A combination of key pharmacodynamic substances from green tea with therapeutic effects on age-related macular degeneration, along with its optimization method and application.

Abstract

The present invention relates to the field of traditional Chinese medicine, and specifically to a combination of key medicinal substances of green tea with the effect of intervening in the treatment of age-related macular degeneration, and a compatibility optimization method and application thereof. The key medicinal substance combination of green tea provided by the present invention is: quinic acid, epicatechin, gallocatechin, epigallocatechin, gallocatechin gallate, and epigallocatechin gallate. The green tea component combination optimization method based on uniform design and AMD zebrafish model provided by the present invention has reasonable process design and strong operability. The present invention can be applied to optimize large-scale Chinese medicine prescriptions and component combinations. Compared with traditional methods, it can significantly improve the optimization efficiency, save optimization costs, and has the characteristics of fast, accurate, and good reproducibility. At the same time, the optimal compatibility combination of effective components of green tea screened out by the present invention has an excellent pharmacodynamic effect of intervening in the treatment of AMD, laying a foundation for the clinical treatment application of AMD and the development of green tea health products.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a combination of key medicinal substances of green tea having the effect of intervening and treating age-related macular degeneration, and a compatibility optimization method and application thereof. Background Art

[0002] Age-related macular degeneration (AMD), with blurred central vision and distorted vision in the early stage and permanent vision loss in the late stage, is the leading eye disease causing blindness in the elderly. AMD is divided into two types: dry (atrophic) and wet (exudative). Exudative AMD is the leading cause of vision loss in people over 50 years old, with a blindness rate of up to 80%. Traditional Chinese medicine classifies AMD as "blurred vision", and clinical symptoms are mostly "phlegm and blood stasis", "blood stasis", and "qi deficiency". Especially for exudative AMD, choroidal neovascularization and hemorrhage lead to decreased vision and "sudden blindness", and macular edema leads to distorted vision and is classified as "seeing straight but curved". At present, the main clinical treatments for AMD are laser surgery and anti-VEGF drugs, but because the AMD patient population is concentrated in the elderly, the high price of current clinical treatments and the high surgical risks have deterred many AMD patients.

[0003] green tea( Camellia Sinensis (L.) Kuntze) belongs to the Theaceae family and the Camellia genus. It is cool in nature, bitter and sweet in taste, and enters the liver, pericardium, heart, lung, spleen, kidney, and stomach meridians. It has the effects of lowering qi and digestion, clearing the head, clearing phlegm, and clearing heat and detoxifying. AMD is classified as "phlegm stasis", "blood stasis", and "qi deficiency" in traditional Chinese medicine. Therefore, it is often used in combination with liver-soothing and kidney-tonifying drugs in clinical practice.

[0004] The compatibility rules of Chinese medicine are the concrete embodiment of the theory of syndrome differentiation and treatment in Chinese medicine. With the development of chemical component separation technology, it is no longer limited to the combination of multiple medicinal flavors. Academician Zhang Boli proposed a new strategy for the study of effective component combinations of modern Chinese medicine based on component combinations. With the study of effective component combinations, Professor Jia Xiaobin proposed the "component structure" theory, believing that the material basis of the efficacy of Chinese medicine is composed of multiple components according to a certain composition structure (content) ratio, and further systematically and deeply proposed a new model for Chinese medicine research with components as functional units. At present, there has been no screening of the optimal compatibility structure of the effective components of green tea, and it is difficult to clarify the pharmacodynamic mechanism of effective component combinations. Therefore, how to give full play to the unique advantages of Chinese medicine compatibility in enhancing efficacy and reducing toxicity, how to combine modern analytical chemistry, molecular biology and other research methods to analyze the compound compatibility enhancing efficacy and reducing toxicity mechanism of key effective components of green tea, and provide reference and basis for the rational use of green tea in clinical practice; is a technical problem that needs to be solved at present. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a combination of key medicinal substances of green tea that has the effect of intervening and treating age-related macular degeneration.

[0006] The present invention also provides a method for optimizing the combination of the above-mentioned key medicinal substances of green tea. The method is based on the component structure theory, and adopts the uniform design method and the AMD zebrafish model to examine the medicinal effects of the effective components of green tea under different compatibility structures, analyze the synergistic effect and matching relationship between the components, and at the same time use network pharmacology and molecular docking technology to explore the potential mechanism of action of the effective components of green tea in intervening AMD, thereby providing a method for optimizing the combination of green tea components with the effect of intervening in the treatment of age-related macular degeneration.

[0007] Another object of the present invention is to provide an application of the above-mentioned combination of key medicinal substances in green tea.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0009] The present invention provides a combination of key medicinal substances of green tea with the effect of intervening and treating age-related macular degeneration. The key medicinal substances of green tea are: quinic acid, epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), gallocatechin gallate (GCG), and epigallocatechin gallate (EGCG).

[0010] Furthermore, the mass ratio of quinic acid: epicatechin: gallocatechin: epigallocatechin: gallocatechin gallate: epigallocatechin gallate is 8-10: 8-10: 3-5: 8-10: 3-5: 3-5.

[0011] Furthermore, the optimal compatibility structure of the combination of key medicinal substances in green tea is quinic acid: epicatechin: gallocatechin: epigallocatechin: gallocatechin gallate: epigallocatechin gallate, with a mass ratio of 8:8:5:10:3:3.

[0012] The present invention also provides a method for optimizing the compatibility of the above-mentioned combination of key medicinal substances in green tea, comprising the following steps:

[0013] (1) Design different combinations of key active ingredients of green tea using uniform design method;

[0014] (2) Using the model organism zebrafish to investigate the maximum dosage of the six component monomers;

[0015] (3) Based on the maximum dosage of the six components obtained from the study, improve U11 (11 6) Uniform design table, using AMD zebrafish model, taking the angiogenesis of zebrafish fundus as the efficacy evaluation index, to examine the efficacy of different combinations;

[0016] (4) Conduct multiple linear regression analysis to determine the optimal combination of green tea active ingredients;

[0017] (5) Using the AMD zebrafish model, the angiogenesis of the zebrafish retina was used as the efficacy evaluation index to verify the efficacy of the optimal combination;

[0018] (6) Based on network pharmacology, explore the potential mechanisms of action of the six effective components in the treatment of AMD, screen key pathways and targets, and verify them using molecular docking technology;

[0019] (7) An AMD zebrafish model was constructed and the optimal combination of components was given for intervention. The qPCR technology was used to detect the expression regulatory effects of the six component combinations on key gene targets, and the key pharmacological substances of green tea after the optimized combination were determined.

[0020] Furthermore, in step (4), the multivariate linear regression analysis uses six components: quinic acid (X1), epicatechin (X2), epigallocatechin gallate (X3), epigallocatechin (X4), epigallocatechin gallate (X5), and epigallocatechin gallate (X6) as independent variables, and the relative inhibition rate of zebrafish fundus vascular growth as the dependent variable (Y).

[0021] Furthermore, in step (6), the targets are TP53, TNF, IL6, STAT3, HIF1A, MYC, CTNNB1, JUN, and EGFR.

[0022] Furthermore, in step (7), the primers used for qPCR detection are:

[0023]

[0024] The present invention also provides the use of the above-mentioned combination of key medicinal substances of green tea in the preparation of a drug for intervention treatment of age-related macular degeneration.

[0025] Among the key medicinal substances of green tea provided by the present invention, the concentration range of quinic acid, EC and EGC is 40-50 μg / mL, and the concentration range of GC, GCG and EGCG is 15-25 μg / mL, which are all effective doses when combined.

[0026] The beneficial effects of the present invention are as follows: the green tea component combination optimization method based on uniform design and AMD zebrafish model provided by the present invention has reasonable process design and strong operability. The present invention can be applied to optimize large-scale Chinese medicine prescriptions and component combinations. Compared with traditional methods, it can significantly improve the optimization efficiency and save the optimization cost. It has the characteristics of rapidity, accuracy, good reproducibility, etc. At the same time, the optimal combination of effective components of green tea screened by the present invention has an excellent pharmacological effect of intervening in the treatment of AMD, laying a foundation for the clinical treatment application of AMD and the development of green tea health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Effects of green tea active ingredients on zebrafish survival rate ( , n =30);

[0028] Figure 2 Fluorescence microscopy images of the fundus blood vessels of zebrafish in different groups ( , n =10);

[0029] Figure 3 Effects of different group combinations on the growth of blood vessels in the zebrafish retina ( , n =10); where B is the fluorescence area of ​​the fundus vessels of each group of zebrafish; C is the relative inhibition rate of fundus vessel growth of each group of zebrafish;

[0030] Figure 4 Effect of the optimal combination of green tea active ingredients on the growth of blood vessels in the zebrafish retina ( , n =10); where A is the fluorescence micrograph of fundus vessels of zebrafish in each group; B is the fluorescence area of ​​fundus vessels of zebrafish in each group;

[0031] Figure 5 This is a Venn diagram of green tea active ingredients and AMD intersection targets;

[0032] Figure 6 It is a “component-disease” target network diagram;

[0033] Figure 7 This is the core target PPI network diagram;

[0034] Figure 8 This is the result of GO enrichment analysis;

[0035] Fig. 9 The results of KEGG (B) enrichment analysis;

[0036] Fig.10It is a molecular docking model diagram; Note: D, E, F, K, L, M, and N are GCG and CTNNB1, EGFR, HIF1A, STAT3, TNF, TP53, and VEGFA targets, respectively; A, G, H, and J are EGCG and AKT, IL6, JUN, and PI3K targets, respectively; I, C, and B are quinic acid and MYC, GC and BCL-2, and EGC and BAX, respectively;

[0037] Fig.11 The expression of key target mRNA in zebrafish eye tissue ( , n =3). DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0039] In some embodiments, the specific compatibility optimization method is:

[0040] 1. Use the uniform design method to design different group matching groups. Since there are six effective components, we design 6 factors and 11 levels of U11 (11 6 ) Uniform design table;

[0041] 2. The maximum dosage of the six components was investigated using the model organism zebrafish, and 2 dpf Flila:EGFP zebrafish were selected for the experiment;

[0042] 3. Based on the maximum dosage of the six components obtained from the investigation, improve U11 (11 6 ) Uniform design table, a total of 11 different group combinations were designed;

[0043] 4. Construct an AMD zebrafish model. Select 2 dpf Flila:EGFP zebrafish and induce intraocular hypoxia with 100 μg / mL cobalt chloride to induce abnormal proliferation of fundus blood vessels, thus constructing an exudative AMD zebrafish model.

[0044] 5. Using the AMD zebrafish model, the angiogenesis of the zebrafish retina was used as the efficacy evaluation index to investigate the efficacy of 11 combinations;

[0045] 6. Using SPSS 23.0 software, multiple linear regression analysis was performed with six components: quinic acid (X1), EC (X2), GC (X3), EGC (X4), GCG (X5), and EGCG (X6) as independent variables and the relative inhibition rate of zebrafish fundus vascular growth as the dependent variable (Y) to determine the optimal combination of effective components of green tea;

[0046] 7. Using the AMD zebrafish model, the angiogenesis of the zebrafish retina was used as the efficacy evaluation index to verify the efficacy of the best combination;

[0047] 8. Based on network pharmacology, explore the potential mechanism of action of six effective components in the treatment of AMD, screen key pathways and targets, and verify them using molecular docking technology;

[0048] 9. Construct an AMD zebrafish model and give the best combination of components for intervention. After the intervention, precisely take the zebrafish eye parts and use qPCR technology to detect the expression regulation effects of the six component combinations on key gene targets.

[0049] Example 1

[0050] 1 Materials and instruments

[0051] 1.1 Animals

[0052] Adult Flila:EGFP vascular fluorescent transgenic zebrafish were provided by the Institute of Pharmacology, Shandong University of Traditional Chinese Medicine, and cultured in a professional zebrafish culture system. The culture environment was maintained at a constant temperature of 28 ℃ and a daily light / dark cycle of 14 h / 10 h. Adult male and female zebrafish naturally mated and laid eggs in a mating tank, which were then collected into culture dishes and incubated in a 28 ℃ constant temperature and light incubator.

[0053] 1.2 Drugs and reagents

[0054] Cobalt chloride (Shanghai MacLean Biochemical Technology Co., Ltd., batch number: C15852024); esculin digoxin eye drops (Germany Vision Turin Pharmaceutical Co., Ltd., batch number: 24A077); quinic acid (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS0713-0020); epicatechin (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS1391-0200); epigallocatechin gallate (Chengdu Pusi Biotechnology Co., Ltd., batch number: PU0554-0025); epigallocatechin (Chengdu Pusi Biotechnology Co., Ltd., batch number: PU0460-0025); epigallocatechin gallate (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS1489-0020); epigallocatechin gallate (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS3007-0050); PrimeScript™ RT reagent Kit (Japan TAKARA, batch number: RR047A), TB Green® Premix Ex Taq™ II (TAKARA, Japan, batch number: RR820A); Animal Tissue RNA Extraction Kit (Shandong Sikoje Biotechnology Co., Ltd., batch number: AC0202).

[0055] 1.3 Instruments

[0056] BP211D electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.); Allegra X-30R high-speed refrigerated centrifuge (Beckman Coulter, Inc., USA); IX73 fluorescence microscope (Olympus Corporation, Japan); BJPX-L200 illumination incubator (Shandong Bo Ke Holding Co., Ltd.); KZ-III-F tissue grinder (Wuhan Sewell Biotechnology Co., Ltd.); Roche LightCycler 96 real-time fluorescence quantitative PCR (Roche, Switzerland); ultra-micro-spectrophotometer (Thermo Scientific, USA); zebrafish culture system (Shanghai Haisheng Biological Experiment Equipment Co., Ltd., model ZA-D5).

[0057] 2. Detection and analysis

[0058] 2.1 Effects of green tea active ingredients on zebrafish survival rate

[0059] Flila:EGFP zebrafish at 2 dpf after fertilization were selected. After demembranation, the normally developed juveniles were screened under a fluorescence microscope and placed in 6-well plates, with 30 fish per well and two replicates per group. The effects of different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL, 1000 μg / mL, 1500 μg / mL) of quinic acid, EC, GC, EGC, GCG, and EGCG on the survival rate of zebrafish were investigated, and the maximum tolerance concentration (MTC) of each component for zebrafish was determined; then the effects of different concentration combinations of each component (MTC, 1 / 2MTC, and 1 / 4MTC) on the survival rate of zebrafish were investigated.

[0060] 2.2 Cobalt chloride-induced AMD zebrafish experiment

[0061] Select Flila:EGFP zebrafish at 2 dpf after fertilization, and screen the juveniles with normal development under a fluorescence microscope after demembraning and place them in a 6-well plate. Select 100 μg / mL cobalt chloride to induce intraocular hypoxia in zebrafish and induce abnormal proliferation of fundus blood vessels to construct an exudative AMD zebrafish model [8] The growth of zebrafish fundus blood vessels was observed under a fluorescence microscope, and the fluorescent area of ​​zebrafish fundus blood vessels was measured using Image J software to confirm that the AMD zebrafish model was successfully established.

[0062] 2.3 Study on the compatibility structure of effective components of green tea

[0063] 2.3.1 Uniform design grouping

[0064] Quinic acid ( X 1 )、EC( X2 )、GC( X 3 )、EGC( X 4 )、GCG( X 5 )、EGCG( X 6 ) as the factors to be investigated, each factor is set to 11 levels, increasing in the dosage range, and the dosage range is set according to the MTC of each component obtained in item "2.1". Select the uniform design table U11 (11 6 ), according to the uniform design table, when the number of factors is 6, the deviation is 0.4942, and columns 1, 2, 3, 4, 5, and 6 are selected (Table 1-1 and Table 1-2).

[0065] Table 1-1 Uniform design table U11 (11 6 )

[0066]

[0067] Table 1-2 Uniform design table U11 (11 6 ) Use table

[0068]

[0069] 2.3.2 Screening of the best combination of green tea active ingredients

[0070] Flila:EGFP zebrafish of 2 dpf after fertilization were selected, and the juveniles with normal development were screened and placed in 6-well plates, with 10 fish in each well. A blank control group (C), a model group (M), a positive control group (Y), and 11 drug-treated groups (S1-S11) were set up. Except for group C, the other groups were given 100 μg / mL cobalt chloride for incubation and modeling. Group Y and the green tea-treated group were given drugs at the same time as modeling. Group Y was given 100 μg / mL esculent diglycosides eye drops. Groups S1-S11 were treated according to the uniform design table U11 (11 6 ) were used for drug intervention with different group combination structures obtained by the method of the present invention. The zebrafish were incubated at 28 ℃ for 72 h, and the survival of the zebrafish was observed. The growth of the fundus blood vessels of the zebrafish was observed under a fluorescence microscope. The fluorescence area of ​​the fundus blood vessels of the zebrafish was measured using Image J software. The inhibition rate and relative inhibition rate of the growth of the fundus blood vessels of the zebrafish in each group were calculated.

[0071] Inhibition rate = (Area drug treatment group - Area model group) / (Area blank control group - Area model group);

[0072] Relative inhibition rate = inhibition rate / [(Area positive control group-Area model group) / (Area blank control group-Area model group)].

[0073] 2.3.3 Analysis of the optimal compatibility structure of green tea active components

[0074] SPSS 23.0 software was used to analyze the quinic acid ( X 1 )、EC( X 2 )、GC( X 3 )、EGC( X 4 )、GCG( X 5 )、EGCG( X 6 ) was the independent variable, and the relative inhibition rate of zebrafish fundus vascular growth was the dependent variable ( Y ), and multiple linear regression analysis was performed to determine the optimal compatibility of the effective components of green tea.

[0075] 2.3.4 Verification Experiment

[0076] Flila:EGFP zebrafish of 2 dpf after fertilization were selected, and the normally developed juveniles were screened and placed in 6-well plates, with 80 in each well. A blank control group (C), a model group (M), a positive control group (Y), and an optimal compatibility group (S0) were set up. According to the experimental method of "2.3.2", 10 zebrafish were selected from each group and photographed under a fluorescence microscope to investigate the effect of the optimal compatibility of green tea effective components on the growth of blood vessels in the fundus of zebrafish.

[0077] 2.4 Network Pharmacology

[0078] 2.4.1 Target screening of green tea active components for intervention of AMD

[0079] The targets of quinic acid, EC, GC, EGC, GCG, and EGCG were searched in the Swiss Target Prediction and PharmMapper databases, and the targets of each component were obtained by removing duplicates and merging. The keywords "Age-related Macular Degeneration" were used to search in the CTD and GeneCards databases, and secondary screening was performed with "relevance score greater than the median" and "inference score greater than the median", and duplicates were removed and integrated to obtain AMD disease targets. The targets of each component and the AMD disease targets were intersected, and the intersection was the target of the effective components of green tea for intervening AMD.

[0080] 2.4.2 Construction of PPI network and screening of core targets

[0081] The selected targets were imported into the STRING database, the species was selected as "Homo sapiens", the minimum interaction threshold was set to "highest confidence", and the free nodes were hidden. CytoScape 3.9.1 was used to construct the "disease-component-target" network diagram and PPI network diagram of green tea effective components-AMD, and the built-in Network Analyzer was used to analyze the network topology parameters of components and targets, and the core targets were screened based on the node degree value (degree).

[0082] 2.4.3 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis

[0083] The potential targets of the screened green tea effective components for intervening AMD were subjected to GO enrichment analysis and KEGG signaling pathway analysis in the DAVID database to predict key pathways and targets.

[0084] 2.4.4 Molecular docking experiment

[0085] In order to analyze the interaction between the screened key targets and the effective components of green tea, quinic acid, EC, GC, EGC, GCG, and EGCG were molecularly docked with the key targets. The sdf structure files of each component were downloaded from the PubChem database, imported into the ChemBio 3D software to adjust the spatial conformation of the compound, minimize energy optimization was performed, and saved in mol2 format. The 3D structure of the key target was then downloaded from the PDB website, and PyMOL was used to remove water molecules and original ligands. Finally, Maestro software was used to dock each component with the key target.

[0086] 2.5 Real-time fluorescence quantitative PCR (qPCR) validation experiment

[0087] The qPCR method was used to detect the effect of the optimal combination of green tea effective components on the expression level of key target genes in zebrafish eye tissue. After the verification experiment of "2.3.4", the zebrafish in each group were taken, and the zebrafish eye parts were precisely cut under a stereo microscope, and the whole was homogenized. The total RNA of zebrafish eye tissue was extracted using an RNA extraction kit. After extraction, the concentration of RNA was detected, and then the RNA was reverse transcribed into cDNA using a fluorescent quantitative PCR instrument, and PCR amplification was performed. The amplification conditions were: 95 ℃ pre-denaturation for 10 min; 95 ℃ denaturation for 10 s, 60 ℃ annealing for 10 s, 72 ℃ extension for 10 s, and 40 cycles. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are shown in Table 2.

[0088] Table 2 Primer sequences

[0089]

[0090] 2.6 Data Analysis

[0091] SPSS 23.0 software was used for statistical analysis of the data, GraphPad Prism7 was used for graphical analysis of the statistical results, and the comparison between the two groups was performed using the one-way ANOVA test method. P <0.05 indicated that the difference was statistically significant.

[0092] 3 Results

[0093] 3.1 Effects of green tea active ingredients on zebrafish survival rate

[0094] Depend on Figure 1 It can be seen that when the concentrations of quinic acid, EC, and EGC were higher than 200 µg / mL, and when the concentrations of GC, GCG, and EGCG were higher than 100 µg / mL, zebrafish began to die, and the number of deaths gradually increased with the increase in concentration. Therefore, the maximum tolerable concentration (MTC) of quinic acid, EC, and EGC was 200 µg / mL, and the MTC of GC, GCG, and EGCG was 100 µg / mL. In order to subsequently compare the pharmacological effects of different compatibility structures of each component on AMD zebrafish, it is necessary to first examine the MTC of each component after combination. Therefore, the effects of MTC, 1 / 2 MTC, 1 / 4 MTC, 1 / 6 MTC, and 1 / 8 MTC combination of each component on the survival rate of zebrafish were investigated. The final results showed that when the components were administered in combination with 1 / 4 MTC, zebrafish began to die, but the number of deaths was small. When the concentration was higher than this, a large number of deaths occurred. Therefore, 1 / 4 MTC (quinic acid: EC: GC: EGC: GCG: EGCG = 50: 50: 25: 50: 25: 25 µg / mL) was finally selected as the maximum dosage concentration.

[0095] 3.2 Compatibility structure experiment of green tea effective components

[0096] 3.2.1 Screening of the best combination of green tea active ingredients

[0097] Based on the uniform design table U11 (11 6 ) and its usage table, a total of 11 groups of different component combinations were designed, namely S1-S11, to conduct the efficacy experiment of intervening AMD zebrafish. The detailed component concentration information and efficacy of each group are shown in Table 3 and Figure 2 and Figure 3 Compared with group C, the blood vessels in the fundus of zebrafish in group M showed significant abnormal growth ( P <0.001); Compared with group M, the abnormal growth of blood vessels in the fundus of zebrafish in group Y and 11 combined drug groups was significantly reduced ( P<0.001), and gradually approached group C. The intervention effect of group S11 was the worst, but it was still significantly different from group M ( P <0.001), the intervention effect of S7 group was the most obvious, the inhibition rate and relative inhibition rate of abnormal fundus vascular growth were the highest, and it was the group with the best efficacy. At this time, the effective component compatibility structure was quinic acid ( X 1 ):EC( X 2 ):GC( X 3 ):EGC ( X 4 ):GCG( X 5 ):EGCG ( X 6 ) = 46:42:24:41:19:18. Since the abnormal formation of blood vessels in the fundus of zebrafish was significantly reduced after administration in the S1-S11 groups, when the concentrations of quinic acid, EC, and EGC were in the range of 40-50 µg / mL, and the concentrations of GC, GCG, and EGCG were in the range of 15-25 µg / mL, they were all effective doses, and the effective ratio of green tea components was quinic acid: EC: GC: EGC: GCG: EGCG = 8-10: 8-10: 3-5: 8-10: 3-5: 3-5.

[0098] Table 3 Structural information and pharmacodynamic effects of different components ( , n =10)

[0099]

[0100] 3.2.2 Analysis and verification of the optimal compatibility structure of green tea active components

[0101] Quinic acid ( X 1 )、EC( X 2 )、GC( X 3 )、EGC( X 4 )、GCG( X 5 )、EGCG( X 6 ) was the independent variable, and the relative inhibition rate of zebrafish fundus vascular growth was the dependent variable ( Y ), and then perform multiple linear regression analysis, and finally get the regression equation as follows: Y =1.80634-0.0098 X 1 -0.0036X 2 +0.00256 X 3 +0.00048 X 4 -0.0031 X 5 -0.0112 X 6 ( R 2 =0.95487, P =0.04139<0.05), when Y When the maximum value is taken, the drug effect is the best. According to the regression equation model, the best component compatibility structure is predicted to be quinic acid ( X 1 ):EC( X 2 ):GC( X 3 ):EGC ( X 4 ):GCG( X 5 ):EGCG ( X 6 )=8:8:5:10:3:3.

[0102] The best combination group (S0) predicted by the model was verified by zebrafish efficacy experiment, and group C, group M, group Y and group S0 were set. Figure 4 As shown in Table 3, the angiogenesis of zebrafish fundus in the S0 group showed a significant decrease compared with that in the M group ( P <0.001), and from the relative inhibition rate of vascular growth, the efficacy of S0 group is very close to that of Y group, and is better than that of S7 group selected under "3.2.1". Therefore, the optimal compatibility structure of green tea active components in intervening AMD is quinic acid: EC: GC: EGC: GCG: EGCG = 8:8:5:10:3:3.

[0103] 3.3 Network pharmacology research

[0104] 3.3.1 Screening of potential targets of green tea active components for intervention of AMD

[0105] By searching the database, we screened the targets of quinic acid, EC, GC, EGC, GCG, and EGCG, and obtained a total of 2199 potential targets after integration and deduplication. Using "Age-related Macular Degeneration" as the keyword, we searched for disease targets in the database, and after integration and deduplication, we screened a total of 2496 targets. Using Venny 2.1, we intersected the drug targets and disease targets and drew a Venn diagram, and obtained a total of 571 common targets, namely the potential targets of green tea active ingredients for intervening in AMD ( Figure 5 ).

[0106] 3.3.2 Construction of “component-disease” target network diagram and PPI network diagram

[0107] The six green tea active ingredient targets and AMD disease targets were combined to construct a "ingredient-disease" target network, which has 577 nodes and 1318 edges ( Figure 6 The more lines between the component and the target, the greater the degree value, indicating that the correlation between the component and the efficacy is stronger. Among the six components, the degree values ​​are EGCG, GCG, GC, EGC, EC, and quinic acid from large to small.

[0108] The String database was used to perform protein interaction analysis on all potential targets of green tea active ingredients in intervening AMD, and Cytoscape 3.9.1 was used for topological analysis to screen core targets and construct a PPI network diagram of core targets. Figure 7 The results showed that the network involved 84 nodes, 2942 edges, and an average degree of 70.048. Nine targets ranked first (degree = 83) were selected based on the node degree ranking, namely TP53, TNF, IL6, STAT3, HIF1A, MYC, CTNNB1, JUN, and EGFR, which were predicted to be the core targets of green tea effective components in intervening in AMD.

[0109] 3.3.3 GO and KEGG enrichment analysis

[0110] The potential targets of green tea active components in intervening AMD were subjected to GO and KEGG enrichment analysis. P Sort by value, filter the first 10 analysis items respectively, see Figure 8In the biological process (BP) classification, the items related to AMD mainly involved negative regulation of apoptosis, hypoxia response, positive regulation of cell proliferation, inflammatory response, and positive regulation of angiogenesis; in the cell composition (CC) classification, the items related to AMD mainly involved extracellular exosomes, macromolecular complexes, mitochondria, and RNA polymerase II transcription factor complexes; in the molecular function (MF) classification, the items related to AMD mainly involved RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, growth factor activity, and peptidase activity.

[0111] Through KEGG enrichment analysis, P <0.05, 6 green tea active components were enriched into 179 signaling pathways, and the top 20 pathways were screened and displayed in a bubble chart. Fig. 9 . The results showed that the top three were the pathogenesis of cancer, lipids and atherosclerosis, and the AGE-RAGE signaling pathway in diabetic complications. It can be seen that the six components may also have anti-tumor and intervention effects on lipid metabolism disorders; among the top 20 signaling pathways, the PI3K-AKT signaling pathway, TNF signaling pathway, Apoptosis and HIF-1 hypoxia signaling pathways are highly related to the occurrence and development of AMD. The key targets involved in the pathways are PI3K, AKT, TNF, BAX, BCL-2, HIF1A and VEGFA. It can be seen that the six effective components of green tea may play a pharmacological role in intervening in the treatment of AMD by affecting the process of these signaling pathways and regulating the expression of key targets.

[0112] 3.3.4 Molecular docking

[0113] The core targets selected from the above PPI network (TP53, TNF, IL6, STAT3, HIF1A, MYC, CTNNB1, JUN, EGFR) and the core targets in the key pathways selected from the KEGG enrichment analysis results (PI3K, AKT, TNF, BAX, BCL, HIF1A, VEGFA) were integrated and deduplicated to perform molecular docking experiments with EGCG, GCG, GC, EGC, EC, and quinic acid. The specific docking results and binding energy information are shown in Fig.10And Table 4. In the molecular docking experiment, the lower the binding energy between the molecule and the target, the more stable the binding between the ligand and the receptor protein will be. When the binding energy is less than -5 kJ / mol, the two have strong binding activity; when the binding energy is less than -7 kJ / mol, the two have strong binding activity. The results showed that the binding energy between the 6 components and the 11 targets was negative, and the binding energy between the components and the targets was less than -5 kJ / mol, indicating that there was a certain binding ability between the components and the targets. Among them, GCG had the best docking activity with CTNNB1, EGFR, HIF1A, STAT3, TNF, TP53, and VEGFA targets, EGCG had the best docking activity with AKT, IL6, JUN, and PI3K targets, and quinic acid, GC, and EGC had the best docking activity with MYC, BCL-2, and BAX targets, respectively. Moreover, among the 11 targets, the binding energies between HIF1A, EGFR, IL6, and VEGFA and the six components were all less than -5 kJ / mol, indicating that there was a strong binding activity between each component and the target, and it is very likely that the pharmacological effect of intervening in AMD can be exerted by regulating these targets.

[0114] Table 4 Binding energy information between green tea active components and core targets

[0115]

[0116] 3.4 Effects of the optimal combination of green tea components on the expression of key target genes in AMD zebrafish eye tissues

[0117] In the above molecular docking experiment, the binding performance of 6 effective components of green tea with 14 key action targets was investigated. In order to further clarify the mechanism of intervention of AMD after component combination, the qPCR method was used to detect the regulatory effect of the optimal component combination on the expression of 14 key action targets. Fig.11 . The results showed that there was no significant difference in the expression of CTNNB1, JUN, and MYC genes in the ocular tissues of the three groups of zebrafish; the expression of AKT, BCL-2, EGFR, HIF1A, PI3K, STAT3, TP53, and TNF genes in the ocular tissues of zebrafish in group M was significantly lower than that in group C, and the expression levels showed a significant upward trend after drug intervention; the expression of BAX, IL6, and VEGFA genes in the ocular tissues of zebrafish in group M was significantly upregulated compared with that in group C, and the expression levels showed a significant downward trend after drug intervention. Therefore, it is concluded that the optimal compatibility structure of green tea components may play a pharmacological role in intervening AMD by regulating the expression of AKT, BCL-2, EGFR, HIF1A, PI3K, STAT3, TP53, BAX, IL6, VEGFA, and TNF genes in zebrafish ocular tissues.

[0118] 4 Conclusion

[0119] In summary, the present invention takes the component structure theory as the core, uses the uniform design test method in combination with the AMD zebrafish animal model, and finds that when quinic acid, EC, and EGC are 40-50 µg / mL, and GC, GCG, and EGCG are 15-25 µg / mL for compatibility, it is the effective dose of green tea components for the intervention treatment of AMD. Therefore, the effective ratio range of green tea components is quinic acid: EC: GC: EGC: GCG: EGCG = 8-10: 8-10: 3-5: 8-10: 3-5: 3-5, and the best compatibility combination is quinic acid: EC: GC: EGC: GCG: EGCG = 8: 8: 5: 10: 3: 3. And through network pharmacology and molecular docking technology, it is preliminarily proved that the effective components of green tea regulate the expression of genes such as PI3K, AKT, TP53, HIF1A, VEGF, activate the PI3K-AKT signaling pathway, HIF-1 hypoxia signaling pathway, Apoptosis and TNF signaling pathway, and finally play an intervention and treatment role in AMD. The present invention combines "precision medicine" with "compatibility structure" to provide a green tea component combination optimization method with intervention and treatment of age-related macular degeneration, providing ideas for the study of the compatibility rules and efficacy and toxicity reduction of traditional Chinese medicine components, and laying a foundation for the research and development of new drugs for green tea components to intervene in AMD.

Claims

1. A combination of key medicinal substances of green tea having an intervention effect in treating age-related macular degeneration, characterized in that: The key medicinal substance combination of green tea is: quinic acid: epicatechin: gallocatechin: epigallocatechin: gallocatechin gallate: epigallocatechin gallate, and the mass ratio is 8-10: 8-10: 3-5: 8-10: 3-5: 3-5: 3-5.

2. The green tea key medicinal substance combination according to claim 1, characterized in that: The key medicinal substance combination of green tea is: quinic acid: epicatechin: gallocatechin: epigallocatechin: gallocatechin gallate: epigallocatechin gallate, and the mass ratio is 8:8:5:10:3:

3.

3. A method for optimizing the compatibility of the combination of key medicinal substances in green tea as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Design different combinations of key active ingredients of green tea using uniform design method; (2) Using the model organism zebrafish to investigate the maximum dosage of the six component monomers; (3) Improve U11-11 based on the maximum dosage of the six components obtained from the investigation 6 The uniform design table uses the AMD zebrafish model and the angiogenesis of the zebrafish fundus as the efficacy evaluation index to examine the efficacy of different combinations; (4) Perform multiple linear regression analysis to determine the optimal combination of green tea active ingredients; (5) Using the AMD zebrafish model, the angiogenesis of the zebrafish retina was used as the efficacy evaluation index to verify the efficacy of the optimal combination; (6) Based on network pharmacology, explore the potential mechanisms of action of the six effective components in the treatment of AMD, screen key pathways and targets, and verify them using molecular docking technology; (7) An AMD zebrafish model was constructed and the optimal combination of components was given for intervention. The qPCR technology was used to detect the expression regulatory effects of the six component combinations on key gene targets, and the key pharmacological substances of green tea after the optimized combination were determined.

4. The compatibility optimization method according to claim 3, characterized in that: In step (4), the multivariate linear regression analysis uses six components: quinic acid X1, epicatechin X2, gallocatechin X3, epigallocatechin X4, gallocatechin gallate X5, and epigallocatechin gallate X6 as independent variables, and the relative inhibition rate of zebrafish fundus vascular growth as the dependent variable.

5. The compatibility optimization method according to claim 3, characterized in that: In step (6), the targets are TP53, TNF, IL6, STAT3, HIF1A, MYC, CTNNB1, JUN, and EGFR.

6. The compatibility optimization method according to claim 3, characterized in that: In step (7), the primers used for qPCR detection are: 。 7. Use of the green tea key medicinal substance combination as claimed in claim 1 or 2 in the preparation of a drug for intervention treatment of age-related macular degeneration.

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