Pharmaceutical composition for preventing myopia and degenerative fundus disease and application

Abstract

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for preventing myopia and degenerative fundus diseases and application. Comprising the following components: peony seed oil, lutein ester and zeaxanthin. The total mass of the lutein ester and the zeaxanthin accounts for 0.3%-1% of the weight of the peony seed oil; the weight ratio of the lutein ester to the zeaxanthin is (2-10): 1. Experiments of a guinea pig high-myopia animal model prove that the pharmaceutical composition can significantly improve the diopter of a guinea pig of the model and reduce the eye axis change of the guinea pig, ERG detection is carried out on the guinea pig retinitis pigmentosa model, and the result shows that the pharmaceutical composition can effectively improve ERG bright adaptation and dark adaptation, and has good clinical application prospects. A retinal angiography experiment is carried out on a mouse wet age-related macular degeneration CNV model, and retinal angiography data of model mice taking the medicine composition are effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a pharmaceutical composition and application thereof for preventing myopia and degenerative fundus diseases. Background Art

[0002] High myopia (HM) refers to myopia with a refractive power higher than 6.00D. As the refractive power increases, the axial length of the eye continues to lengthen, and the risk of HM eye complications will continue to increase, such as vitreous degeneration, chorioretinal atrophy, retinal holes, choroidal neovascularization, macular hemorrhage, concurrent cataracts, secondary glaucoma, strabismus, amblyopia, etc. HM is a highly blinding eye disease. In recent years, the incidence rate has continued to increase and is showing a trend of younger age. It is estimated that by 2050, the number of HM patients worldwide will reach 938 million, and 10.1% of HM patients will become blind or have impaired vision. The prevalence of HM among adolescents in my country is as high as 6.69%-38.4%, threatening human visual health and quality of life.

[0003] Retinitis pigmentosa (RP) is a kind of ophthalmic genetic disease and also a degenerative disease. Its main clinical manifestations are night blindness, visual field loss, retinal function decline, vision loss, and even blindness. Its pathological characteristics are the progressive disappearance of photoreceptor cells (rods and cones). Its condition often develops progressively and is a blinding eye disease. According to the data of RP reports in recent years, it is found that one in every 3,000-7,000 people in the world suffers from the disease, with a total of more than 1 million people; and among the people with visual impairment in my country, about 400,000 people have lost their vision due to RP.

[0004] Age-related macular degeneration: Retinal AMD is a disease that increases in incidence with age. The main clinical manifestations are metamorphopsia, central scotoma, and decreased central vision. It often occurs in the elderly over 50 years old, with both eyes affected successively or simultaneously, and vision progressively deteriorates. AMD is an age-related eye disease caused by age-related changes in the retinal pigment epithelium (RPE) and retina. The disease is the primary cause of irreversible decline or even loss of central vision in middle-aged and elderly people. AMD is currently clinically divided into "dry" and "wet". Wet AMD develops rapidly, with a sudden decrease in vision in the affected eye, accompanied by metamorphopsia or central scotoma. The fundus is manifested by the formation of choroidal neovascularization (CNV) under the fovea of ​​the macula, and bleeding, exudation, edema and scarring of the RPE due to vascular damage. However, due to the rapid onset of wet AMD, patients' vision can be severely and irreversibly reduced or even lost in a short period of time. Although wet AMD only accounts for 20% of all AMD cases, the blindness rate can be as high as 80%, which has adverse consequences on the quality of life of patients and their families. There is currently no clinical treatment for AMD that can completely cure it or prevent recurrence, so the treatment of wet AMD is particularly important.

[0005] With respect to the above-mentioned comprehensive eye diseases, there is no drug for prevention and treatment with good comprehensive effect in the prior art. Summary of the invention

[0006] The purpose of the present invention is to overcome the defect of poor prevention and treatment effect of myopia in the prior art, and to provide a pharmaceutical composition and application for preventing myopia and degenerative fundus disease.

[0007] In a first aspect, the present invention provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising the following ingredients: peony seed oil, lutein ester and zeaxanthin; the total mass of the lutein ester and the zeaxanthin accounts for 0.3%-1% of the weight of the peony seed oil; the weight ratio of the lutein ester to the zeaxanthin is (2-10):1.

[0008] Further preferably, the total weight percentage of the lutein ester and the zeaxanthin to the peony seed oil is 0.3%-0.8%.

[0009] Preferably, the total weight percentage of the lutein ester and the zeaxanthin to the peony seed oil is 0.4%-0.6%.

[0010] Preferably, the weight ratio of the lutein ester to the zeaxanthin is (4-8):1.

[0011] More preferably, the weight ratio of the lutein ester to the zeaxanthin is (4-6):1.

[0012] The pharmaceutical composition of the invention comprises three effective ingredients: peony seed oil, lutein ester and zeaxanthin.

[0013] As a new resource food, peony seed oil is an oil product extracted from peony seeds through a certain process. It can be used as daily oil and has considerable medicinal value. Peony seed oil is rich in ω-3 unsaturated fatty acids and α-linolenic acid, has high nutritional value and is safe and non-toxic. It can play an antioxidant, lipid-lowering, UV-resistant, anti-inflammatory, cardiovascular and nerve-protecting role, and is known as "plant brain gold".

[0014] Lutein ester is an important carotenoid fatty acid ester, which is decomposed into free lutein after being absorbed by the human body. It has the basic function of crystalline lutein to supplement the lutein lost by the human body.

[0015] Zeaxanthin is a fat-soluble pigment component of lutein and a dihydroxy derivative of β-carotene. A large number of studies have shown that zeaxanthin has health benefits such as anti-oxidation, prevention of macular degeneration, treatment of cataracts, prevention of cardiovascular diseases, enhancement of immunity, and slowing down of atherosclerosis, and is closely related to human health.

[0016] The retina receives light stimulation for a long time and undergoes the photoelectric signal conversion process, especially in the photoreceptor cell layer, where the metabolism is very active. Even in a dark environment, the dark current maintenance process on the photoreceptor cells requires a large amount of oxygen consumption. The high metabolism and high oxygen consumption of the retina make it vulnerable to oxidative stress damage, causing lipid peroxidation (LPO) on the cell membrane, thereby producing a variety of aldehydes. When there is excessive accumulation of aldehydes in the cells or they cannot be cleared in time, a variety of cell damage links will be activated, including endoplasmic reticulum stress (ERS) reaction, cell autophagy, and cell death mediated by apoptosis, causing corresponding tissue and organ damage. RP can cause photoreceptor cells to die and disappear; it is generally believed that rods and cones disappear mainly through apoptosis; therefore, ERS reactions may exist in the pathogenesis of RP.

[0017] The endoplasmic reticulum stress (ERS) channel mainly includes three pathways, namely, the inositol-requiring protein-1 (IRE-1) pathway, the activating transcription factor-6 (ATF-6) pathway, and the protein kinase-like endoplasmic reticulum kinase (PKR-related ER kinase, PERK) pathway; when ERS occurs, a variety of upstream and downstream related signal molecules also undergo corresponding changes. In the technical scheme of the present invention, glucose regulated protein 78 (GRP78) and cysteine-containing aspartate proteinase 12 (Caspase12) are selected as detection indicators reflecting the upstream and downstream molecules of ERS.

[0018] At present, in the construction of inducible RP animal models, the retinitis pigmentosa animal model established with N-methyl-N-nitrosourea (MNU) as an inducer has been widely studied and has a high success rate. Therefore, in the technical scheme of the present invention, the MNU-induced mouse RP model is selected, and the mouse ERG and retinal HE staining confirm the success of the disease model. After establishment, cell biology technology is used to detect the changes in proteins related to the retinal endoplasmic reticulum stress response pathway, as well as the treatment improvement of RP mice after intervention with the drug combination of the present invention.

[0019] Oxidative stress (OS) is the result of an imbalance between oxidation and antioxidant defense mechanisms when the body is subjected to harmful stimuli. It is considered to be a key common pathway for a variety of diseases such as glaucoma, age-related macular degeneration, dry eye, keratoconus and myopia. OS can cause inflammation and cell death, and can also damage cell function by destroying lipids, proteins, etc. When the body is subjected to harmful stimuli, products such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulate in large quantities in the retina and choroid, causing lipid peroxidation, activating the redox pathway in the cell, inducing scleral cell apoptosis, destroying the normal growth regulation mechanism of the eyeball, inducing endoplasmic reticulum stress, causing excessive eye growth, and forming myopia. The technical solution of the present invention constructs a guinea pig high myopia model by form deprivation method, measures the guinea pig eye axis, changes in diopter, and studies the mechanism of the pathway proteins related to retinal endoplasmic reticulum stress, and clarifies the efficacy of the pharmaceutical composition of the present invention on high myopia.

[0020] Oxidative damage in the human body will produce too many reactive oxygen free radicals and reactive nitrogen free radicals, which exceed the body's ability to clear them in time, thus causing damage. The main feature of early AMD is the reduction of rod cells. As light is absorbed, the rod cells wither and die. The retina is exposed to light for a long time throughout a person's life. The retina contains a large amount of photosensitizer lipofuscin, which consumes a lot of oxygen and is more susceptible to oxidative damage. Oxidative stimulation can produce reactive oxygen. Oxidative damage to the retina causes apoptosis of retinal cells and lipid peroxidation, activates the redox pathway in the cell, and induces endoplasmic reticulum stress.

[0021] The present invention uses a laser-induced wet age-related macular degeneration CNV model in mice, and uses retinal angiography to observe the mechanism of neovascularization in mice to study the proteins in the retinal endoplasmic reticulum stress-related pathway, thereby clarifying the efficacy of the drug combination of the present invention on laser-induced wet age-related macular degeneration CNV in mice.

[0022] The composite composition of the present invention is used in preparing a drug for promoting the expression of ATF6 protein, GRP78 protein and Caspase12 protein, and in preparing a drug for inhibiting the expression of IRE1 protein and PERK protein.

[0023] The composite composition of the invention is used in preparing a drug for inhibiting the expression of IRE1 mRNA, PERK mRNA, GRP78 mRNA and Caspase-12 mRNA, and in preparing a drug for promoting the expression of ATF6 mRNA.

[0024] The three substances are combined in an optimized ratio to form the composite composition of the present invention. The composite composition of the present invention has the effect of preventing and treating diseases such as high myopia, retinitis pigmentosa and wet age-related macular degeneration CNV. Through in-depth research on the drug mechanism, it is found that the drug combination of the present invention can promote the expression of ATF-6 protein, GRP78 protein and Caspase12 protein, and can inhibit the expression of IRE1 protein and PERK protein.

[0025] The pharmaceutical composition of the present invention can also be used to prepare health products or foods for treating and preventing myopia and degenerative fundus diseases. It can be used as a health product for daily prevention of high myopia or degenerative fundus diseases. Foods include solid powder beverages.

[0026] Preferably, the pharmaceutical composition can be added with pharmaceutically acceptable carriers and / or excipients, such as starch, dextrin, microcrystalline cellulose, compressible starch, magnesium stearate, micropowdered silica gel, sucrose, sodium carboxymethyl starch, hydroxypropyl methylcellulose, vegetable oil, lecithin, polyethylene glycol, propylene glycol, ethyl paraben, etc., to prepare pharmaceutically suitable dosage forms, such as capsules, emulsions, oral liquids, dripping pills, water-flooded pills, powders, etc.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The drug combination provided by the present invention comprises three effective ingredients, namely peony seed oil, lutein ester and zeaxanthin. Among them, peony seed oil, as a new resource food, is a fat product extracted from peony seeds through a certain process. It can be used as oil for daily life and also has considerable medicinal value.

[0029] Peony seed oil is rich in ω-3 unsaturated fatty acids and α-linolenic acid, has high nutritional value and is safe and non-toxic. It can play an antioxidant, lipid-lowering, anti-ultraviolet, anti-inflammatory, cardiovascular and nerve protection role, and is known as "plant brain gold."

[0030] Lutein ester is an important carotenoid fatty acid ester. After being absorbed by the human body, it is decomposed into free lutein. It has the basic function of crystalline lutein to supplement the lutein lost by the human body. Zeaxanthin is a fat-soluble pigment component of lutein and a dihydroxy derivative of β-carotene.

[0031] It has health benefits such as anti-oxidation, prevention of macular degeneration, treatment of cataracts, prevention of cardiovascular diseases, enhancement of body immunity, and slowing down of atherosclerosis. This drug combination can effectively prevent and relieve myopia. After taking this drug combination, the refractive value of guinea pigs' eyes in the fourth week was restored to a refractive value close to that of healthy guinea pigs' eyes -3.52±0.75 &&** .

[0032] It also has a certain inhibitory and therapeutic effect on diseases such as retinitis pigmentosa and age-related macular degeneration. EDG detection of mice found that by taking this drug combination to the mice in the model group, the EDG light-adapted 3.0b amplitude and dark-adapted 3.0b amplitude values ​​were restored to a level close to that of healthy mice: 37.6±6.3 && 、292±28 && .

[0033] The retinal angiography data showed that the retinal angiography data level of wet age-related macular degeneration CNV model mice could be restored to a level close to that of healthy mice after taking the drug combination of the present invention.

[0034] The drug combination of the present invention can promote the expression of ATF6 protein, GRP78 protein and Caspase12 protein, and inhibit the expression of IRE1 protein and PERK protein.

[0035] The drug combination of the present invention can inhibit the expression of IRE1mRNA, PERKmRNA, GRP78mRNA and Caspase-12mRNA, and promote the expression of ATF6 mRNA. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0037] Example 1

[0038] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 2000 parts of peony seed oil, 1.2 parts of zeaxanthin, and 4.8 parts of lutein ester.

[0039] Example 2

[0040] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 1500 parts of peony seed oil, 1.2 parts of zeaxanthin, and 6 parts of lutein ester.

[0041] Example 3

[0042] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 1170 parts of peony seed oil, 1 part of zeaxanthin, and 6 parts of lutein esters.

[0043] Example 4

[0044] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 1200 parts of peony seed oil, 1.2 parts of zeaxanthin, and 3.6 parts of lutein ester.

[0045] It is stipulated that lutein ester is A, zeaxanthin is B, and peony seed oil is C;

[0046] Example 5

[0047] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 1500 parts of peony seed oil, 1 part of zeaxanthin, and 7 parts of lutein esters.

[0048] Example 6

[0049] This embodiment provides a pharmaceutical composition for preventing myopia and degenerative fundus diseases, comprising 1500 parts of peony seed oil, 0.82 parts of zeaxanthin, and 8.2 parts of lutein ester.

[0050] Table 1 is a summary table of the ratios and contents of the pharmaceutical compositions of Examples 1 to 4.

[0051]

[0052] Comparative Example 1

[0053] This comparative example 1 compares the efficacy of single-ingredient peony seed oil, wherein two different peony seed oil contents are used, 1500 parts in comparative example 1-1 and 2000 parts in comparative example 1-2.

[0054] Comparative Example 2

[0055] This comparative example 2 compares the efficacy of a combination of lutein ester and zeaxanthin without peony seed oil. Specifically, in this comparative example:

[0056] The drug composition of Comparative Example 2-1 is: 4.8 parts of lutein ester and 1.2 parts of zeaxanthin.

[0057] The drug composition of Comparative Example 2-1 is 6 parts of lutein ester and 1.2 parts of zeaxanthin.

[0058] Comparative Example 3

[0059] This comparative example is to investigate the therapeutic effect of peony seed oil and compare the efficacy by replacing other oils.

[0060] The drug composition of Comparative Example 3-1 is 1500 parts of peanut oil;

[0061] The drug component of Comparative Example 3-2 is 1500 parts of walnut oil.

[0062] Comparative Example 4

[0063] In order to investigate the important role of peony seed oil in the pharmaceutical composition, this comparative example replaced it with other oils to compare the efficacy, specifically:

[0064] The pharmaceutical composition of Comparative Example 4-1 comprises 1500 parts of peanut oil, 6 parts of lutein ester and 1.2 parts of zeaxanthin.

[0065] The pharmaceutical composition of Comparative Example 4-2 comprises 1500 parts of walnut oil, 6 parts of lutein ester and 1.2 parts of zeaxanthin.

[0066] Comparative Example 5

[0067] In order to investigate the contribution rate of lutein ester and zeaxanthin in the drug combination, this comparative example 5 provides drug compositions with different proportions for comparison, specifically:

[0068] The pharmaceutical composition of this comparative example comprises 1500 parts of peony seed oil, 1.2 parts of lutein ester and 6 parts of zeaxanthin.

[0069] Table 2 is a summary of the drug ratios and contents of Comparative Examples 1 to 5

[0070]

[0071]

[0072] Experimental example:

[0073] 1. Experimental subjects:

[0074] Mice: Clean grade healthy male C57BL / 6J mice, 6-8 weeks old, weighing 16-20g. Adaptive feeding for 14 days (D), barrier environment: day and night cycle of 12 / 12h, room temperature maintained at 22-24℃, no restriction on mice food and water.

[0075] Guinea pigs: 170 healthy British tricolor male guinea pigs, 3 weeks old and weighing 160g-220g, were selected for the intervention experiment. Healthy guinea pigs with no anterior segment disease, transparent refractive media without turbidity and no refractive error were selected. The internal temperature was controlled at 22-24℃, and the guinea pigs were not restricted in eating and drinking.

[0076] Animal experiments were performed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology (One-way analysis of variance, ARVO).

[0077] 2. The animal models include guinea pig high myopia model, mouse retinitis pigmentosa model, and mouse wet age-related macular degeneration CNV model.

[0078] The high myopia model (defined as model 1) was established as follows:

[0079] The experimental modeling started after 48 hours of adaptive feeding. The right eye of the guinea pig was covered as the modeling eye to establish the form deprivation myopia (FDM) model. The left eye as the control eye was not intervened. After the experiment started, the blank control group was not treated in any way, and the guinea pigs in the other groups were covered with a trimmed latex balloon mask. A 10-inch latex balloon was cut into a mask that covered the right eye and completely exposed the left eye, mouth, nose and ears, and then put on the guinea pig's head. The position was adjusted to ensure that the right eye was completely covered without compressing the eyeball, and the left eye and mouth and nose were completely exposed. The right eye of the guinea pig in the implementation group was covered for 6 weeks, and the refractive power of the right eye of the modeling group was induced to -6.00D or above, which was a successful modeling. If the position of the headgear was found to be abnormal during the modeling period, it should be adjusted in time. Check the balloon mask covering every day to ensure that the right eye is completely covered and there is no pressure on the eyeball.

[0080] The mouse retinitis pigmentosa model (defined as model 2) was constructed as follows:

[0081] C57BL / 6J mice were randomly divided into a normal group (N) and a model group (M). Normal saline and 60 mg / kg MNU were intraperitoneally injected, respectively. Seven days later, the mice were examined by electroretinogram (ERG) and the eyeballs were removed for HE-stained tissue sections. It was observed that the ERG was extinguished and the photoreceptor cells in the outer nuclear layer of the retina on the HE slide were apoptotic, indicating that the mouse RP model was successfully established.

[0082] Laser-induced wet age-related macular degeneration CNV model in mice (designated as model 3) was performed as follows:

[0083] Male C57BL / 6J mice were anesthetized intraperitoneally and dilated, then placed on a slit lamp platform and anesthetized with 0.5% propacaine hydrochloride eye drops (Alcon Laboratories). Laser photocoagulation was performed bilaterally on each mouse (577 nm krypton laser, 150 mW, 10 ms, 10 um spot). Laser photocoagulation was performed on about 6 points of the retinal large blood vessel space 2 PD away from the actual plate. The generation of small bubbles indicated that Bruch's membrane was ruptured, which was considered as an effective laser spot. Mice with vitreous / retinal hemorrhage after photocoagulation were eliminated.

[0084] 3. Treatment of drug compound compositions

[0085] The drugs were mixed in a predetermined ratio. The above examples and comparative examples are all for adults. The adult consumption was converted to the mouse consumption and then gavaged once a day. The specific conversion ratio is 1:12.

[0086] 4. Experimental Grouping:

[0087] 340 mice and 170 guinea pigs were randomly divided into a blank group, a model group, experimental groups 1-4 (Examples 1-4), and control groups 1-5 (Comparative Examples 1-5), with 10 mice in each group, and the right ears were marked with ear tags.

[0088] See the table below:

[0089] In order to further study the technical effect of the drug combination of the present invention, the following experimental studies were carried out:

[0090] 5. Experimental methods

[0091] 5.1 Refractive power and axial length experiment (for testing animal models of high myopia)

[0092] The refractive power and axial length of guinea pigs in each group of model 1 were measured at the 0th, 2nd and 4th week of the experiment.

[0093] For the measurement of refraction, turn off the indoor light source 30 minutes in advance, use compound tropicamide eye drops 4 times, with an interval of 5 minutes between each time, wait for the pupil to be fully dilated and have it fixed by an assistant, hold a strip retinoscope at arm's length (about 50 cm) for retinoscopy, take the horizontal and vertical principal meridians for inspection, and include astigmatism in the test result at half the equivalent spherical lens. Repeat the measurement 3 times to calculate the average value.

[0094] For the measurement of the axial length, 0.5% proparacaine hydrochloride eye drops were first used for local anesthesia of both eyes, and then the ophthalmic A-type ultrasound instrument was used for measurement under natural light. The probe was gently aligned with the center of the cornea and perpendicular to the corneal surface. The measurement was performed 5 times in a row, and the values ​​with severe deviation were eliminated and the average value was calculated. After the measurement was completed, levofloxacin eye drops were used to prevent infection.

[0095] Diopter ( D) Experimental results are shown in Table 3

[0096] Table 3 is the experimental data of the diopter of guinea pigs in each group of model 1

[0097]

[0098]

[0099] Note: Compared with the blank group, * indicates P < 0.05, ** indicates P < 0.001;

[0100] Compared with the model group, & indicates P < 0.05, && indicates P < 0.001.

[0101] By analyzing the refractive test results in Table 3, it can be seen that the pharmaceutical compositions in Examples 1-8 of the present invention can significantly improve the refractive power of the guinea pigs in the high myopia model of guinea pigs after acting on the model, while in the 9 comparative examples, changing the composition and ratio of the pharmaceutical compositions will lead to a decrease in the improvement effect on the refractive power of guinea pigs; in particular, from the analysis of the data of Comparative Examples 1-1 to Comparative Example 1-1, and Comparative Example 1-2 and Example 1, it can be seen that the peony seed oil itself has no obvious effect on improving the refractive power of guinea pigs, but has a synergistic effect after being compounded with lutein esters and zeaxanthin, and can significantly improve the refractive power of guinea pigs.

[0102] Changes in the right eye axis ( mm) The experimental results are shown in Table 4:

[0103] Table 4 shows the data of eye axis changes in each group of guinea pigs in model 1

[0104]

[0105]

[0106] Note: Compared with the blank group, * indicates P < 0.05, ** indicates P < 0.001;

[0107] Compared with the model group, & indicates P < 0.05, && indicates P < 0.001.

[0108] By analyzing the experimental results of axial length change in Table 4, it can be seen that the pharmaceutical compositions in Examples 1-6 of the present invention can control the axial length change of guinea pigs after acting on the guinea pig high myopia model, while in the comparative example, the control effect of axial length change is not very obvious by changing the composition and ratio of the Chinese medicine compound composition.

[0109] 5.2 ERG detection (for the detection of animal models of retinitis pigmentosa)

[0110] The mice were placed in a dark environment for dark adaptation for no less than 2 hours. After the dark adaptation, the mice were anesthetized by intraperitoneal injection of 0.01 ml / mouse Lumianning and 0.3 ml / 100 g sodium pentobarbital solution to anesthetize the mice. At the same time, compound tropicamide eye drops were used to dilate the pupils of the mice. After the mice were anesthetized successfully, they were fixed on the experimental table. The recording electrode, reference electrode and ground electrode were installed in the center of the left cornea, the left cheek and the subcutaneous part of the tail of each mouse. Physiological saline was dripped on the corneal surface to prevent drying. All processes were performed under a weak red light source in a dark room. The dark adaptation 0.01, 3.0, and ops wave reactions of the electroretinogram were detected respectively, and the light adaptation 3.0 and Flicker reactions were detected respectively; after the dark adaptation test was completed, the light adaptation test was performed again after 10 minutes of exposure to standard strong light. Mice were randomly selected for testing, and the data of each individual were kept separately, and the relative ERG amplitude was recorded.

[0111] The ERG test results are shown in Table 5 below

[0112] Table 5 is a summary table of ERG test data

[0113]

[0114] By analyzing the above data obtained from the retinitis pigmentosa animal model test, it can be seen that the amplitude of light adaptation and dark adaptation of mice taking the drug combination of the present application is close to the normal level. The amplitude of the mice in the model group is smaller, and the amplitude of the mice in the control group has a certain improvement, but the effect is not as significant as that of Examples 1-6.

[0115] 5.3 Retinal angiography (mouse CNV model of wet age-related macular degeneration)

[0116] After the mouse fundus photography was performed in vivo, 10% sodium fluorescein (injection dose of 0.1 mL / 10 g) was injected intraperitoneally, and fundus fluorescein angiography (FFA) was performed to observe the changes in retinal blood vessels in vivo. In vivo, tortuous and dilated retinal blood vessels were observed, and the non-perfused area was noted, so that retinal vascular lesions could be dynamically detected. The retinal vascular density, porosity, and number of connection points were analyzed using Angio Tool software. The vascular density is defined as the ratio of the vascular area to the total area, which can reflect the changes in retinal microvessels and the severity of the damage to the blood-retinal barrier. The porosity is defined as the percentage of the pore volume to the total volume, which reflects the heterogeneity of the blood vessels and indirectly reflects the size of the non-perfused area. The number of connection points refers to the number of vascular branching points within the analysis image range, which indirectly reflects the branching of retinal blood vessels.

[0117] The retinal angiography data are shown in Table 6 below:

[0118] Table 6 shows the retinal angiography data

[0119]

[0120]

[0121] By analyzing the retinal angiography data of the wet age-related macular degeneration CNV model of mice, it can be seen that the values ​​of various indicators of the model group are higher, while the retinal angiography data indicators of the mice corresponding to Examples 1-6 are reduced and close to those of normal mice because they take the drug combination of the present invention, which indicates that the drug combination of the present invention has a certain therapeutic effect on wet age-related macular degeneration. In the comparative example, although the various indicators of retinal angiography can be controlled, the control effect is not as good as that of the drug combination of the present invention.

[0122] 6. Experiment on the expression of IRE1, ATF6, PERK, GRP78 and Caspase-12 proteins in the retina

[0123] aSample preparation and total protein extraction

[0124] Place mouse retinal tissue in a grinding tube, add lysis buffer, and lyse at 4°C. After centrifugation, take the supernatant and measure the protein concentration using a BCA protein quantification kit.

[0125] b. Sample determination

[0126] Dilute the protein sample with the lysis buffer and mix it with the lysis buffer and BCA working solution. Measure the light absorbance of the sample at a wavelength of 562nm and determine the protein content of the sample based on the standard curve.

[0127] Denaturation and electrophoresis of c-protein

[0128] After the protein was denatured, it was loaded and electrophoresed at 180 V until the bromophenol blue dye reached the bottom of the separation gel.

[0129] d. Transfer and blocking

[0130] Cut the PVDF membrane and 6 filter papers to match the size of the separation gel, soak them in the transfer solution for 10 minutes, and fully wet them. Stack them in the order of sponge → 3 layers of filter paper → membrane → gel → 3 layers of filter paper → sponge, and use a glass rod to remove bubbles between the layers. During the transfer process, connect the gel and PVDF membrane to the negative electrode and the positive electrode respectively, and transfer the membrane at 200mA for 1 to 2 hours after power is turned on. After completion, seal the PVDF membrane.

[0131] eIncubation with antibody

[0132] Primary antibody incubation: The PVDF membrane was incubated in the primary antibody solution (HIF-1α concentration 1:1000, tubulinβ1 concentration 1:5000), placed at 4°C, and gently shaken on a shaker overnight; then the PVDF membrane was washed 3 times with TBST solution, each time for 5 minutes. Secondary antibody incubation: The membrane was transferred to the secondary antibody solution (dilution ratio 1:5000), incubated on a shaker at room temperature for 2-3 hours, and then rinsed as described above.

[0133] fDevelopment and fixing

[0134] The optical density value was measured using Tianneng GIS chassis control software V2.0 to calculate the relative expression level of the target protein.

[0135] Table 7 is the experimental data on the expression of IRE1, ATF6, PERK, GRP78, and Caspase-12 proteins in the retina of the guinea pig model of form-stripping high myopia

[0136]

[0137] Table 8 shows the experimental data of the expression of IRE1, ATF6, PERK, GRP78 and Caspase-12 proteins in the retina of the retinitis pigmentosa mouse model

[0138]

[0139]

[0140] Table 9 shows the experimental data of the expression of IRE1, ATF6, PERK, GRP78 and Caspase-12 proteins in the retina of the choroidal neovascularization model of age-related macular degeneration

[0141]

[0142]

[0143]

[0144] In order to explore the mechanism of the drug combination of the present invention, the protein expression levels of IRE1, ATF6, PERK, GRP78 and Caspase-12 in the retina were respectively carried out on three models. The experimental data showed that the drug combination of the present invention had a promoting effect on the ATF6 protein, GRP78 protein and Caspase-12 protein in the three animal models, and had a significant inhibitory effect on the IRE1 protein and PERK protein.

[0145] 7. Real-time fluorescence quantitative PCR experiment

[0146] Total RNA extraction from sample A

[0147] ① Before the first use, add 24 mL of anhydrous ethanol to the binding solution (2.4 mL / 24 mL for 5T / 50T, respectively) and 52 mL of anhydrous ethanol to the rinsing solution (5.2 mL / 52 mL for 5T / 50T, respectively), and mix thoroughly.

[0148] ② Take 10-20 mg of fresh sample into an EP tube and add 350 μL of lysis buffer LB, and crush it with a high-speed low-temperature tissue grinder.

[0149] ③ Transfer the evenly ground homogenate to a DNA removal / RNA adsorption universal column (the column is placed in a 2 mL collection tube), centrifuge at 13,000 rpm for 1 min, and collect the filtrate containing RNA.

[0150] ④ Add a binding solution of equal volume to the above filtrate, and mix gently by pipetting immediately.

[0151] ⑤ Add all the above mixed solutions into a new DNA removal / RNA adsorption universal column, centrifuge at 13000rpm for 30s, and discard the filtrate.

[0152] ⑥ Add 700 μL of deproteinization solution, centrifuge at room temperature for 30 seconds, and centrifuge at 13,000 rpm for 30 seconds. Discard the filtrate and put the DNA removal / RNA adsorption universal column back into the 2 mL collection tube.

[0153] ⑦ Add 500 μL of rinse solution, centrifuge at 13000 rpm for 30 seconds, and discard the filtrate.

[0154] ⑧Repeat step 7.

[0155] ⑨ Centrifuge the empty column at 13000rpm for 2min to remove the remaining rinse solution.

[0156] ⑩Put the DNA removal / RNA adsorption universal column into a new 1.5mL centrifuge tube, add 30-50μL RNase-free H2O in the center of the membrane, and leave it at room temperature for 1min. Then centrifuge at 13000rpm for 1min, collect the filtrate, which is the RNA solution, to be tested or stored at -80℃.

[0157] B. Removal reaction of genomic DNA:

[0158] Table 10 Genomic DNA removal reaction system

[0159] Reagents Volume (μL) 5×gDNAEraserBuffer 2 gDNAEraser 1 Total RNA 2 RNaseFreedH2O 5 Total 10

[0160] Note: After adding each reagent in turn, place in thermal cycler, 42°C, 2min

[0161] C reverse transcription reaction

[0162] Table 11 Reverse transcription reaction system

[0163] Reagents Volume (μL) 5×PrimeScriptBuffer2 4 PrimeScriptRTEnzymeMixI 1 RTPrimerMix 1 RNA 10 RNaseFreedH2O 4 Total 20

[0164] Note: Add each reagent in turn and place in a thermal cycler, 37℃, 15min; 85℃, 5sec

[0165] Real-time fluorescence quantitative polymerase chain reaction (qPCR)

[0166] a Primer design

[0167] The complete gene sequence was retrieved from the National Center for Biotechnology Information (NCBI) database, and specific primers were designed for each gene using PrimerPremier software. The design and synthesis of primers were undertaken by Shanghai Shenggong Biotechnology Technology Service Co., Ltd., and the primers were purified using the ULTRAPAGE method. (See Table 3).

[0168] Table 12 Primers and base sequences used in this test (mouse)

[0169]

[0170] b Real-time fluorescence quantitative PCR reaction (Table 13, Table 14):

[0171] Table 13 PCR reaction system

[0172] Reagents Volume (μL) 2×RealPCREasyTMMix-SYBR 10.0 Forward Primer (10 μM) 0.8 Reverse Primer (10 μM) 0.8 Template(DNA) 2.0 <![CDATA[ddH2O]]> 6.4 Total 20.0

[0173] Table 14 PCR reaction system

[0174] Reaction temperature time Remark 95℃ 30s Pre-denaturation 95℃ 5s transsexual 55℃ 30s annealing 72℃ 30s Fully extend to collect fluorescence

[0175] Note: 45 cycles

[0176] c QuantStudioTM Design&Analysis SE Software was used to analyze the CT (Threshold cycle) value of each test sample during the PCR process.

[0177] The results of real-time fluorescence quantitative PCR experiments are shown in the following table:

[0178] Among them, Table 15 is the experimental data of the expression of IRE1, ATF6, PERK, GRP78, and Caspase-12 mRNA in the retina of the form-stripping high myopia guinea pig model:

[0179]

[0180]

[0181] Among them, Table 16 is the experimental data of the expression of IRE1, ATF6, PERK, GRP78, and Caspase-12 mRNA in the retina of the retinitis pigmentosa mouse model:

[0182]

[0183]

[0184] Among them, Table 17 is the experimental data of the expression of IRE1, ATF6, PERK, GRP78, and Caspase-12 mRNA in the retina of the choroidal neovascularization model of age-related macular degeneration:

[0185]

[0186]

[0187]

[0188] Note: Compared with the blank group, * indicates P < 0.05, ** indicates P < 0.001;

[0189] Compared with the model group, & indicates P < 0.05, && indicates P < 0.001.

[0190] In order to explore the mechanism of the drug combination of the present invention, the expression level experiments of IRE1, ATF6, PERK, GRP78 and Caspase-12 mRNA in the retina were also carried out on three models. The experimental data showed that the drug combination of the present invention has a promoting effect on ATF6 mRNA in the three animal models, and has a significant inhibitory effect on IRE1mRNA, PERK mRNA, GRP78mRNA and Caspase-12mRNA.

[0191] The Chinese medicine compound compositions in Examples 1-8 of the present invention can significantly promote the expression of ATF6 mRNA in mice.

[0192] 9. HE retinal and choroidal total thickness test

[0193] The specific experimental method is as follows: the fixed tissue of the eyeball is dehydrated and embedded in an automatic dehydrator, and the following operations are performed after slicing: dewaxing the slices to water → hematoxylin staining for 10-20 minutes → rinsing with tap water for 1-3 minutes → hydrochloric acid alcohol differentiation for 5-10 seconds → hydrochloric acid alcohol differentiation for 5-10 seconds → rinsing with tap water for 1-3 minutes → placing in 50°C warm water or weak alkaline aqueous solution to return to blue → rinsing with tap water for 1-3 minutes → placing in 85% alcohol for 3-5 minutes → eosin staining for 3-5 minutes → washing with water for 3-5 seconds → gradient alcohol dehydration → xylene transparent → neutral gum sealing. The entire process follows the standard operating procedure (SOP) of pathological examination, and after a series of steps such as dehydration, trimming, embedding, slicing, staining, and sealing, the slices are finally examined under a microscope; the slices are imaged using a Pannoramic250 digital slice scanner. The entire tissue is observed at a low magnification, and then a specific area adjacent to the optic disc is selected for image acquisition at a magnification of 200 times. Caseviewer image analysis software was used to accurately measure the collected images. For each numbered slice, a total of 10 sets of data were measured and their average values ​​were calculated for subsequent analysis.

[0194] The test results are shown in Table 18 below:

[0195] Table 18 shows the experimental data of the total thickness of the retina and choroid of model 1 after 6 weeks of intervention

[0196]

[0197]

[0198] Note: Compared with the blank group, * indicates P < 0.05, ** indicates P < 0.001;

[0199] Compared with the model group, & indicates P < 0.05, && indicates P < 0.001.

[0200] Result analysis:

[0201] By analyzing the experimental data results of the total thickness of the retina and choroid in Table 18, it can be seen that the drug combination of Examples 1-6 of the present invention can significantly inhibit the thinning of the thickness of the choroid of guinea pigs, while in Comparative Examples 1-5, the composition and ratio of the drug combination are changed, and the inhibitory effect on the thinning of the thickness of the choroid of guinea pigs is significantly reduced; in particular, analysis of the data of Comparative Example 6 and Example 1 shows that peony seed oil itself has no inhibitory effect on the thinning of the thickness of the choroid of guinea pigs, but has a synergistic effect when combined with lutein esters and zeaxanthin, and can significantly inhibit the thinning of the thickness of the choroid of guinea pigs.

[0202] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition for preventing myopia and degenerative fundus disease, characterized in that: Includes the following ingredients: Peony Seed Oil, Lutein Esters and Zeaxanthin; The total weight of the lutein ester and the zeaxanthin accounts for 0.3% to 1% of the weight of the peony seed oil; The weight ratio of the lutein ester to the zeaxanthin is (2-10):

1.

2. The pharmaceutical composition for preventing myopia and degenerative fundus disease according to claim 1, characterized in that: The total weight of the lutein ester and the zeaxanthin accounts for 0.3%-0.8% of the weight of the peony seed oil.

3. The pharmaceutical composition for preventing myopia and degenerative fundus disease according to claim 2, characterized in that: The total weight of the lutein ester and the zeaxanthin accounts for 0.4%-0.6% of the weight of the peony seed oil.

4. The pharmaceutical composition for preventing myopia and degenerative fundus disease according to claim 1, characterized in that: The weight ratio of the lutein ester to the zeaxanthin is (4-8):

1.

5. The pharmaceutical composition for preventing myopia and degenerative fundus disease according to claim 1, characterized in that: The weight ratio of the lutein ester to the zeaxanthin is (4-6):

1.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in preventing myopia.

7. Use of the pharmaceutical composition according to any one of claims 1 to 5 in preventing and treating degenerative fundus diseases.

8. Use of the pharmaceutical composition according to claim 6 or 7, characterized in that: The invention can be used in the preparation of drugs for promoting the expression of ATF6 protein, GRP78 protein and Caspase12 protein, and in the preparation of drugs for inhibiting the expression of IRE1 protein and PERK protein.

9. Use of the pharmaceutical composition according to claim 6 or 7, characterized in that: The invention can be used in the preparation of drugs for inhibiting the expression of IRE1 mRNA, PERK mRNA, GRP78 mRNA and Caspase-12 mRNA, and in the preparation of drugs for promoting the expression of ATF6 mRNA.

10. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of health products or foods for treating and preventing myopia and degenerative fundus diseases.

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