Application of guaijaverin in preparation of eye protection product

Abstract

The invention discloses application of guaijaverin in preparation of eye protection products, and belongs to the technical field of biological medicine. The invention provides application of guaijave glycoside in preparation of eye protection products, a human retina primary Muller cell illumination damage model constructed in vitro in the embodiment, a zebra fish blue-light eye damage model and a mouse retina light damage model, and provides application of guaijave glycoside in preparation of eye protection products. After guaijave glycoside is given to the cell model, the zebra fish animal model and the mouse retina light injury model, guaijave glycoside is found to have a protective effect on cell viability decline of human retina primary Muller cells and cell apoptosis of the animal model, and the retina light injury resisting effect of guaijave glycoside is defined. Based on the eye protection effect of the guaijave glycoside, the guaijave glycoside can be further applied to the development of functional products for treating retinal light injury diseases, relieving visual fatigue and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of guajava glycosides in the preparation of eye protection products. Background Art

[0002] Eye health is an important part of national health, involving people of all ages throughout their lives. However, as electronic products gradually penetrate people's lives, excessive use of eyes has become the norm, troubling people from children to the elderly. Myopia problems in children and adolescents, high blue light exposure problems in young and middle-aged people, and macular degeneration problems in the elderly are all related to varying degrees of retinal light damage. In particular, age-related macular degeneration (AMD) is a difficult-to-treat disease that seriously damages vision and is one of the leading causes of blindness in adults over 50 years old worldwide. With the aging of the population, the incidence of AMD is also increasing year by year. It is estimated that the number of AMD patients worldwide will reach 288 million by 2040, and there is currently no clinically effective drug treatment except for anti-VEGF drugs. It can be seen that related retinal diseases and even visual impairment caused by retinal light damage have a very serious impact on the physical and mental health and quality of life of the people, and increase the burden on families and society. Therefore, preventing retinal light damage and promoting visual health have become important issues that need to be urgently addressed in Healthy China. Summary of the invention

[0003] The purpose of the present invention is to provide an application of guava glycosides in the preparation of eye protection products, and to propose for the first time that guava glycosides have a protective effect on retinal light damage.

[0004] The present invention provides application of guajava glycosides in preparing functional food for alleviating visual fatigue.

[0005] The present invention provides application of guajaroside in preparing eye protection medicine.

[0006] The present invention provides the use of guajaroside in preparing a medicine for preventing and / or treating retinal light damage.

[0007] Preferably, the drug has at least one of the following effects: (1) restoring the vitality of Müller cells damaged by light stress;

[0008] (2) Reduce ocular cell apoptosis;

[0009] (3) Relieve visual fatigue;

[0010] (4) Reduce ocular oxidative stress;

[0011] (5) Reduce ocular inflammation;

[0012] (6) Reduce retinal damage;

[0013] (7)Improve the thinning of the inner and outer nuclear layer thickness of the retina caused by light damage.

[0014] The present invention provides a functional food for relieving visual fatigue, comprising guaijaverin and a food-acceptable excipient.

[0015] Preferably, the concentration of guaijaverin in the functional food is not less than 10 μM.

[0016] The present invention provides an eye-protecting drug, comprising guaijaverin and a pharmaceutically acceptable excipient.

[0017] Preferably, the concentration of guaijaverin in the drug is not less than 10 μM.

[0018] The present invention provides a drug for preventing and / or treating retinal light damage, comprising guaijaverin and a pharmaceutically acceptable excipient.

[0019] Preferably, the concentration of guaijaverin in the drug is not less than 10 μM.

[0020] Beneficial effects: The present invention provides the application of guaijaverin in the preparation of eye-protecting products. In the examples, a light damage model of human retinal primary Müller cells, a blue light eye damage model of zebrafish, and a retinal light damage model of mice were constructed in vitro. After administering guaijaverin to the cell model, zebrafish animal model, and mouse model, it was found that guaijaverin has a protective effect on the decrease in cell viability of human retinal primary Müller cells and apoptosis of ocular cells in the animal model, and the anti-retinal light damage effect of guaijaverin was clarified. Based on the eye-protecting effect of guaijaverin, the present invention can be further applied to the development of drugs for treating retinal light damage diseases and functional products such as relieving visual fatigue. Description of the Drawings

[0021] Figure 1 It is a research result diagram of the anti-light damage effect of 10 μM guaijaverin on human primary Müller cells; when processing data, the statistical processing results are expressed as mean±SE, and statistical analysis is performed using GraphPad Prism (Version 9.3.1) software. A paired t-test is used to compare the statistical differences between groups; *P<0.05, **P<0.01; # P<0.05; * indicates the statistical difference of the same group under different light intensity conditions; # It indicates the statistical difference between the guaijaverin administration group and the control group under strong light irradiation conditions;

[0022] Figure 2Figure showing the results of the study on the anti-photodamage effect of 30 μM guaijaverin on human primary Müller cells; the results of statistical analysis are expressed as mean ± SE, and GraphPad Prism (Version 9.3.1) software was used for statistical analysis. Paired t-tests were used to compare the statistical differences between groups; ****P<0.0001; #### P<0.0001; * indicates the statistical difference within the same group under different light intensity conditions, and ns represents no statistical difference; # Indicates the statistical difference between the guaijaverin administration group and the control group under strong light irradiation conditions;

[0023] Figure 3 Typical figure of the fluorescence intensity of apoptotic cells in the eyes of zebrafish after treatment with guaijaverin and lutein. The green fluorescent particles indicated by the red arrows in the figure are apoptotic cells;

[0024] Figure 4 Quantitative figure of the fluorescence intensity of apoptotic cells in the eyes of zebrafish after treatment with guaijaverin and lutein; SPSS 26.0 software was used for statistical analysis, **P<0.01; ***P<0.001; ****P<0.0001; # P<0.05; ## P<0.01; ### P<0.001; #### P<0.0001; * indicates the statistical difference between the model group and the normal control group, # Indicates the statistical difference between different administration groups and the model group, and ns indicates no significant difference compared with the model group; the same below;

[0025] Figure 5 H&E staining figure of mouse retina; A in the figure represents the H&E staining figure of mouse retina tissue and the thickness figures of the outer nuclear layer and inner nuclear layer of the retina, scale bar: 500 μm and 20 μm; B and C represent the quantitative figures of the thickness of the outer nuclear layer and inner nuclear layer of the retina, number of mice (n = 6);

[0026] Figure 6 TUNEL staining figure of apoptotic cells in mouse retina; A in the figure represents the TUNEL fluorescence staining figure of mouse retina, DAPI blue fluorescence represents the nucleus, and TUNEL red fluorescence represents apoptotic cells, scale bar: 500 μm and 20 μm; B represents the quantitative figure of the fluorescence intensity of apoptotic cells in the retina, number of mice (n = 6 - 7);

[0027] Figure 7 Figure showing the results of the total antioxidant capacity (T-AOC) of mouse plasma, number of mice (n = 4);

[0028] Figure 8It is the result graph of IL-6 in mouse plasma, and the number of mice is (n = 6).

[0029] Figure 9 It is the graph of the expression level of HO-1 protein in mouse retinal tissue; in the figure, A represents the Western blot detection graph of the expression of HO-1 protein and the internal reference GAPDH protein; B represents the quantitative graph of HO-1 protein expression, and the number of mice is (n = 4). Specific implementation manners

[0030] The present invention provides the application of guaijaverin in the preparation of a functional food for relieving visual fatigue.

[0031] The functional foods referred to in the present invention include: 1) ordinary foods that have been experimentally proven to have the function of regulating the physiological functions of the body and enhancing the health of the body, but cannot be classified as health foods; 2) health foods with health functions such as being able to improve the health status of the body.

[0032] The present invention has no special limitation on the source of the guaijaverin (Guaijaverin, molecular formula: C 20 H 18 O 11 , molecular weight: 434.35.). It can be extracted by existing methods or obtained by purchase. For example, in the examples, it was purchased from Chengdu Mansite Biotechnology Co., Ltd. (extraction source: guava leaves), with the number A1157. In the examples of the present invention, cell models and animal models were used, and through experimental verification, it was found that guaijaverin has a protective effect on the decrease in cell viability and apoptosis of primary human retinal Müller cells. The present invention first proposes that guaijaverin has a protective effect on the light damage of retinal cells.

[0033] The present invention provides the application of guaijaverin in the preparation of a drug for protecting eyes.

[0034] The application described in the present invention is preferably the same as the above, and will not be elaborated here. The drug described in the present invention preferably has at least one of the following effects: (1) restoring the viability of Müller cells damaged by light stress;

[0035] (2) reducing apoptosis of eye cells;

[0036] (3) relieving visual fatigue;

[0037] (4) reducing oxidative stress response in the eyes;

[0038] (5) reducing inflammatory response in the eyes;

[0039] (6) reducing retinal damage;

[0040] (7) improving the thinning of the inner and outer nuclear layers of the retina caused by light damage.

[0041] In the embodiments of the present invention, experiments were conducted using cell models, zebrafish models, and mouse models, all of which confirmed that guaijaverin can reduce retinal damage and retinal cell apoptosis. In the mouse experiments of the present invention, it was also confirmed that guaijaverin can enhance antioxidant capacity and reduce retinal oxidative stress, thus reducing ocular oxidative stress response; and guaijaverin can reduce the secretion of inflammatory factors and lower the inflammation level, so it can also reduce ocular inflammatory response.

[0042] The present invention provides the use of guaijaverin in the preparation of a drug for preventing and / or treating retinal light damage.

[0043] The use of the present invention is preferably the same as described above and will not be elaborated here.

[0044] The present invention provides a functional food for relieving visual fatigue, comprising guaijaverin and a food-acceptable excipient.

[0045] The present invention does not particularly limit the type of the functional food, such as eye-care gel gummies, chewable tablets, (soft) capsules, solid tablets, or oral liquids, etc. Through cell experiment concentration verification in the embodiments, the concentration of guaijaverin in the functional food is preferably not less than 10 μM.

[0046] The present invention provides an eye-care drug, comprising guaijaverin and a pharmaceutically acceptable excipient.

[0047] The dosage form of the drug of the present invention preferably includes solid preparations, capsule preparations, or granule preparations. The concentration of guaijaverin in the drug of the present invention is preferably not less than 10 μM.

[0048] The present invention provides a drug for preventing and / or treating retinal light damage, comprising guaijaverin and a pharmaceutically acceptable excipient.

[0049] The dosage form of the drug of the present invention preferably includes solid preparations, capsule preparations, or granule preparations. The concentration of guaijaverin in the drug of the present invention is preferably not less than 10 μM.

[0050] To further illustrate the present invention, the following examples are used to describe in detail the use of guaijaverin provided by the present invention in the preparation of eye-care products, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1 Effects of Guaijaverin on the White Light Damage Model of Human Retinal Primary Müller Cells

[0052] 1. Experimental method:

[0053] 1.1 Culturing and subculturing of human retinal primary Müller cells

[0054] Primary human retinal Müller cells were obtained from the retinal tissues of donors and cultured.

[0055] Obtaining primary human Müller cells: The retina was separated from the retinal pigment epithelium-choroid-sclera eyecup using surgical scissors and forceps. A 1-cm 2 sized retinal tissue was placed into a T25 culture flask containing 5 mL of complete DMEM medium. The culture flask was wrapped with aluminum foil and then placed in an environment at 4 °C overnight. The trypsin digestion solution was preheated in a 37 °C water bath. The retinal tissue was transferred to a new culture flask containing 5 mL of preheated trypsin digestion solution and incubated in a 37 °C, CO₂ incubator for 60 min. The digested retina was transferred to a cell culture dish containing 5 mL of complete DMEM medium using sterile forceps and cut into small pieces (1×1 mm) under a dissecting microscope. These small pieces of retinal tissue were transferred back to the T25 culture flask together with the complete DMEM medium. The small retinal tissues were evenly distributed at the bottom of the culture flask using an 18G needle with a 90-degree bend at the top and pressed onto the bottom of the culture flask under the microscope. Subsequently, 2 mL of complete DMEM medium was carefully added. The culture flask was vertically placed in a cell incubator (37 °C, CO₂) for 15 min to allow the retinal fragments to better adhere to the bottom of the T25 culture flask, and then the T25 culture flask was placed horizontally and cultured in the incubator. When cultured to the 7th day, 2 mL of complete DMEM medium was supplemented. The entire process minimized interference with the culture flask.

[0056] Culturing primary human Müller cells: On the 10th day of culture, 4 mL of fresh complete DMEM medium was replaced, and then the medium was changed twice a week. It took about 2-3 weeks for primary human Müller cell colonies to appear from the tissue, and another 2-3 weeks to reach a density of 80%-90%.

[0057] Subculture of human primary Müller cells: The digestion time of human primary Müller cells is longer than that of general cell lines. Initially, subculture is carried out at a ratio of 1:1, and after passage 2 (P2), subculture can be performed at a ratio of 1:2 to 1:3. Usually, P3 cells can be used for cell experiments. Although it is generally possible to passage up to P10, it also depends on the situation of different donors. The specific subculture steps are as follows: Remove the culture medium and rinse with 3 mL of sterile PBS solution. Remove the PBS solution and add 2 mL of trypsin digestion solution to each T25 culture flask for digestion (37 °C, 6 - 8 min). When more than half of the cells detach from the bottom of the culture flask, add 2 mL of complete DMEM medium to terminate the digestion. Pipette to dissociate all the cells and transfer all the liquid to a 15 mL centrifuge tube. Precipitate human primary Müller cells by centrifugation (200 g, 5 min, 20 °C). Resuspend the cell pellet with 1 mL of complete DMEM medium and transfer it to a new T25 culture flask containing 3 mL of complete DMEM culture, then return it to the incubator for culture.

[0058] Cryopreservation and resuscitation of human primary Müller cells: Digest and collect human primary Müller cells as described above. Resuspend the cell pellet with 1 mL of cryopreservation solution and transfer it to a cryotube. Transfer the cryotube-containing cell program cooling box to an -80 °C refrigerator for cryopreservation, and transfer the cryotube to a liquid nitrogen tank for storage the next day. When resuscitating the cells, thaw the cryotube in a pre-warmed 37 °C water bath. After it has completely melted, add the suspension to 4 mL of pre-prepared complete DMEM culture medium, mix well, and centrifuge at 200 g for 5 min at room temperature. Then discard the supernatant, resuspend the cells with 4 mL of complete DMEM culture medium, and transfer them to a cell culture flask. Place it in a 37 °C, 5% CO2 incubator for culture.

[0059] 1.2 Photostress modeling and guaiacin drug treatment of human retinal primary Müller cells

[0060] Seed human primary Müller cells in the logarithmic growth phase at 5,000 cells / well in a 96-well plate and culture them in a 37 °C, 5% CO2 incubator for 24 h. Aspirate the culture medium. Set up 6 replicates for the guaiacin drug solution group, and add 100 μL of guaiacin drug solution diluted with DMEM solution to each well at concentrations of 10 μM and 30 μM respectively, with DMSO as the solvent. The control group is added with 100 μL of DMEM solution. Use a self-made strong light irradiation system in the laboratory to perform photostress modeling on human retinal primary Müller cells cultured in vitro. The strong light irradiation group is irradiated with strong light of 32 k Lux for 4 h, and the weak light irradiation group is irradiated with weak light of 5 k Lux for 4 h. Then use the AlamarBlue kit to detect cell viability.

[0061] 1.3 Cell viability detection

[0062] After the cells were stimulated by light, the cell viability was detected using the AlamarBlue kit. The specific steps were as follows: Discard the cell culture supernatant, wash twice with 100 μL of PBS, add 100 μL of AlamarBlue reagent diluted 1:10 with DMEM solution, and incubate in a 37 °C, 5% CO2 incubator for 4 h. Read the fluorescence of each well using a microplate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0063] 2. Experimental results:

[0064] The above method was used to construct a light stress model of human primary Müller cells derived from peripheral retinal tissue, and the effects of different concentrations of guaijaverin on the viability of Müller cells after strong light irradiation were explored. Figure 1 As can be seen, after 4 h of strong light irradiation, the viability of human primary Müller cells decreased significantly. However, after pre-incubation with 10 μM of guaijaverin for 24 h, the cell viability of Müller cells could be significantly restored, indicating that it could protect Müller cells from strong light damage. At the same time, Figure 2 as can be seen, after pre-incubation with 30 μM of guaijaverin for 24 h, the cell viability of Müller cells decreased after strong light modeling could be significantly restored, and the cell viability of Müller cells after strong light stress could be restored to the level under weak light irradiation conditions. It shows that guaijaverin has a very significant protective effect on Müller cells against light stress damage.

[0065] Example 2 Effects of Guaijaverin on the Blue Light Eye Injury Model of Zebrafish

[0066] This experiment was completed by Hangzhou Huante Biotechnology Co., Ltd. (Project No.: 8254).

[0067] 1. Experimental method:

[0068] 1.1 Experimental animals

[0069] Zebrafish were all raised in fish culture water at 28 °C (Water quality: Add 200 mg of instant sea salt to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L CaCO3). They were provided by the fish breeding center of Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK(Zhe)2022 - 0004. The feeding management met the requirements of international AAALAC certification (Certification No.: 001458), and the IACUC ethical review number was: IACUC - 2024 - 8254 - 01.

[0070] 1.2 Instruments, consumables and reagents

[0071] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China); Electric focusing continuously variable fluorescence microscope (AZ100, Nikon, Japan); Blue light instrument (50w 450nm, China).

[0072] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); Pronase E (batch number G12511Y118034, Shanghai Yuanye Bio-Technology Co., Ltd., China); Acridine orange (AO, batch number C12894919, Shanghai Macklin Biochemical Co., Ltd., China); Methyl cellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0073] 1.3 Determination of the maximum tested concentration (MTC)

[0074] Wild-type AB strain zebrafish at 1 day post-fertilization (1 dpf) were randomly selected after hatching and irradiated with blue light to establish a zebrafish blue light eye injury model. At 3 dpf, model zebrafish with good developmental status were randomly selected and distributed into 6-well plates, with 30 zebrafish in each well (experimental group). Different concentrations of guaijaverin were dissolved in water and administered respectively (concentrations are shown in Table 1). At the same time, a normal control group and a model group were set up, and the volume of each well was 3 mL. After treatment at 28 °C for 1 day, the MTC of guaijaverin on model zebrafish was determined.

[0075] 1.4 Evaluation of the anti-blue light eye protection effect

[0076] Wild-type AB strain zebrafish at 1 dpf were randomly selected after hatching and irradiated with blue light to establish a zebrafish blue light eye injury model. At 3 dpf, model zebrafish with good developmental status were randomly selected and distributed into 6-well plates, with 30 zebrafish in each well (experimental group). Different concentrations of guaijaverin were dissolved in water and administered respectively (concentrations are shown in Table 2), and the positive control lutein was at a concentration of 62.5 μg / mL. At the same time, a normal control group and a model group were set up, and the volume of each well was 3 mL. After treatment at 28 °C for 1 day, the zebrafish in each experimental group were stained with AO in the dark for 30 min. After washing 3 times with standard dilution water, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photographing. Image J software was used to analyze and collect data, and the fluorescence intensity of apoptotic cells in the zebrafish eyes was analyzed and statistically analyzed. The anti-blue light eye protection effect of guaijaverin was evaluated based on the statistical analysis results of the above indicators. The statistical processing results were expressed as mean ± SE. SPSS 26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0077] Table 1 Results of the experiment on exploring the concentration of the anti-blue light and eye protection effect of guaijaverin (n = 30)

[0078]

[0079]

[0080] 2. Experimental results:

[0081] 2.1 MTC

[0082] Under the conditions of this experiment, the MTC of guaijaverin against blue light and eye protection effect on zebrafish was 150 μg / mL.

[0083] 2.2 Evaluation of anti-blue light and eye protection effect

[0084] Under the conditions of this experiment, the blue light-induced eye damage model in zebrafish showed apoptosis of ocular cells. After treatment with different concentrations of guaijaverin, the apoptosis of ocular cells in zebrafish could be significantly reduced, indicating that guaijaverin has the eye protection effect of anti-ocular blue light damage. Moreover, guaijaverin had the same significant eye protection effect as lutein at a dose of 125 μg / mL, and guaijaverin had a more significant eye protection effect than lutein at a dose of 150 μg / mL. See Table 2 Figure 3 and Figure 4 .

[0085] Table 2 Results of the experiment on evaluating the anti-blue light and eye protection effect of guaijaverin (n = 10)

[0086]

[0087] Example 3 Effect of guaijaverin on the light-induced retinal damage model in mice

[0088] 1. Experimental method:

[0089] 1.1 Experimental animals

[0090] 8-week-old male BALB / c mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Before the start of the experiment, all mice were adaptively fed under a 12:12 h light / dark cycle in an animal room at a certain temperature and humidity for 1 week, and provided with normal food and drinking water every day. All experimental operations were carried out in accordance with the guidelines for animal care and use of the Animal Research Committee of Zhejiang University.

[0091] 1.2 Construction of the mouse light damage model

[0092] A self-made light box with a length of 108 cm, a width of 50 cm, and a height of 72 cm was used. The light intensity was detected by a lux meter and was 8500 - 10000 Lux. After eye examination of the mice, 60 mice with normal eyes were randomly divided into a control group, a model group, a lutein group (100 mg / kg), a low-dose guaijaverin group (50 mg / kg), and a high-dose guaijaverin group (100 mg / kg), with 12 mice in each group. Lutein and guaijaverin were suspended in a 5% sodium carboxymethylcellulose (CMC-Na) solution and administered by gavage once a day. The mice in the blank and model groups were gavaged with an equal volume of 5% CMC-Na. The administration was continued for 10 days. After 10 days, the mice in the model group, lutein group, low-dose guaijaverin group, and high-dose guaijaverin group were dark-treated for 36 h, then atropine ophthalmic gel was applied to both eyes of the mice to dilate the pupils. The mice were placed in a self-made light device with a light intensity of 8000 - 10000 Lux (the mice in the blank group were raised in a normal environment) and given continuous light for 24 h. After the light exposure, blood was collected by enucleating the eyeballs of each group of mice, and the eyeball tissues were collected for relevant tests in subsequent experiments.

[0093] Table 3 Experimental instruments involved in this example

[0094]

[0095]

[0096] Table 4 Experimental consumables and reagents involved in this example

[0097]

[0098]

[0099] 1.3 Histopathological examination

[0100] For each group, 6 mouse eyeball tissues were taken for hematoxylin-eosin staining (HE) and TUNEL fluorescence staining of apoptotic cells, and the histopathological changes of the mouse eyeball tissues were observed using an optical microscope (completed by Wuhan Sevier Biotechnology Co., Ltd.).

[0101] 1.3.1 Experimental procedure for H&E staining

[0102] a) Dewax the paraffin sections to water: sequentially place the sections in environmental protection type dewaxing solution Ⅰ for 20 min - environmental protection type dewaxing solution Ⅱ for 20 min - absolute ethanol Ⅰ for 5 min - absolute ethanol Ⅱ for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0103] b) Pretreatment: Immerse the sections in the hematoxylin-eosin (H&E) high-definition constant staining pretreatment solution for 1 min.

[0104] c) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0105] d) Eosin staining: Dehydrate the sections in 95% gradient alcohol for 1 min and then stain them in eosin solution for 15 s.

[0106] e) Dehydration and sealing: the sections were sequentially placed in anhydrous ethanol I for 2 min-anhydrous ethanol II for 2 min-anhydrous ethanol III for 2 min-n-butanol I for 2 min-n-butanol II for 2 min-xylene I for 2 min-xylene II for 2 min, and sealed with transparent neutral gum.

[0107] f) Microscope examination, image acquisition and analysis.

[0108] 1.3.2 TUNEL staining experimental steps

[0109] a) Dewaxing of paraffin sections: sequentially place the sections into environmentally friendly dewaxing solution I for 10 min - environmentally friendly dewaxing solution II for 10 min - environmentally friendly dewaxing solution I and II for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and wash with distilled water.

[0110] b) Proteinase K repair: After the slices are slightly dried, use a tissue pen to draw a circle around the tissue (to prevent the liquid from flowing away), add proteinase K working solution to the circle to cover the tissue, and incubate in a 37°C incubator for 20 minutes. Place the slide in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, each time for 5 minutes. (Protease K working solution preparation method, stock solution: PBS = 1:9).

[0111] c) Membrane permeabilization: After the slices are slightly dried, drip the membrane permeabilization working solution into the circle to cover the tissue, incubate at room temperature for 20 minutes, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The membrane permeabilization solution is 0.1% triton. The preparation method is triton stock solution: PBS = 1:1000).

[0112] d) Room temperature equilibrium: After the slices are slightly dried, add buffer to cover the tissue and incubate the buffer at room temperature for 10 minutes.

[0113] e) Add reaction solution: according to the number of slices and tissue size, take appropriate amount of TDT enzyme, dUTP and buffer in the tunel kit and mix them in the ratio of 1:5:50, add them to the circle to cover the tissue, lay the slices flat in a humidified box, incubate at 37℃ for 1 hour, and add a small amount of water to the humidified box to maintain humidity.

[0114] f) DAPI counterstaining of cell nuclei: Wash the sections with PBS (PH7.4) for 3 times, 5 min each time. After removing PBS, add DAPI staining solution in the circle and incubate at room temperature for 10 min away from light.

[0115] g) Sealing: Place the slides in PBS (PH7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time. After the sections are slightly dried, seal them with anti-fluorescence quenching sealing medium.

[0116] h) Microscopic examination and photography: The sections were observed and images were collected under a fluorescence microscope. (DAPI ultraviolet excitation wavelength is 330-380nm, emission wavelength is 420nm, emitting blue light; TMR excitation wavelength is 510-561nm, emission wavelength is 590nm, emitting red light.

[0117] i) Interpretation of Tunel red fluorescence results: The cell nuclei stained with DAPI are blue under ultraviolet excitation. The Tunel kit is labeled with TMR fluorescein, and the positive apoptotic cell nuclei are red.

[0118] 1.4 Western Blot detection of related protein expression levels

[0119] Take the mouse retina, add 100-150μL of RIPA lysis buffer for every 10mg tissue, and use the BCA protein quantification kit to detect the protein content after sufficient lysis. Add an appropriate amount of loading buffer to the protein lysis buffer and heat at 95℃ for 10min to prepare the protein loading sample. Add an appropriate amount of sample to 10% SDS-PAGE gel, transfer to PVDF membrane after electrophoresis. After the transfer, use 5% skim milk to block at room temperature for 1h, then add the corresponding primary antibody and incubate overnight in a 4℃ refrigerator. After incubating the PVDF membrane with the secondary antibody of the corresponding species at room temperature for 1h, develop it in a red laser two-color imaging system, use the red laser two-color imaging system supporting software for band analysis, and use GAPDH protein expression as an internal reference.

[0120] 1.5 Determination of IL-6 levels in mouse plasma

[0121] The mouse blood was collected in an anticoagulant tube, centrifuged at 3000 RPM for 10 min at 4°C, and the upper plasma was collected. The IL-6 content was determined according to the method provided by the kit manufacturer.

[0122] 1.6 Determination of T-AOC levels in mouse plasma

[0123] The mouse blood was collected in an anticoagulant tube, centrifuged at 3000 RPM for 10 min at 4°C, and the upper plasma was collected. The content of T-AOC was determined according to the method provided by the kit manufacturer.

[0124] 1.7 Statistical analysis

[0125] Graphs were plotted and statistical analyses were performed using Prism 8 software. All data were expressed as mean ± standard error. Independent sample t-tests were used to analyze the differences between the two groups, and P < 0.05 was considered statistically significant.

[0126] 2. Experimental results

[0127] 2.1 H&E histopathological evaluation

[0128] The results are as Figure 5 shown. From the H&E staining results of the retina, it can be seen that light damage led to morphological changes such as loosening and reduced thickness in the outer nuclear layer and inner nuclear layer of the retina in the model group mice, indicating that light caused damage to retinal cells ( Figure 5 A in Figure 5 ). However, both low-dose and high-dose guaijaverin under light conditions could well improve the morphological changes in the outer nuclear layer and inner nuclear layer of the retina and increase the thickness of the outer nuclear layer and inner nuclear layer (

[0129] B - C in

[0130] ), indicating that guaijaverin has a good effect on improving retinal light damage. And compared with the positive drug lutein, the high dose of guaijaverin has a better anti-light damage and eye protection effect than lutein. Figure 6 According to the TUNEL fluorescence staining results of apoptotic cells in the retina tissue, it can be seen that light caused a large number of apoptotic retinal cells in the model group mice (red fluorescence, Figure 6 A in

[0131] ), and both low-dose and high-dose guaijaverin could well improve the cell apoptosis in the retina caused by light. Moreover, the protective effect of low-dose guaijaverin on retinal cell apoptosis is equivalent to that of the positive drug lutein, and the high dose of guaijaverin has a better protective effect than lutein. (

[0132] A - B in Figure 7 ).

[0133] 2.4 Determination of plasma total antioxidant capacity

[0134] Plasma total antioxidant capacity (T-AOC) refers to the total antioxidant level composed of various antioxidant substances and antioxidant enzymes, etc. To protect cells from oxidative stress damage caused by reactive oxygen free radicals, T-AOC can be used to evaluate the total antioxidant capacity of bioactive substances. In the present invention, light led to a decrease in the T-AOC level in the model group mice, while both low-dose and high-dose guaijaverin could significantly increase the T-AOC level, indicating that guaijaverin can improve the total antioxidant capacity in vivo ( Figure 7 ).

[0133] 2.4 Determination of plasma interleukin-6 (IL-6) level

[0134] Interleukin refers to cytokines produced by white blood cells and playing a regulatory role among white blood cells. It plays an important role in transmitting information, activating and regulating immune cells, mediating the activation, proliferation and differentiation of T and B cells, and in inflammatory responses. In the present invention, light caused an increase in the secretion of IL-6 in the mice of the model group, indicating that light led to an increase in the inflammatory level in the mice, while low and high doses of guaijaverin could significantly reduce the level of IL-6 in the body, indicating that guaijaverin could significantly reduce the inflammatory level in the body( Figure 8 ).

[0135] 2.5 Detection of the protein expression level of heme oxygenase-1 (HO-1) in the retina

[0136] The expression of HO-1 protein can be upregulated after oxidative stress and cell damage. The upregulation of HO-1 level is a characteristic of the downstream effects of oxidative stress, especially in a pro-oxidative state. In the present invention, it was detected by Western blot experiment that the expression of HO-1 protein in the retina tissue of the mice in the model group was upregulated after light damage, indicating that significant oxidative stress reactions occurred in the retinas of the mice in the model group, while the high dose of guaijaverin significantly reduced the expression level of HO-1 protein in the retina tissue, indicating that guaijaverin reduced the retinal oxidative stress reaction( Figure 9 in A-B). GAPDH was used as an internal reference for protein expression.

[0137] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of guaijaverin in the preparation of a functional food for relieving visual fatigue.

2. Use of guaijaverin in the preparation of a drug for eye protection.

3. Use of guaijaverin in the preparation of a drug for preventing and / or treating retinal light damage.

4. The application according to claim 2 or 3, characterized in that The drug has at least one of the following effects: (1) restoring the viability of Müller cells damaged by light stress; (2) reducing apoptosis of ocular cells; (3) relieving visual fatigue; (4) reducing oxidative stress response in the eye; (5) reducing inflammatory response in the eye; (6) reducing retinal damage; (7) improving the thinning of the inner and outer nuclear layers of the retina caused by light damage.

5. A functional food for relieving visual fatigue, characterized in that, It includes guaijaverin and a food-acceptable excipient.

6. The functional food according to claim 5, characterized in that, The concentration of guaijaverin in the functional food is not less than 10 μM.

7. A drug for protecting eyesight, characterized in that, It includes guaijaverin and a pharmaceutically acceptable excipient.

8. The drug according to claim 7, wherein The concentration of guaijaverin in the drug is not less than 10 μM.

9. A drug for preventing and / or treating retinal light damage, characterized in that, It includes guaijaverin and a pharmaceutically acceptable excipient.

10. The drug according to claim 9, wherein, The concentration of guaijaverin in the drug is not less than 10 μM.

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