiment, the effective concentration of the tilapia skin peptide is 500 μg / mL.

[0103] Example 5

[0104] An embodiment of the dry eye prevention and treatment product of the present invention is different from Embodiment 2 only in that in this embodiment, the effective concentration of the tilapia skin peptide is 250 μg / mL.

[0105] Comparative Example 1

[0106] The only difference between this comparative example and Example 2 is that the eye drops described in Comparative Example 1 do not contain tilapia skin peptide (an equal amount of physiological saline is used to replace the tilapia skin peptide in the eye drops described in Example 2).

[0107] Comparative Example 2

[0108] This comparative example is the positive medicine eye drops used in Test Example 2, namely, sodium hyaluronate eye drops (sodium hyaluronate concentration is 0.1%).

[0109] Test Example 4

[0110] The mice were randomly divided into 7 groups according to the method described in Test Example 2. One group of mice was selected as a blank control group and only used physiological saline eye drops; the remaining groups used 0.3% BAC to induce dry eyes in mice and at the same time used the eye drops described in Examples 2 to 5 and Comparative Examples 1 to 2 to treat the mice. 5 μL of eye drops were applied to each eye of each mouse, twice a day, for 14 days.

[0111] The effects of the eye drops described in each embodiment and comparative example are shown in Table 1. Compared with the blank control group, # p<0.05; ## p<0.01, compared with comparative example 1, * p<0.05; ** p<0.01.

[0112] Table 1 Test results of the tested samples for preventing and treating dry eyes (n=12, )

[0113]

[0114]

[0115] As shown in Table 1, the tilapia skin peptide eye drops prepared in the present invention (Examples 2-5) all have the effect of preventing and treating dry eyes. Among them, when the eye drops of Example 3 of the present invention are used, the tear secretion of mice can reach 4.17±0.27mm, and its comprehensive effect of preventing and treating dry eyes is the best, which is better than the positive drug group (Comparative Example 2).

[0116] In summary, the tilapia skin peptide of the present invention has a good effect of improving dry eyes, and can be used to prepare products for preventing and treating dry eyes, wherein the products for preventing and treating dry eyes contain 1 to 2000 μg / mL of the tilapia skin peptide prepared in Example 1 of the present invention. The products for preventing and treating dry eyes, regardless of how their administration methods and dosage forms change, can play a role in improving dry eyes as long as they contain the tilapia skin peptide of the present invention.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a small molecule active peptide in the preparation of a product for preventing and treating dry eyes, characterized in that: The small molecule active peptide is tilapia skin peptide; the molecular weight of the small molecule active peptide is 0.40 kDa~2.00 kDa; The preparation method of the tilapia skin peptide comprises the following steps: (a) Sample pretreatment: descaling, defatting and drying the tilapia skin to obtain tilapia skin dry powder; (b) dissolving the tilapia skin dry powder, performing enzymatic hydrolysis, and centrifuging to obtain the supernatant; (c) using an ultrafiltration membrane to ultrafilter the supernatant obtained in step (b), and then concentrating and freeze-drying to obtain the tilapia skin peptide; In step (b), the enzymatic hydrolysis operation is as follows: adding 5 times the volume of ultrapure water to soak the tilapia skin dry powder, adding 3% of the weight of the fish skin dry powder neutral protease, maintaining the pH of the solution system at 7.0±0.5, and performing enzymatic hydrolysis at 50±5°C for 2 hours, adding 0.3% of the weight of the fish skin dry powder alkaline protease, maintaining the pH of the solution system at 9.0±0.2, and performing enzymatic hydrolysis at 50±5°C for 2 hours.

2. The use of the small molecule active peptide according to claim 1 in the preparation of a product for preventing and treating dry eyes, characterized in that: The products for preventing and treating dry eyes include eye injections, eye drops, eye tablets, eye ointments, eye sprays or eye patches.

3. The use of the small molecule active peptide according to claim 2 in the preparation of a product for preventing and treating dry eyes, characterized in that: The product for preventing and treating dry eyes is eye drops; in the eye drops, the concentration of the small molecule active peptide is 1-2000 μg / mL.

4. The use of the small molecule active peptide as claimed in claim 3 in the preparation of a product for preventing and treating dry eyes, characterized in that: In the eye drops, the concentration of the small molecule active peptide is 250-1000 μg / mL.

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