Application of tea

Abstract

The invention discloses application of tea. The solid composition is applied to preparation of a medicine for preventing and / or treating form deprivation myopia. The solid composition comprises Pu'er tea powder, Baxian tea powder and dried orange peel. The invention also discloses an application of the tea in preparation of a medicine for preventing and / or treating shape deprivation myopia. The tea is prepared from Pu'er tea powder, Baxian tea powder and pericarpium citri reticulatae according to the mass ratio of 5: 3: 2. Main components of tea are identified, possible toxic reactions of the main components are explored, a C57BL / 6 mouse form deprivation myopia model is used for evaluating the improvement effect of the tea on myopia symptoms, HKS can regulate form deprivation induced MMP-2 and TIMP-2 protein expression level abnormity, and the HKS can be used for improving myopia symptoms. HKS is prompted to improve myopia symptoms induced by form deprivation by regulating extracellular matrix homeostasis.

Description

Technical Field

[0001] The invention relates to the application of tea, and more specifically to the application of tea in preparing medicine for preventing and / or treating form deprivation myopia. Background Art

[0002] The occurrence and development of myopia involves many factors, including genetics, environment, and behavioral habits. Recent studies have found that the formation of myopia may be related to insufficient choroidal blood supply, which leads to hypoxia in the sclera and retinal microenvironment. At present, there are relatively mature and diverse methods for myopia correction and prevention and control in clinical practice, but the causes of the occurrence and development of myopia are not yet clear. Tea is rich in flavonoids and phenolic acid compounds. Studies have shown that flavonoids can improve the antioxidant and free radical scavenging capabilities of animal bodies. Flavonoids can combine with peroxyl radicals to generate flavonoid free radicals due to the hydrogen atoms on the phenolic hydroxyl group, and then react with other free radicals to terminate the free radical chain reaction. There are many compounds with medicinal value in flavonoids. These compounds are used to prevent and treat cardiovascular and cerebrovascular diseases, such as reducing the fragility of blood vessels, improving vascular permeability, lowering blood lipids and cholesterol, preventing and treating hypertension, cerebral hemorrhage, coronary heart disease, angina pectoris, dilating coronary blood vessels, and increasing coronary flow. Many flavonoids have antitussive, expectorant, antiasthmatic and antibacterial activities. They also have liver protection, liver detoxification, antifungal, treatment of acute and chronic hepatitis, liver cirrhosis, and anti-free radical and antioxidant effects. In addition, flavonoids have the same effects as phytoestrogens. In animal production in animal husbandry, the application of flavonoids can significantly improve animal production performance, improve animal disease resistance, and improve animal immune function. Studies in recent years have shown that phenolic compounds have antioxidant, anti-inflammatory, and cardiovascular disease risk reduction effects. They also have certain pharmacological properties, such as anti-infection, anti-virus, anti-bacterial, anti-allergic, anti-bleeding, and immunity enhancement.

[0003] Tea is a solid beverage composed of Pu'er tea powder, Baxian tea powder and tangerine peel. Summary of the invention

[0004] The object of the present invention is to provide the application of tea.

[0005] The present invention firstly identifies the main components of tea, explores its possible toxic reactions, and uses the C57BL / 6 mouse form deprivation myopia model to evaluate the improvement effect of tea on myopia symptoms.

[0006] The present invention provides an application of a solid composition in preparing a drug for preventing and / or treating form deprivation myopia;

[0007] The solid composition comprises Pu'er tea powder, Baxian tea powder and tangerine peel.

[0008] In the above application, the mass ratio of the Pu'er tea powder, the Eight Immortals tea powder and the tangerine peel can be 4.5-7:2-5:2.

[0009] In the above application, the mass ratio of the Pu'er tea powder, the Eight Immortals tea powder and the tangerine peel can be 5:3:2.

[0010] In the above application, the Pu'er tea powder is obtained by grinding Pu'er tea;

[0011] The Eight Immortals Tea powder is obtained by grinding Eight Immortals Tea.

[0012] Use of the tea of ​​the present invention in preparing medicine for preventing and / or treating form deprivation myopia;

[0013] The tea is composed of Pu'er tea powder, Baxian tea powder and tangerine peel in a mass ratio of 5:3:2.

[0014] The present invention further provides a drug for preventing and / or treating form deprivation myopia, the active ingredient of which is the solid composition or the extract obtained by brewing the tea with water.

[0015] The present invention has the following beneficial effects:

[0016] The present invention uses LC-MS technology and ophthalmic OCTA technology to study the main chemical composition, safety and effect of tea (abbreviated as HKS) on improving the myopia symptoms induced by form deprivation in C57BL / 6 mice, and explores the changes in the expression of related proteins during the occurrence and development of myopia. The main chemical components of tea (HKS) are phenolic acids and flavonoid compounds such as (+)-catechins, myricetin, puer tea extract, kaempferol, ferulic acid, and tangeretin. The maximum daily intragastric administration of the tea composition to mice is equivalent to 840 times the recommended drinking amount of 2g / 70kg for adults, indicating that the tea composition has no acute toxic reaction and is highly safe when taken orally. HKS has a protective effect on the myopia symptoms induced by form deprivation in C57BL / 6 mice, and the minimum effective dose is 2080mg / kg. HKS can regulate the abnormal expression levels of MMP-2 and TIMP-2 proteins induced by form deprivation, indicating that HKS may improve the myopia symptoms induced by form deprivation by regulating the homeostasis of the extracellular matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the total ion chromatogram of HKS crude extract in positive ion mode.

[0018] Figure 2 The main compound structure.

[0019] Figure 3 is the average weight of mice before and after oral administration (g), Figure 3 (a) shows before intragastric administration, and (b) shows after intragastric administration.

[0020] Figure 4 Pathological examination of mice (HE staining, 100×), Figure 4 (a) shows the results of female mice, and (b) shows the results of male mice.

[0021] Figure 5 is the mouse organ coefficient, where Figure 5 (a) shows the results of male mice, and (b) shows the results of female mice.

[0022] Figure 6 Serum biochemical indexes of mice.

[0023] Figure 7 For ophthalmic OCTA imaging measurement results, Figure 7 Compared with the normal control group, # P<0.05, ## P<0.01, ### P<0.001; compared with the form deprivation group, * P<0.05, ** P<0.01, *** P<0.001.

[0024] Figure 8 (a) is an example of ophthalmic OCTA imaging, and (b) is an example of retinal imaging.

[0025] Fig. 9 (a) is HE staining of the eyeball, (b) is HE staining of the posterior wall of the eyeball, Fig. 9 The layers of the posterior wall of the eyeball are clearly visible in each of the images, from top to bottom, they are sclera-choroid-retinal pigment epithelium-outer segment-inner segment-outer nuclear layer-outer plexiform layer-inner nuclear layer-inner plexiform layer-ganglion cell layer-nerve fiber layer. The scale bar is 200 μm.

[0026] Fig.10 The expression of MMP-2 protein.

[0027] Fig.11 The expression of MMP-14 protein.

[0028] Fig.12 The expression of TIMP-2 protein. DETAILED DESCRIPTION

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example

[0032] 1. Mass Spectrum Analysis of the Main Chemical Components of Tea

[0033] 1. Experimental Materials and Instruments

[0034] The tea is in brown powder form, consisting of Pu'er tea powder (50%), Eight Immortals tea powder (30%), and tangerine peel (20%); it was purchased from Guangzhou Huiyanshi Health Industry Co., Ltd., batch number 2023061701.

[0035] 2. Experimental Methods

[0036] The crude tea extract (i.e., the extract obtained by brewing tea with boiling water, concentrating and drying) was dissolved in methanol, centrifuged and passed through a membrane, and mass spectrometry data was collected in the positive ion mode.

[0037] Chromatographic conditions:

[0038] Chromatographic column: ACQUITY UPLC(R)BEH C18 (2.1mm×50mm, 1.7μm); detection wavelength: 210, 254nm; injection volume: 3μL; column temperature: 35℃; flow rate: 0.3mL / min; mobile phase: acetonitrile (A, with 0.1% formic acid)-water (B, with 0.1% formic acid). Elution method: 0-2min, 10% (A); 2-23min, 10%-95% (A); 23-24min, 95%-10% (A); 24-26min, 10% (A).

[0039] Mass spectrometry conditions:

[0040] The chromatographic analysis was performed by UPLC-Q-TOF-MS / MS (Waters, USA) in positive ion mode, and the time-of-flight mass spectrometer was controlled by the G2-XS QTOF system. The positive ion mode was selected, ESI was used as the ionization source, the desolvation temperature was 450°C, the gas flow rate was 700L / h, the ionization source temperature was 80°C, leucine enkephalin was selected as the mass lock standard ([M+H]+=556.2771), the concentration was set to 200mL / mL, and the injection flow rate was 10μL / min. The original data was processed in Continuum mode, the mass spectrometry data acquisition range was m / z 50-1500, the capillary voltage value was 2.0kV, the cone voltage value was 40V, the collision energy was 6eV, the energy scanning range was 20-40eV, and the data were processed by MassLynx 4.1 software.

[0041] 3. Experimental results

[0042] The total ion chromatogram of UPLC-Q-TOF-MS / MS in positive ion mode is shown in Figure 1 As shown, the mass spectrometry results are shown in Table 1, and the identified compounds are shown in Table 1, Figure 2 shown.

[0043] Table 1 Basic information of identified compounds in HKS crude extract (positive ion mode structural analysis)

[0044]

[0045]

[0046]

[0047] 4. Summary

[0048] A total of 21 compounds were identified in the HKS crude extract, including phenolic acids and flavonoids such as (+)-catechin, myricetin, puer tea extract, kaempferol, ferulic acid, and tangeretin. Dehydration and RDA cleavage were the main cleavage modes of flavonoid compounds.

[0049] 2. Acute toxicity test of tea composition on mice

[0050] 1. Experimental Materials and Instruments

[0051] 1.1 Experimental animals

[0052] In this experiment, 18-20 g ICR mice were used (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number NO: 1110322241100197083).

[0053] 1.2 Rearing conditions

[0054] Animals were raised in the SPF-level experimental animal center of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. The animal experimental facilities continuously maintained barrier environment standards. The control range of the main environmental indicators: room temperature 20-26°C, daily temperature difference ≤4°C. Relative humidity 40-70%. Minimum ventilation rate 15 times / hour, light illumination: dark = 12h, light = 12h, 5 animals per box, and the breeding space complies with the provisions of the National Standard of the People's Republic of China GB14925-2010 on the minimum space required for experimental animals. All animals were raised and managed by qualified personnel, and the animals were kept free to eat and move during the entire breeding process.

[0055] 1.3 Quarantine and acclimatization process

[0056] The quarantine period for newly stored animals is 3 to 7 days. During the quarantine period, observe the animals' drinking water, food intake, health status, and whether there are signs of disease and death. Healthy animals have smooth fur, are sensitive, and have no abnormal secretions. Any abnormal phenomenon must be reported to the subject leader and clinical veterinarian. Under the guidance of the veterinarian and with the confirmation of the subject leader, sick animals are removed and replaced with healthy animals. Animals can only be used after they are confirmed to be healthy and disease-free. Experiments can only be carried out after the animals have been adaptively raised for 1 week.

[0057] 1.4 Animal Welfare

[0058] The animals used in this experiment and related treatments must comply with animal welfare requirements and must be reviewed and approved by the Institutional Animal Care and Welfare Committee (IACUC) of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences / Peking Union Medical College before the experiment is carried out.

[0059] 1.5 Experimental instruments

[0060] Fully automatic biochemical analysis system (AU480), Beckman Coulter Co., Ltd.

[0061] 2. Experimental Methods

[0062] 2.1 Basis for dosage design

[0063] With reference to the technical guidelines for acute toxicity studies of traditional Chinese medicine and natural medicine, the maximum dosage is the dose at which the test article can be fully dissolved and successfully administered orally.

[0064] In this embodiment, the test product is tea, and the dosage is 8, 4, 2, and 0 g of tea per kilogram of mice (the corresponding dosage groups in subsequent experiments are recorded as HKS (8 g / kg), HKS (4 g / kg), HKS (2 g / kg), and Control, respectively); according to 40 ml of drug solution per kilogram of mouse gavage, the above-mentioned dose of tea is brewed with 100°C water, and 8, 4, 2, and 0 g of tea are brewed with 40 ml of boiling water, respectively, to obtain drug concentrations for different doses of gavage (the doses involved below are all calculated according to the method herein), as shown in Table 2.

[0065] Table 2 Maximum dosage of intragastric tea for gavage, experimental dosage design and grouping

[0066]

[0067] 2.2 Mouse LD50 Preliminary Experiment

[0068] Twenty mice were randomly selected, half of them were male and half were female, housed in separate cages, and fasted (with free access to water) for 12 h.

[0069] The mice were given the maximum dosage of tea (8 g solid powder / kg mouse) at a dosage volume of 0.4 mL / 10 g for 3 times within 24 h. The mice were observed for 24 hours and the number of mouse deaths was recorded.

[0070] 2.3 Maximum dosage experiment in mice

[0071] 40 mice were taken, half male and half female. After fasting (free drinking water) for 12 hours, each mouse in the drug group was gavaged with 0.8mL / 20g of tea (8g solid powder / kg water). The same dose was repeated 3 times within 24 hours, and the mice were kept and observed for 13 consecutive days. The toxicity of the mice was observed every day, and the observation indicators are shown in Table 3. The mice were weighed 1 day before gavage, the day of gavage, the 1st day after gavage, the 3rd day after gavage, the 5th day after gavage, the 7th day after gavage, the 9th day after gavage, the 11th day after gavage, and the 13th day after gavage. On the 14th day after gavage, the mice were killed. At the end of the experimental observation period, all experimental mice were grossly dissected to observe the changes in the main organs and tissues such as the heart, liver, brain, lungs, kidneys, and spleen. Animals that were killed due to dying during the experiment and dead animals should be grossly dissected and observed in time.

[0072] Table 3 Observation indexes of the maximum tolerated dose experiment of tea composition in mice

[0073]

[0074] 2.4 Statistical analysis

[0075] GraphPad prism10.0 statistical software was used for statistical analysis of data. The normal distribution of the data was tested by D'Agostino & Pearson comprehensive analysis. The normal subset was analyzed by one-way analysis of variance (ANOVA) for multiple group comparisons. The normal distribution of the data was tested by D'Agostino & Pearson comprehensive analysis. The statistical differences between the two groups were analyzed by nonparametric two-tailed Mann-Whitney U test for nonnormal subsets. P < 0.05 was considered statistically significant, and all results were expressed as mean ± SD.

[0076] 3. Experimental results

[0077] 3.1 Mouse LD 50 Numeric

[0078] After mice were given tea (8g solid powder / kg mouse, 8g solid powder brewed with 40ml water) 0.4mL / 10g three times a day, no acute toxicity symptoms were observed in the mice. Due to the limitation of the dosing volume and drug dosage of mice, the LD50 value of mice was not obtained, so the maximum dosage experiment was carried out with the maximum dosage (8g / kg).

[0079] 3.2 Changes in body weight of mice before and after administration

[0080] After administration, the mice's activities were relatively reduced and their diet was reduced. They recovered after 3 to 5 hours. There was no significant difference in other indicators compared with before administration. All indicators returned to normal 24 hours after administration. No death was found in the continuous observation for 13 days. There was no significant change in the behavior and mental state of the mice compared with before administration. The body weight of mice in each group increased significantly. The results are shown in Figure 3 The weight loss on the first day was due to fasting for 12 hours before drug administration.

[0081] 3.3 Pathological staining of mouse organs and changes in organ coefficients

[0082] At the end of the experimental observation period, all experimental mice were grossly dissected, and all organs were thoroughly observed with the naked eye, and no abnormalities were found. HE-stained sections of the liver and kidneys were observed, and it was found that the liver cells were intact in morphology and normal in size, with no obvious pigmentation and material deposition in the cells, no enlargement of the nuclei, and intact liver lobule structure. The renal mesangial cells and endothelial cells proliferated or hypertrophied, and the glomeruli were intact in morphology, normal in structure, with clear boundaries and obvious cystic cavities, without swelling. No crescents were formed in the glomeruli, and the renal tubules were intact in morphology and normal in cavity structure. The results are shown in Figure 4 .

[0083] At the same time, the main organs, such as the heart, liver, spleen, lungs, kidneys and brain, were weighed and the organ coefficients were calculated. It was found that there was no significant difference in the organ coefficients of female and male mice between the two groups. Figure 5 .

[0084] 3.4 Changes in serum biochemical parameters of mice

[0085] Serum biochemical indicators of mice in each group were as follows Figure 6 As shown, for female and male mice, there were no significant differences in ALT, AST, CK, Cre, LDH and Urea levels between the drug-treated groups and the blank group, and they were within the normal range.

[0086] 3.5 Maximum tolerance multiple

[0087] The maximum oral dosage for tea mice is 24g solid powder / kg. The recommended daily drinking dosage for adults is 2g / 70kg. The maximum tolerance multiple of mice = tolerance dose per mouse / average weight of mice × average weight of adults (70kg) / daily dosage for adults = 840 times.

[0088] 4. Summary

[0089] The acute toxicity preliminary experiment in mice did not obtain the median lethal dose (LD50). During the maximum dosage method experimental observation period, the mice did not show obvious toxic reactions, and no significant lesions were observed in various organs and tissues after dissection. There was no significant difference in the relative weight and organ mass of the animals in the control group and the treatment group. The blood biochemical indexes were all within the normal range, there were no significant lesions in the main organs, and no animal deaths were observed. The maximum daily dosage for mice is 24g / kg, which is equivalent to 840 times the recommended daily dose of 2g / 70kg for adults. In summary, tea has no acute toxic effects and is highly safe.

[0090] 3. Evaluation of the protective effect of tea composition on form deprivation myopia in mice

[0091] 1. Experimental Materials and Instruments

[0092] 1.1 Experimental animals

[0093] In this experiment, 3-week-old healthy male C57BL / 6 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd., certificate number NO: 110322241101147938, NO: 110322241101767748) were used.

[0094] 1.2 Rearing conditions

[0095] Animals were raised in the SPF-level experimental animal center of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. The animal experimental facilities continuously maintained barrier environment standards. The control range of the main environmental indicators: room temperature 20-26°C, daily temperature difference ≤4°C. Relative humidity 40-70%. Minimum ventilation rate 15 times / hour, light illumination: dark = 12h, light = 12h, 5 animals per box, and the breeding space complies with the provisions of the National Standard of the People's Republic of China GB14925-2010 on the minimum space required for experimental animals. All animals were raised and managed by qualified personnel, and the animals were kept free to eat and move during the entire breeding process.

[0096] 1.3 Quarantine and acclimatization process

[0097] The quarantine period for newly stored animals is 3 to 7 days. During the quarantine period, observe the animals' drinking water, food intake, health status, and whether there are signs of disease and death. Healthy animals have smooth fur, are sensitive, and have no abnormal secretions. Any abnormal phenomenon must be reported to the subject leader and clinical veterinarian. Under the guidance of the veterinarian and with the confirmation of the subject leader, sick animals are removed and replaced with healthy animals. Animals can only be used after they are confirmed to be healthy and disease-free. Experiments can only be carried out after the animals have been adaptively raised for 1 week.

[0098] 1.4 Animal Welfare

[0099] The animals used in this experiment and related treatments must comply with animal welfare requirements and must be reviewed and approved by the Institutional Animal Care and Welfare Committee (IACUC) of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences / Peking Union Medical College before the experiment is carried out.

[0100] 1.5 Experimental instruments

[0101] The electronic balance was purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.;

[0102] The retinal imaging system (OPTO-RIS) was purchased from Optoprobe Science LTD;

[0103] The visual electrophysiological instrument (OPTO-ERG) was purchased from Optoprobe Science LTD;

[0104] The ophthalmic ultramicroscopy system (ISOCT) was purchased from Optoprobe Science LTD.

[0105] 2. Experimental Methods

[0106] 2.1 Experimental Grouping

[0107] Fifty-six C57BL / 6 mice were randomly divided into eight groups.

[0108] Form deprivation myopia model: Cover one eye of the mouse with a cloth (or sew the upper and lower eyelids of the mouse) for two weeks, and keep the other eye normal. After two weeks, test whether the eye axis has lesions. The number of experimental mice in each group is n.

[0109] (1) Normal control group: neither left nor right eyes underwent eyelid suture (n=8).

[0110] (2) Form deprivation group: The right eye was used as the form deprivation eye and underwent eyelid suture (n=8).

[0111] (3) HKS treatment group (260 mg / kg): 260 mg / kg was administered by gavage after one week of form deprivation (n=8).

[0112] (4) HKS treatment group (520 mg / kg): 520 mg / kg was administered orally after one week of form deprivation (n=8).

[0113] (5) HKS treatment group (1040 mg / kg): 1040 mg / kg was administered orally after one week of form deprivation (n=8).

[0114] (6) HKS treatment group (2080 mg / kg): 2080 mg / kg was administered orally after one week of form deprivation (n=8).

[0115] (7) Methylcobalamin treatment group (14.95 mg / kg): 14.95 mg / kg was administered orally after one week of form deprivation (n=8).

[0116] Note: The above dosage is the amount of HKS or methylcobalamin administered per kilogram of mice (mg). The amount of HKS or methylcobalamin administered per kilogram of mice was obtained by brewing with 40 ml of boiling water.

[0117] 2.2 Ophthalmic OCTA imaging

[0118] Using ophthalmic OCTA technology, the eyeballs of C57BL / 6 mice deprived of form for 21 days were imaged to measure the axial length, anterior chamber depth, lens thickness, and vitreous cavity depth. Retinal imaging was then performed on the mice to measure the thickness of the retina and choroid at the posterior pole.

[0119] 2.3 Hematoxylin-eosin (HE) staining

[0120] The paraffin sections of the posterior wall of the eyeball were stained with HE, and the histopathological changes were observed under a 40× lens.

[0121] 2.4 Immunohistochemical staining

[0122] The paraffin sections of the posterior wall of the eyeball were used for immunohistochemical staining of MMP-2, MMP-14, and TIMP-2 proteins, and the expression of the target proteins in different tissues was observed under a 40× lens.

[0123] 2.5 Statistical analysis

[0124] GraphPad prism 10.0 statistical software was used to analyze the data.

[0125] The data were analyzed by D'Agostino & Pearson comprehensive analysis of normal distribution. The normality subset was analyzed by one-way analysis of variance (ANOVA) for multiple group comparisons. The data were analyzed by D'Agostino & Pearson comprehensive analysis of normal distribution. The statistical differences between the two groups were analyzed by nonparametric two-tailed Mann-Whitney U test for nonnormal subsets. P < 0.05 was considered statistically significant, and all results are expressed as mean ± SD.

[0126] 3 Experimental results

[0127] 3.1 Effect of tea composition on axial length of form-deprivation myopic mice

[0128] The experimental results are as follows Figure 7As shown in the figure, compared with the normal control group, the axial length of the deprived eyes in the model group increased significantly (P < 0.001), the lens length increased significantly (P < 0.01), the vitreous cavity length decreased significantly (P < 0.01), and the anterior chamber length decreased significantly (P < 0.05). The posterior pole retinal thickness and choroidal thickness decreased significantly (P < 0.05). Compared with the deprived eyes in the model group, the axial length of the deprived eyes in the HKS (2080 mg / kg) group and the methylcobalamin (14.95 mg / kg) group decreased significantly (P < 0.05), the lens length decreased significantly (P < 0.05), the vitreous cavity length increased significantly (P < 0.05), there was no significant difference in the anterior chamber length (P > 0.05), and there was no significant difference in the posterior pole retinal thickness and choroidal thickness (P > 0.05). Examples of ophthalmic OCTA imaging and retinal imaging are shown in Figure 8 In summary, HKS can significantly inhibit the axial length growth, lens thickening, and vitreous cavity depth increase induced by form deprivation in mice, and has a protective effect on form deprivation myopia in C57BL / 6 mice, and the minimum effective dose is 2080 mg / kg.

[0129] 3.2 Effects of tea composition on the ocular structure of form-deprivation myopic mice

[0130] like Fig. 9 As shown, HE staining of paraffin sections of the eyeballs revealed a significant increase in the axial length of the eye in the form deprivation group of mice under light microscopy. Compared with the control eyes, the retina and sclera of the form deprivation eyes were thinner, the fibroblasts were disordered, the collagen fibers were disordered, and the thickness was uneven. The retina of the form deprivation eyes was thinner, especially the inner nuclear layer and the inner plexiform layer. The choroid of the form deprivation eyes was thinner. Compared with the form deprivation group, the retina, choroid, and sclera of the form deprivation eyes in the HKS (2080 mg / kg) and methylcobalamin (14.95 mg / kg) groups were thickened.

[0131] 3.3 Effects of tea composition on the expression of MMP-2, MMP-14 and TIMP-2 in form-deprivation myopic mice

[0132] The expression changes of MMP-2, MMP-14 and TIMP2 proteins in the form-deprived eyes of each group of mice were shown in Figure 10-12 . The figure shows that in the normal control group, MMP-2, MMP-14, and TIMP2 positive proteins are expressed in the retina, showing brown-yellow granules with obvious staining. After form deprivation, MMP-2 protein expression was upregulated, cytoplasmic staining increased, TIMP-2 protein expression was downregulated, cytoplasmic staining decreased, and MMP-14 protein expression did not change significantly. Compared with the form deprivation group, the HKS (2080 mg / kg) and methylcobalamin (14.95 mg / kg) groups had downregulated MMP-2 protein expression, and the HKS (2080 mg / kg) group had upregulated TIMP-2 protein expression.

[0133] 4. Summary

[0134] HKS has a protective effect on form deprivation myopia in C57BL / 6 mice, with the lowest effective dose being 2080 mg / kg. HKS can regulate the abnormal expression of MMP-2 and TIMP-2 proteins induced by form deprivation, indicating that HKS may improve form deprivation-induced myopia symptoms by regulating the homeostasis of the extracellular matrix.

[0135] discuss

[0136] 1. The above experiments show that the main chemical components of the tea composition are phenolic acids and flavonoids such as (+)-catechin, myricetin, puer tea extract, kaempferol, ferulic acid, and tangeretin.

[0137] 2. Acute toxicity experiment is one of the important methods to evaluate the safety of clinical drugs. The results of the acute toxicity preliminary experiment of the tea composition (HKS) showed that the mice were in good condition and no deaths occurred after oral gavage at the maximum dosage. The LD50 value of mice was not obtained, so the maximum dosage experiment method was selected for the acute toxicity experiment of HKS. The experimental results showed that HKS had no significant changes in the activity changes, mental state, and diet of mice, and had no significant effects on the body weight, morphology of major organs, pathological examination of liver and kidney sections, organ coefficients, and serum biochemical indicators of mice. According to the guidelines for acute toxicity experiments, the maximum tolerated dose of mice is more than 100 times the clinical dose for adults to be safe. The maximum daily gavage dose of HKS for mice is 24g solid powder / kg, which is equivalent to 840 times the recommended drinking dose of 2g / 70kg for adults. It indicates that HKS has no acute toxic reaction when taken orally and is relatively safe.

[0138] 3. The efficacy experiment of HKS on the protective effect of form deprivation myopia in C57BL / 6 mice was carried out using a form deprivation myopia model, with the right eye as the deprivation eye and the left eye as the control eye. HKS (260 mg / kg, 520 mg / kg, 1040 mg / kg, 2080 mg / kg) and the positive drug methylcobalamin (14.95 mg / kg) were administered by gavage 7 days after form deprivation. The experimental results showed that HKS (2080 mg / kg) and the positive drug methylcobalamin (14.95 mg / kg) could significantly inhibit the growth of the eye axis, lens thickening, and increase in the depth of the vitreous cavity induced by form deprivation in mice, down-regulate the expression of MMP-2, and up-regulate the expression of TIMP-2. In summary, the tea composition has a certain effect in improving the symptoms of form deprivation myopia.

[0139] 4. The matrix metalloproteinase (MMP) family is a family of proteolytic enzymes that rely on metal zinc ions. It plays an important role in the degradation of the extracellular matrix (ECM), tissue reconstruction, and the regulation of various soluble factors in cells. It is closely related to tissue stability, angiogenesis, and wound healing. MMP-2 is an enzyme related to axial growth, which plays a role in scleral remodeling. In the development of myopia, MMP-2 may be involved in the extension of the axial length, but its exact mechanism needs further study. MMP-14 is a membrane-type matrix metalloproteinase. In the occurrence of myopia, MMP-14 may be related to the remodeling of ocular tissues, but its specific role in the development of myopia is not yet fully understood. TIMP-2 is an endogenous inhibitor of MMPs, which affects the metabolism of the extracellular matrix and tissue remodeling by regulating the activity of MMPs. TIMP-2 may participate in the growth of the axial length and changes in the fundus by affecting the activity of MMPs. The function of TIMP-2 is not limited to its inhibitory effect on MMPs. It may also participate in the regulation of cell behavior through non-MMP-dependent pathways. In addition to MMPs and TIMPs, there are many other proteins and molecular pathways that may be involved in the development of myopia and may become new targets for myopia treatment, such as transcription factor MAF, bone morphogenetic proteins (BMPs) and 7-methylxanthine (7-MX). HKS can regulate the abnormal expression of MMP-2 and TIMP-2 proteins induced by form deprivation, indicating that HKS may improve form deprivation-induced myopia symptoms by regulating extracellular matrix homeostasis.

[0140] In this experiment, the main chemical composition, safety and effect of HKS on improving the myopia symptoms induced by form deprivation in C57BL / 6 mice were studied by LC-MS technology and ophthalmic OCTA technology, and the changes in the expression of related proteins during the development of myopia were explored. Common animal models of myopia include form deprivation myopia model, optical defocus-induced myopia model, light environment-induced myopia model and behavioral myopia model. The form deprivation myopia model (FDM) is easy to operate and has a high modeling rate. Its modeling mechanism is to block the central visual pathway, so that the retina of the experimental animals loses form stimulation in childhood, and the axial length of the eye grows abnormally under the influence of emmetropization, forming myopia. This modeling method can produce a higher degree of myopia, causing the entire axial length including the equator to be extended. The refractive state of the occluded eye of the experimental animal cannot be measured during the experiment. The minimum effective dose of HKS to improve the symptoms of form deprivation-induced myopia is 2080 mg / kg. During the production process, it is possible to consider purifying its main components or concentrating the preparation.

[0141] Summarize

[0142] The main chemical components of the tea composition (HKS) are phenolic acids and flavonoids such as (+)-catechins, myricetin, puer tea extract, kaempferol, ferulic acid, and tangeretin. The maximum daily oral administration of the tea composition to mice is equivalent to 840 times the recommended drinking amount of 2g / 70kg for adults, indicating that the tea composition has no acute toxic reaction and is highly safe when taken orally. HKS has a protective effect on form deprivation-induced myopia symptoms in C57BL / 6 mice, with the minimum effective dose being 2080mg / kg. HKS can regulate the abnormal expression levels of MMP-2 and TIMP-2 proteins induced by form deprivation, indicating that HKS can improve form deprivation-induced myopia symptoms by regulating the homeostasis of the extracellular matrix.

Claims

1. Use of a solid composition in the preparation of a drug for preventing and / or treating form deprivation myopia; The solid composition comprises Pu'er tea powder, Baxian tea powder and tangerine peel.

2. The use according to claim 1, characterized in that: The mass ratio of the Pu'er tea powder, the Eight Immortals tea powder and the tangerine peel is 4.5-7:2-5:

2.

3. The use according to claim 1 or 2, characterized in that: The mass ratio of the Pu'er tea powder, the Eight Immortals tea powder and the tangerine peel is 5:3:

2.

4. The use according to any one of claims 1 to 3, characterized in that: The Pu'er tea powder is obtained by grinding Pu'er tea; The Eight Immortals Tea powder is obtained by grinding Eight Immortals Tea.

5. Use of tea in the preparation of drugs for preventing and / or treating form deprivation myopia; The tea consists of Pu'er tea powder, Eight Immortals tea powder and tangerine peel in a mass ratio of 5:3:

2.

6. A drug for preventing and / or treating form deprivation myopia, characterized in that: The active ingredient is the solid composition according to any one of claims 1 to 4 or the extract obtained by brewing the tea according to claim 5 with water.

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