Chrysanthemum plant-derived traditional Chinese medicine monomer composition for treating diabetic retinopathy as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine monomer composition derived from chrysanthemum plants and used for treating diabetic retinopathy as well as a preparation method and application of the traditional Chinese medicine monomer composition. The traditional Chinese medicine monomer composition prepared from the chrysanthemum plant as the raw material is high in safety, small in side effect and definite in component and is prepared according to a specific ratio, and pharmacodynamic experiments prove that compared with an ethanol extract, the traditional Chinese medicine monomer composition is more remarkable in curative effect on diabetic retinopathy, and the curative effect of the traditional Chinese medicine monomer composition on diabetic retinopathy is better than that of a traditional Chinese medicine composition on diabetic retinopathy. The composition can improve the pathological state of diabetic retinopathy from multiple targets and multiple ways, specifically can inhibit body mass increase caused by high glucose, reduce blood glucose level, improve retinal tissue structure damage, recover retinal tissue thickness, inhibit retinal cell apoptosis and inflammatory response, reduce the expression level of HIF-1alpha and VEGF protein in retinal tissue, and can be used for treating diabetic retinopathy. Scientific basis and technical support are provided for research and development of novel drugs for treating the diabetic retinopathy, and important clinical significance and market application prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a traditional Chinese medicine monomer composition derived from chrysanthemum plant for treating diabetic retinopathy, its preparation method, and its application. Background Technology

[0002] Diabetic retinopathy (DR) is a leading cause of blindness and visual impairment, and the number of people with DR worldwide is projected to increase from 103 million in 2020 to 161 million in 2045. Despite increasing research into DR treatment, effective treatment in clinical practice remains challenging due to the complex pathogenesis of the disease.

[0003] Currently, the main treatments for diabetic retinopathy (DR) in clinical practice fall into two categories: drug therapy and surgical treatment. Drug therapy primarily includes hypoglycemic agents and anti-VEGF drugs. Hypoglycemic agents are used to control basal blood glucose levels to slow the progression of DR, while anti-VEGF drugs (such as aflibercept and bevacizumab) are administered intraocularly to inhibit abnormal angiogenesis and alleviate fundus lesions. When DR progresses to an advanced stage, patients require surgical treatments such as panretinal laser photocoagulation and vitrectomy to control the condition by sealing leaking blood vessels, clearing vitreous hemorrhage, or repositioning detached retina. However, these treatments can lead to side effects such as hypoglycemia, gastrointestinal discomfort, decreased renal function, and peripheral visual field loss.

[0004] Traditional Chinese medicine (TCM) has demonstrated significant clinical efficacy in slowing the progression of diabetic retinopathy (DR), reducing the incidence of blindness, and improving patients' quality of life, with the advantage of low toxicity. In recent years, it has become an important direction for the research and development of new drugs for DR treatment. Currently, most TCM used in clinical practice and research for DR treatment are single-herb extracts or compound extracts. Although they have been proven to have certain preventive and therapeutic effects on DR, the chemical composition of these extracts is complex, and their specific therapeutic targets and molecular mechanisms are difficult to elucidate. Furthermore, they suffer from difficulties in quality control, large batch-to-batch differences in efficacy, and low purity, which limit their widespread clinical application and further research.

[0005] Chrysanthemum, the dried capitulum of *Chrysanthemum morifolium* Ramat., a plant in the Asteraceae family, is a major traditional Chinese medicine used for both food and medicine. It is believed to have effects such as dispelling wind-heat, calming the liver and improving eyesight, and detoxifying and reducing swelling. Modern research shows that chrysanthemum contains abundant flavonoids, phenolic acids, volatile oils, and other chemical components, exhibiting various pharmacological activities including antioxidant, anti-inflammatory, antibacterial, hypoglycemic, and hepatoprotective effects. Existing research has confirmed that the chemical components and pharmacological effects of chrysanthemum stems, leaves, roots, and inflorescences are similar, and they all possess potential for development and utilization. However, current pharmacological research on chrysanthemum mainly focuses on its total flavonoid extract; there are no publicly available documents or patent reports on the therapeutic effects of specific herbal monomer compositions derived from chrysanthemum on diabetic retinopathy. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a traditional Chinese medicine monomer composition derived from chrysanthemum plants for the treatment of diabetic retinopathy. This traditional Chinese medicine monomer composition has clearly defined components and a scientific ratio, with each monomer compound acting synergistically. It has a definite curative effect in treating diabetic retinopathy with few side effects, providing a scientific basis and technical support for the development of new therapeutic drugs for diabetic retinopathy.

[0007] This invention is achieved through the following technical solution:

[0008] On one hand, the present invention provides a traditional Chinese medicine monomer composition derived from chrysanthemum plant for treating diabetic retinopathy, wherein the traditional Chinese medicine monomer composition comprises, by mass ratio, 3.88~6.99: 78.77~154.32: 73.54~125.46: 53.94~90.27: 49.93~80.96: 264.90~450.42: 53.37~107.93: 50.61~89.82: 4.52~8.15: 2.56~4.47 ∶25.45~44.12∶9.69~17.25∶29.82~64.36∶8.43~13.55∶5.80~14.40∶0.95~3.27∶3.45~5.71∶269.42~414.94∶3170.57~5685.61∶1 chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, Tuberonic acid Composed of apigenin glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

[0009] In a preferred embodiment of the present invention, the herbal monomer composition comprises herbs in a mass ratio of 4.4~5.9: 85.6~105.7: 80.4~104.3: 58.3~79.5: 52.1~66.4: 280.7~312.9: 79.5~88.1: 62.9~78.3: 4.9~6.7: 2.8~3.7: 30.1~36.4: 10.9~ Chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, and Tuberonic acid are present in a 15.7:33.7~45.9:8.8~10.6:7.4~11.4:1.1~2.6:3.6~4.8:279.8~318.7:3495.2~3583.3:1 ratio. Composed of apigenin glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

[0010] As a further preferred embodiment of the present invention, the herbal monomer composition comprises chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, and tuberonic acid in a mass ratio of 5.1:98.8:89.3:68.4:57.7:291.2:81.6:70.5:5.3:3.3:33.7:12.2:39.3:9.2:8.8:1.7:4.1:293.4:3512.1:1. It is composed of glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

[0011] On the other hand, the present invention also provides a method for preparing the above-mentioned traditional Chinese medicine monomer composition, comprising the following steps:

[0012] (1) Add the chrysanthemum plant raw material to the extraction solvent and heat and reflux to extract. Filter and concentrate the extract, add an appropriate amount of distilled water, filter under vacuum to obtain the sample solution;

[0013] (2) The sample solution was adsorbed through macroporous resin, eluted with distilled water to remove impurities, and eluted with elution solvent. The eluent was collected, concentrated, and dried to obtain the total flavonoid chemical fraction.

[0014] (3) The lens was isolated from the mouse eyeball, added to PBS buffer solution, homogenized, and the supernatant was collected. The crude enzyme extract was obtained by adjusting the concentration of ammonium sulfate solution in the supernatant in turn. The purified enzyme solution was obtained by dialysis.

[0015] (4) Mix the total flavonoid chemical fraction solution with the purified enzyme solution, incubate in a water bath at 37 °C for 20-40 min, centrifuge, add PBS buffer solution to remove compounds that have not been bound to the enzyme, add 50%-80% methanol solution, incubate at room temperature and centrifuge to obtain the traditional Chinese medicine monomer composition.

[0016] Preferably, in step (1), the chrysanthemum plant raw material includes chrysanthemum inflorescence, chrysanthemum stems and leaves or chrysanthemum roots; the extraction solvent is distilled water or 30%~85% alcohol solution; the material-to-liquid ratio is 1:10~20 g / mL, the extraction time is 1~3 h, and the extraction is performed 1~2 times.

[0017] Preferably, in step (2), the mass concentration of the loading solution is 25~125 mg / mL; and / or

[0018] The volume of the sample loading solution is 10-110 mL; and / or

[0019] The macroporous resin is any one of AB-8, NKA-9, HPD-500, DM-130, or DM-301; and / or

[0020] The volumetric flow rate of the sample loading solution is 0.5~2.0 mL / min; and / or

[0021] The column volume of the distilled water is 1~2 BV; and / or

[0022] The elution solvent is a 60%~80% ethanol solution; and / or

[0023] The volume of the elution solvent is 2-4 BV.

[0024] Preferably, in step (3), the dialysis is performed using a 1000 Da dialysis bag for 24 hours, with the dialysis solution being changed every 8 hours during the process.

[0025] Preferably, in step (4), the mass concentration of the total flavonoid chemical fraction solution is 0.5~2 mg / mL, the mass concentration of the purified enzyme solution is 1~3.5 mg / mL, and the volume ratio of the total flavonoid chemical fraction solution to the purified enzyme solution is 1:1~3.

[0026] In another aspect, the present invention provides the application of the above-mentioned traditional Chinese medicine monomer composition in the preparation of a medicament for treating diabetic retinopathy.

[0027] Pharmacodynamic studies of this invention have shown that the traditional Chinese medicine monomer composition plays a role in the preparation of drugs for treating diabetic retinopathy by inhibiting body weight gain caused by high blood sugar, reducing blood sugar levels, improving retinal tissue structure damage, restoring retinal tissue thickness, inhibiting retinal cell apoptosis and inflammatory response, and reducing the expression levels of HIF-1α and VEGF proteins in retinal tissue.

[0028] Finally, the present invention also provides a medicament for treating diabetic retinopathy, the medicament comprising an effective amount of the above-mentioned traditional Chinese medicine monomer composition and pharmaceutically acceptable excipients.

[0029] The pharmaceutically acceptable excipients described in this invention include, but are not limited to: solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, humectants, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0030] The dosage forms of the drugs described in this invention include any one of the following: decoctions, oral liquids, patches, tablets, granules, capsules, ointments, pills, powders, elixirs, injections, or sustained-release formulations. The preparation methods for the above-mentioned drugs are not strictly limited and can be carried out according to conventional methods in the art.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] This invention utilizes chrysanthemum plants as raw materials to prepare a traditional Chinese medicine monomer composition. This composition exhibits high safety, minimal side effects, and clearly defined components in a specific ratio. Pharmacological experiments have demonstrated that, compared to ethanol extracts, the traditional Chinese medicine monomer composition of this invention is more effective in treating diabetic retinopathy. It can improve the pathological state of diabetic retinopathy through multiple targets and pathways. Specifically, it can inhibit weight gain caused by high blood sugar, lower blood sugar levels, improve retinal tissue structure damage, restore retinal tissue thickness, inhibit retinal cell apoptosis and inflammatory responses, reduce the expression levels of HIF-1α and VEGF proteins in retinal tissue, and slow disease progression. This provides a scientific basis and technical support for the development of novel therapeutic drugs for diabetic retinopathy, and has significant clinical significance and market application prospects. Attached Figure Description

[0033] Figure 1 The effect of ethanol concentration on the total flavonoid content of chrysanthemum (compared to 70% ethanol concentration). ** p < 0.01, *** p < 0.001);

[0034] Figure 2 The effects of different types of resins on the adsorption / desorption capacity (mg / g) and desorption rate (%) of total flavonoids in chrysanthemum (compared with AB-8 resin). ** p < 0.01, *** p < 0.001; ## p < 0.01, ### p < 0.001; & p < 0.05, &&& p < 0.001);

[0035] Figure 3 The effect of different loading solution concentrations on the adsorption rate (%) and desorption rate (%) of total flavonoids from chrysanthemum (compared with a loading solution concentration of 75 mg / mL) was investigated. * p < 0.05, # p < 0.05, ## (p < 0.01)

[0036] Figure 4 The effect of different loading liquid flow rates on the adsorption capacity (mg / g) and adsorption rate (%) of total flavonoids from chrysanthemum (compared with a loading liquid flow rate of 1.0 mL / min) was investigated. * p < 0.05, ** (p < 0.01)

[0037] Figure 5 The effect of sample loading amount on the total flavonoid concentration in the effluent;

[0038] Figure 6 The effect of water purification volume on the proportion of total flavonoids in the effluent (compared to 1 BV water purification volume). *** p < 0.001);

[0039] Figure 7 The effect of ethanol concentration on the desorption capacity (mg / g) and desorption rate (%) of total flavonoids in chrysanthemum (compared to 70% ethanol concentration). * p < 0.05, ** p < 0.01; ## p < 0.01, ### p < 0.001);

[0040] Figure 8 The effect of elution solvent dosage on the desorption capacity (mg / g) and desorption rate (%) of total flavonoids from chrysanthemum (compared with 2 BV of elution solvent). *** p < 0.001, # p < 0.05, ### p < 0.001);

[0041] Figure 9 The effect of traditional Chinese medicine monomer composition on body weight of DR mice (n=6) (compared with the control group, *** p < 0.001; compared with the Model group, ### p < 0.001);

[0042] Figure 10 The effect of traditional Chinese medicine monomer composition on fasting blood glucose levels in DR mice (n=6, compared with the control group, *** p < 0.001; compared with the Model group, ## p < 0.01, ### p < 0.001);

[0043] Figure 11 The effect of a traditional Chinese medicine monomer composition on the total retinal thickness of DR mice (n=5). Compared with the Control group, *** p < 0.001; compared with the Model group, ### p < 0.001.

[0044] Figure 12 The effect of traditional Chinese medicine monomer composition on the thickness of the retinal INL layer in DR mice (n=5, compared with the control group, *** p < 0.001; compared with the Model group, # p < 0.05, ### p < 0.001

[0045] Figure 13 The effect of traditional Chinese medicine monomer composition on the thickness of the ONL layer in the retina of DR mice (n=5, compared with the control group, *** p < 0.001; compared with the Model group, # p < 0.05, ### p < 0.001);

[0046] Figure 14 The effect of a traditional Chinese medicine monomer composition on the serum TNF-α expression level in DR mice (n=5, compared with the Control group, *** p < 0.001; compared with the Model group, ### p < 0.001);

[0047] Figure 15 The effect of a traditional Chinese medicine monomer composition on the serum IL-6 expression level in DR mice (n=5, compared with the control group, *** p < 0.001; compared with the Model group, ### p < 0.001);

[0048] Figure 16 The effect of a traditional Chinese medicine monomer composition on the expression level of IL-1β in the serum of DR mice (n=5 compared with the control group, *** p < 0.001; compared with the Model group, ### p < 0.001);

[0049] Figure 17 The effect of traditional Chinese medicine monomer composition on retinal cell apoptosis in DR mice (n=5, compared with the control group, *** p <

[0050] 0.001; compared with the Model group, ### p < 0.001);

[0051] Figure 18 The effect of traditional Chinese medicine monomer composition on HIF-1α expression in the retina of DR mice (n=5, compared with the control group, *** p < 0.001; compared with the Model group, ### p < 0.001);

[0052] Figure 19 The effect of a traditional Chinese medicine monomer composition on VEGF expression in the retina of DR mice (n=5, compared with the control group, *** p < 0.001; compared with the Model group, ##p < 0.01, ### p < 0.001). Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Example 1: Process optimization of total flavonoids from chrysanthemum

[0055] 1. Investigation of extraction solvents

[0056] Six chrysanthemum samples of a certain mass were weighed and added to water and ethanol solutions of different volume fractions (30%, 50%, 70%, 95%, and 100%), respectively. The samples were extracted twice by heating and reflux, 2 hours each time. The samples were filtered while hot, and the two filtrates were combined. The total flavonoid content in the filtrates was determined. Figure 1 As shown, the total flavonoid content in chrysanthemum was highest when extracted with 70% ethanol, therefore 70% ethanol was chosen as the optimal extraction solvent.

[0057] 2. Optimal Resin Selection

[0058] Take 3 g each of pretreated AB-8, NKA-9, HPD-500, DM-130, DM-301 macroporous adsorption resins and polyamide resin, and place them in separate 50 mL Erlenmeyer flasks. Add 20 mL of 75 mg / mL sample loading solution and shake at 120 rpm for 8 h at 25 ℃. After adsorption equilibrium, collect the supernatant and determine the concentration of total flavonoids in the solution. Wash the resin with 30 mL of distilled water, then add 60 mL of 70% ethanol solution and shake at 120 rpm for 8 h at 25 ℃. Collect the supernatant and determine the concentration of total flavonoids in the solution. Calculate the adsorption capacity, desorption capacity, and desorption rate of different resins.

[0059] like Figure 2 As shown, AB-8 and NKA-9 resins exhibited higher adsorption capacities than other resins, but NKA-9 resin showed lower desorption capacity and desorption rate compared to AB-8 resin. In the desorption experiment, DM-301, DM-130, and AB-8 resins showed higher desorption rates than other resins, but the adsorption and desorption capacities of DM-301 and DM-130 resins were both lower than those of AB-8 resin. Therefore, AB-8 resin was selected for the enrichment and purification of total flavonoid fractions from chrysanthemum.

[0060] 3. Investigation of the mass concentration of the sample loading solution

[0061] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto a column using a wet method. Samples were loaded with different mass concentrations of loading solution to systematically evaluate the effect of loading solution mass concentration on the adsorption and desorption rates of total chrysanthemum flavonoids.

[0062] like Figure 3 As shown, with the increase of the mass concentration of the loading solution, the adsorption rate of total flavonoids from chrysanthemum showed an upward trend and gradually approached [the value].

[0063] The total flavonoids from chrysanthemum were stable; the desorption rate was highest when the sample concentration was 75 mg / mL. Therefore, a sample concentration of 75 mg / mL was chosen for the enrichment and purification of total chrysanthemum flavonoids.

[0064] 4. Investigation of sample loading volumetric flow rate

[0065] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto a column using a wet method. Dynamic adsorption was performed using different sample liquid volume flow rates. The effect of sample liquid volume flow rate on the adsorption capacity and adsorption rate of total chrysanthemum flavonoids was systematically evaluated.

[0066] As shown in Figure 4, the adsorption capacity and adsorption rate of total chrysanthemum flavonoids gradually decreased with increasing sample loading flow rate. When the sample loading flow rate increased from 0.5 mL / min to 1.0 mL / min, there was no significant change in the adsorption capacity and adsorption rate of total chrysanthemum flavonoids. Therefore, considering both purification efficiency and adsorption effect, the optimal sample loading flow rate was determined to be 1.0 mL / min.

[0067] 5. Maximum sample loading investigation

[0068] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto the column using a wet method. A certain volume of loading solution was added for dynamic adsorption. The total flavonoid concentration in the effluent was detected, and the effect of loading amount on the enrichment effect of total flavonoids from chrysanthemum was systematically evaluated.

[0069] As shown in Figure 5, when the sample loading volume reached 100 mL, the total flavonoid concentration in the effluent was less than 5% of the total flavonoid concentration in the sample solution, indicating that the leakage point had not been reached. When the sample loading volume reached 110 mL, the total flavonoid concentration in the effluent exceeded 5% of the total flavonoid concentration in the sample solution, indicating that the leakage point had been reached. To avoid the loss of total flavonoids and improve purification efficiency, 100 mL was selected as the maximum sample loading volume for enriching chrysanthemum total flavonoids.

[0070] 6. Investigation of water impurity removal volume

[0071] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto a column using a wet method. Distilled water of different column volumes (BV) was used to remove impurities. The total flavonoid content in the effluent was determined and its proportion was calculated.

[0072] As shown in Figure 6, when 1 BV of distilled water was used for impurity removal, the proportion of total flavonoids in the effluent was relatively low. However, when the water impurity removal volume exceeded 1 BV, the proportion of total flavonoids in the effluent increased significantly and was consistently above 65%. Therefore, to reduce sample loss, 1 BV was selected as the optimal water impurity removal volume.

[0073] 7. Investigation of the volume fraction of ethanol as eluent

[0074] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto a column using a wet method, and desorption was performed using ethanol solutions of different concentrations. The effect of ethanol volume fraction on the desorption capacity and desorption rate of total flavonoids from chrysanthemum was systematically evaluated.

[0075] like Figure 7 As shown, with increasing ethanol concentration, the desorption capacity and desorption rate of total chrysanthemum flavonoids both showed a trend of first increasing and then decreasing, reaching their highest values ​​at an ethanol concentration of 70%. Therefore, 70% ethanol was selected as the optimal elution solvent.

[0076] 8. Analysis of Elution Solvent Dosage

[0077] 15.0 g of pretreated AB-8 macroporous adsorption resin was loaded onto a column using a wet method, and different volumes of 70% ethanol solution were used for elution. The effect of the amount of elution solvent on the desorption capacity and desorption rate of total flavonoids from chrysanthemum was systematically evaluated.

[0078] As shown in Figure 8, with the increase of the eluent volume, the desorption capacity and desorption rate of total chrysanthemum flavonoids both showed an upward trend and gradually stabilized. However, when the volume of the eluent was between 2 BV and 4 BV, there was no significant change in the desorption capacity and desorption rate of total chrysanthemum flavonoids. Therefore, 2 BV was selected as the optimal eluent volume for subsequent experiments.

[0079] In summary, the optimal purification conditions of this invention are: extraction with 70% ethanol under reflux, enrichment with AB-8 macroporous adsorption resin, a sample concentration of 75 mg / mL, a sample flow rate of 1.0 mL / min, a maximum sample volume of 100 mL, an optimal water removal volume of 1 BV, and 70% ethanol as the elution solvent with a volume of 2 BV. After purification using the above optimal process, the purity of total flavonoids in the chrysanthemum flavonoid fraction reached 75.81%, an increase of 52.29% compared to before purification.

[0080] Example 2: Preparation of a Traditional Chinese Medicine Monomer Composition

[0081] Weigh 500.0 g of chrysanthemum sample and add 70% ethanol solution at material-to-liquid ratios of 1:20 and 1:15 (g / mL), respectively. Heat and reflux to extract twice, 2 h each time. Filter while hot, combine the two filtrates, recover ethanol under reduced pressure, and concentrate until there is no alcohol odor, which is used as the loading solution.

[0082] An AB-8 macroporous adsorption resin column was used. The sample concentration was 75 mg / mL, the sample flow rate was 1.0 mL / min, the sample volume was 100 mL, the sample was eluted with 1 BV of distilled water and 2 BV of 70% ethanol solution. The eluent was collected, the ethanol was recovered under reduced pressure, concentrated until no alcohol odor was detected, and then lyophilized to obtain a total flavonoid chemical fraction with a purity of 75.81%.

[0083] From 7-week-old SPF-grade male db / db mice (BKS.Cg-Dock7) m + / + Lepr db The lens was isolated from the eyeball, and 0.1 mol / L PBS buffer was added. The mixture was homogenized, and the supernatant was collected. 100% saturated ammonium sulfate solution was added to the supernatant to adjust the ammonium sulfate saturation to 40%. The mixture was mixed, allowed to stand for 20 min, and centrifuged at 13000 rpm for 20 min. The supernatant was retained. 100% saturated ammonium sulfate solution was added to adjust the ammonium sulfate saturation to 50%. The mixture was mixed, allowed to stand for 20 min, and centrifuged at 13000 rpm for 20 min. The supernatant was retained. Finally, 100% saturated ammonium sulfate solution was added to adjust the ammonium sulfate saturation to 75%. The mixture was mixed, allowed to stand for 20 min, and centrifuged at 13000 rpm for 20 min. The precipitate was retained. The precipitate was reconstituted with 2 times (v / w) PBS solution and dialyzed using a 1000 Da dialysis bag for 24 h, with the dialysate changed every 8 h, to obtain the purified enzyme solution.

[0084] Weigh an appropriate amount of total flavonoid chemical fraction powder and dissolve it in PBS buffer solution to prepare a total flavonoid chemical fraction solution with a concentration of 1 mg / mL. Take 100 μL of the 1 mg / mL total flavonoid chemical fraction solution and mix it with 100 μL of purified enzyme solution with a concentration of 3 mg / mL. Incubate in a water bath at 37 ℃ for 30 min. After incubation, transfer the mixed solution to a 10 kDa ultrafiltration centrifuge tube and centrifuge at 10000 rpm for 20 min. Add 200 μL of 0.1 mol / L PBS buffer solution to remove compounds that have not bound to the enzyme. Repeat this operation 3 times. Then add 200 μL of 50% methanol solution to the ultrafiltration centrifuge tube, incubate at room temperature for 10 min, centrifuge at 10000 rpm for 20 min, and repeat this operation twice. Combine the ultrafiltrates to prepare the traditional Chinese medicine monomer composition.

[0085] The chemical components in the traditional Chinese medicine monomer composition were characterized using UPLC-Q-TOF / MS.

[0086] Chromatographic conditions: A Waters Acquity UPLC HSST3 column (100 mm × 2.1 mm, 1.8 μm) was used; mobile phase: 0.1% formic acid water (A) - 0.1% formic acid acetonitrile (B); gradient elution: 0–2 min, 5%–7% B; 2–5 min, 7% B; 5–6 min, 7%–15% B; 6–7 min, 15% B; 7–18 min, 15%–35% B; 18–20 min, 35%–67% B; 20–24 min, 67%–100% B; flow rate: 0.4 mL / min; column temperature: 30 ℃; injection volume: 2 μL.

[0087] Mass spectrometry conditions: Electrospray ionization (ESI) was used in negative ion mode, with a mass scan range of 100–1600 Da (m / z). The ion source temperature was 120 °C, capillary voltage was 2.2 kV, cone voltage was 40 V, desolvation gas temperature was 400 °C, and desolvation gas flow rate was 800 L / h. Real-time mass correction was performed using leucine enkephalin.

[0088] The identification results are shown in Table 1. The 20 monomeric compounds are chlorogenic acid, cryptochlorogenic acid, quercetin-3,7-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, tuberonic acid glucoside, apigenin-6,8-di-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolinoside, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, and geraniol-7-O-β-D-glucoside. - Glucoside, geraniol-7-O-6″-malonyl glucoside, farnesin-7-O-β-D-glucoside and genistein, the corresponding ratios of the compounds are: 5.1∶98.8∶89.3∶68.4∶57.7∶291.2∶81.6∶70.5∶5.3∶3.3∶33.7∶12.2∶39.3∶9.2∶8.8∶1.7∶4.1∶293.4∶3512.1∶1.

[0089] Table 1. Identification results of 20 monomeric compounds in the composition

[0090] Compound Name Retention time / min Molecular formula chlorogenic acid 6.76 <![CDATA[C 16 H 18 O9]]> cryptochlorogenic acid 6.98 <![CDATA[C 16 H 18 O9]]> Quercetin-3,7-di-O-β-D-glucoside 7.02 <![CDATA[C 27 H 30 O 17 ]]> Luteolin-7,4'-di-O-β-D-glucoside 7.25 <![CDATA[C 27 H 30 O 16 ]]> Tuberonic acid glucoside 7.49 <![CDATA[C 18 H 28 O9]]> Apigenin-6,8-di-C-glucoside 7.82 <![CDATA[C 27 H 30 O 15 ]]> 1,3-O-dicaffeoylquinic acid 8.18 <![CDATA[C 25 H 24 O 12 ]]> Apigenin-6-C-glucoside-8-C-arabinoside 8.87 <![CDATA[C 26 H 28 O 14 ]]> Sacred herbol-7-O-β-D-glucoside 10.21 <![CDATA[C 21 H 22 O 11 ]]> Isoquercitrin 10.40 <![CDATA[C 21 H 20 O 12 ]]> Luteolin 10.59 <![CDATA[C 21 H 20 O 11 ]]> Isochlorogenic acid B 11.25 <![CDATA[C 25 H 24 O 12 ]]> 1,5-O-dicaffeoylquinic acid 11.49 <![CDATA[C 25 H 24 O 12 <!-- 7 -->]]> Isochlorogenic acid A 11.60 <![CDATA[C 25 H 24 O 12 ]]> Apigenin-7-O-β-D-glucoside 11.94 <![CDATA[C 21 H 20 O 10 ]]> Isochlorogenic acid C 12.32 <![CDATA[C 25 H 24 O 12 ]]> Geraniol-7-O-β-D-glucoside 12.61 <![CDATA[C 22 H 22 O 11 ]]> Geraniol-7-O-6″-malonyl glucoside 14.24 <![CDATA[C 25 H 24 O 14 ]]> Acacia-7-O-β-D-glucoside 15.67 <![CDATA[C 22 H 22 O 10 ]]> Gentian root 17.26 <![CDATA[C 16 H 12 O5]]>

[0091] Example 3: Preparation of Monomer Compositions of Traditional Chinese Medicine

[0092] Weigh an appropriate amount of chrysanthemum stem and leaf sample, add 70% ethanol solution at a material-to-liquid ratio of 1:15 and 1:10 (g / mL), respectively, heat and reflux to extract twice, 2 h each time, filter while hot, combine the two filtrates, recover ethanol under reduced pressure, concentrate until there is no alcohol odor, and use as the loading solution.

[0093] The total flavonoid chemical fraction was prepared by using an NKA-9 macroporous adsorption resin column with a sample concentration of 70 mg / mL, a sample flow rate of 0.5 mL / min, a sample loading volume of 90 mL, elution with 1.5 BV of distilled water for impurity removal, and elution with 2 BV of 70% ethanol solution. The eluent was collected, ethanol was recovered under reduced pressure, concentrated until no alcohol odor was detected, and then lyophilized.

[0094] From 7-week-old SPF-grade male db / db mice (BKS.Cg-Dock7) m + / + Lepr db The lens was isolated from the eyeball, and 0.1 mol / L PBS buffer solution was added. The mixture was homogenized, and the supernatant was collected. The crude enzyme extract was obtained by adjusting the concentration of ammonium sulfate solution in the supernatant sequentially (the specific operation is the same as in Example 2). The extract was dialyzed for 24 h using a 1000 Da dialysis bag, and the dialysis solution was changed every 8 h during the process to obtain a purified enzyme solution.

[0095] Weigh an appropriate amount of total flavonoid chemical fraction powder and dissolve it in PBS buffer solution to prepare a total flavonoid chemical fraction solution with a concentration of 1.2 mg / mL. Take 100 μL of the 1.2 mg / mL total flavonoid chemical fraction solution and mix it with 200 μL of purified enzyme solution with a concentration of 3 mg / mL. Incubate in a water bath at 37 ℃ for 40 min. After incubation, transfer the mixed solution to a 10 kDa ultrafiltration centrifuge tube and centrifuge at 15000 rpm for 15 min. Add 200 μL of 0.1 mol / L PBS buffer solution to remove compounds that have not bound to the enzyme. Repeat this operation 3 times. Then add 200 μL of 50% methanol solution to the ultrafiltration centrifuge tube, incubate at room temperature for 15 min, centrifuge at 10000 rpm for 20 min, and repeat this operation twice. Combine the ultrafiltrates to prepare the traditional Chinese medicine monomer composition.

[0096] The chemical components of the herbal monomer composition were characterized by UPLC-Q-TOF / MS under the same chromatographic and mass spectrometric conditions as in Example 1. The results showed that the 20 monomeric compounds were chlorogenic acid, cryptochlorogenic acid, quercetin-3,7-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, tuberonic acid glucoside, apigenin-6,8-di-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolinoside, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, and geraniol-7-O-β-D- The ratios of the compounds—glucosinolate, geraniol-7-O-6″-malonyl glucoside, farnesin-7-O-β-D-glucosinolate, and genistein—are as follows: 5.6∶104.9∶87.1∶62.5∶58.4∶290.3∶86.8∶67.2∶5.1∶3.2∶30.3∶12.6∶38.8∶9.9∶8.4∶1.5∶3.9∶281.7∶3526.2∶1.

[0097] Example 4: Preparation of Monomer Compositions of Traditional Chinese Medicine

[0098] Weigh an appropriate amount of chrysanthemum root sample and add 85% ethanol solution at material-to-liquid ratios of 1:20 and 1:15 (g / mL), respectively. Heat and reflux to extract twice, 2 h each time. Filter while hot, combine the two filtrates, recover ethanol under reduced pressure, and concentrate until there is no alcohol odor, which is then used as the loading solution.

[0099] The total flavonoid chemical fraction was prepared by using an AB-8 macroporous adsorption resin column with a sample concentration of 100 mg / mL, a sample flow rate of 2.0 mL / min, a sample volume of 95 mL, elution with 1 BV of distilled water for impurity removal, and elution with 4 BV of 70% ethanol solution. The eluent was collected, ethanol was recovered under reduced pressure, concentrated until no alcohol odor was detected, and then lyophilized.

[0100] From 7-week-old SPF-grade male db / db mice (BKS.Cg-Dock7) m + / + Lepr db The lens was isolated from the eyeball, and 0.1 mol / L PBS buffer solution was added. The mixture was homogenized, and the supernatant was collected. The crude enzyme extract was obtained by adjusting the concentration of ammonium sulfate solution in the supernatant sequentially (the specific operation is the same as in Example 2). The extract was dialyzed for 24 h using a 1000 Da dialysis bag, and the dialysis solution was changed every 8 h during the process to obtain a purified enzyme solution.

[0101] Weigh an appropriate amount of total flavonoid chemical fraction powder and dissolve it in PBS buffer solution to prepare a total flavonoid chemical fraction solution with a concentration of 1.8 mg / mL. Take 200 μL of the 1.8 mg / mL total flavonoid chemical fraction solution and mix it with 300 μL of purified enzyme solution with a concentration of 3.5 mg / mL. Incubate in a water bath at 37 ℃ for 40 min. After incubation, transfer the mixed solution to a 10 kDa ultrafiltration centrifuge tube and centrifuge at 12000 rpm for 20 min. Add 200 μL of 0.1 mol / L PBS buffer solution to remove compounds that have not bound to the enzyme. Repeat this operation 3 times. Then add 200 μL of 70% methanol solution to the ultrafiltration centrifuge tube, incubate at room temperature for 10 min, centrifuge at 12000 rpm for 15 min, and repeat this operation twice. Combine the ultrafiltrates to prepare the traditional Chinese medicine monomer composition.

[0102] The chemical components of the herbal monomer composition were characterized by UPLC-Q-TOF / MS under the same chromatographic and mass spectrometric conditions as in Example 1. The results showed that the 20 monomeric compounds were chlorogenic acid, cryptochlorogenic acid, quercetin-3,7-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, tuberonic acid glucoside, apigenin-6,8-di-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolinoside, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, and geraniol-7-O-β-D- The ratios of the compounds—glucosinolate, geraniol-7-O-6″-malonyl glucoside, farnesin-7-O-β-D-glucosinolate, and genistein—are as follows: 5.8∶103.5∶101.7∶71.3∶64.6∶293.2∶83.9∶73.5∶5.6∶3.4∶34.1∶13.3∶42.7∶9.6∶8.5∶1.8∶4.6∶313.6∶3500.3∶1.

[0103] Comparative Example 1:

[0104] Weigh 500.0 g of chrysanthemum sample and add 70% ethanol solution at material-to-liquid ratios of 1:20 and 1:15 (g / mL), respectively. Heat and reflux to extract twice, 2 h each time. Filter while hot, combine the two filtrates, recover ethanol under reduced pressure, and concentrate until no alcohol odor is detected to obtain 70% ethanol extract of chrysanthemum.

[0105] Experimental Example 1: Pharmacological Evaluation of Traditional Chinese Medicine Monomer Combinations in the Treatment of Diabetic Respiratory Disorders (DR)

[0106] 1. Reagents and Materials

[0107] The hematoxylin-eosin high-resolution constant staining kit, TUNEL cell apoptosis detection kit, HIF-1α antibody and VEGF antibody were all purchased from Wuhan Saiweier Biotechnology Co., Ltd.; the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) ELISA kits were all purchased from Shanghai Weiao Biotechnology Co., Ltd.; the traditional Chinese medicine monomer composition prepared in Example 2.

[0108] 2. Animal grouping and administration

[0109] Thirty 7-week-old SPF-grade male db / db mice and ten littermate wild-type male mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., license number SCXK (Su) 2023-0009.

[0110] db / db mice were randomly divided into three groups of 10 mice each: a model group, a 70% ethanol extract group of chrysanthemum (CF-Eth), and a combination group containing 20 herbal monomers (20-Com); ten age-matched wild-type mice served as the control group. After acclimatization, the mice were administered the drug by gavage at 9:00 AM daily for eight weeks. Mice in the drug administration groups received the corresponding drug via gavage, while mice in the control and model groups received an equal volume of physiological saline. After eight weeks of administration, the mice were anesthetized with 3% isoflurane inhalation, and the eyeballs were removed, and blood samples were collected.

[0111] 3. Statistical Analysis

[0112] Statistical analysis was performed using GraphPad Prism 9.1.0 software. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Results are presented as follows: ±SE indicates that p < 0.05 is statistically significant.

[0113] 4. Effects of traditional Chinese medicine monomer compositions on body weight of DR mice

[0114] After drug administration, the fasting body weight of mice in each group was measured using an electronic balance. For example... Figure 9 As shown, the body weight of mice in the model group was significantly higher than that of mice in the normal group (p < 0.001). Compared with the model group, the body weight of mice in the herbal monomer composition group was significantly lower (p < 0.001), and the reduction in body weight was significantly greater than that in the 70% ethanol extract of chrysanthemum group. These results indicate that the herbal monomer composition can effectively reduce the body weight gain induced by diabetes mellitus (DR) in mice.

[0115] 5. Effects of traditional Chinese medicine monomer compositions on blood glucose levels in DR mice

[0116] After drug administration, fasting blood glucose levels in each group of mice were measured using a blood glucose meter. Figure 10 As shown, the fasting blood glucose level of the model group mice was significantly higher than that of the normal group mice (p < 0.001). Compared with the model group mice, the fasting blood glucose level of the mice in the herbal monomer composition group was significantly lower (p < 0.001), and the degree of reduction in fasting blood glucose level was greater than that in the 70% ethanol extract of chrysanthemum group. These results indicate that the composition containing 20 herbal monomers can effectively inhibit blood glucose levels in DR mice.

[0117] 6. Effects of traditional Chinese medicine monomer compositions on pathological changes in retinal tissue of DR mice

[0118] The pathological changes in the retina of mice in each group were detected by H&E staining, and the total retinal thickness and the thickness of the INL and ONL layers were calculated for each group. Figure 11-13 As shown, compared with the normal group mice, the model group mice exhibited significant retinal structural damage, with a significant decrease in total retinal thickness, INL layer thickness, and ONL layer thickness (p < 0.001). Compared with the model group, the herbal monomer composition could improve the retinal pathological damage in DR mice to a certain extent, significantly increasing the total retinal thickness and INL and ONL layer thickness (p < 0.001), and the improvement effect was more significant than that of 70% ethanol extract of chrysanthemum. These results indicate that the herbal monomer composition can effectively improve the structural damage of the retina in DR mice.

[0119] 7. Effects of traditional Chinese medicine monomer compositions on the expression levels of inflammatory cytokines in the serum of DR mice

[0120] The serum levels of TNF-α, IL-6, and IL-1β in mice of each group were detected using ELISA kits. All procedures were strictly performed according to the kit instructions. Figure 14-16 As shown, compared with the normal group mice, the expression levels of TNF-α, IL-6, and IL-1β in the serum of the model group mice were significantly increased (p < 0.001). Compared with the model group mice, the expression levels of TNF-α, IL-6, and IL-1β in the serum of the herbal monomer composition group were significantly decreased (p < 0.001), and the degree of decrease was greater than that in the 70% ethanol extract of chrysanthemum. These results indicate that the herbal monomer composition can effectively inhibit the inflammatory response in DR mice.

[0121] 8. Effects of traditional Chinese medicine monomer compositions on retinal cell apoptosis in DR mice

[0122] The apoptosis level of retinal cells in each group of mice was detected by TUNEL staining, and the TUNEL-positive cell rate in the retina of each group of mice was calculated. Figure 17 As shown, compared with the normal group mice, the number of TUNEL-positive cells in the retina of the model group mice was significantly increased (p < 0.001), and retinal cells underwent significant apoptosis. Compared with the model group, the herbal monomer composition significantly downregulated the apoptosis level of retinal cells in DR mice (p < 0.001), and the downregulation degree was higher than that of 70% ethanol extract of chrysanthemum. These results indicate that the herbal monomer composition has a significant anti-apoptotic effect on the retina of DR mice.

[0123] 9. Effects of traditional Chinese medicine monomer compositions on the expression levels of HIF-1α and VEGF proteins in the retinal tissue of DR mice.

[0124] Immunohistochemical staining was used to determine the percentage of positive retinal area in each group of mice, and the expression levels of HIF-1α and VEGF proteins in the retinal tissue of each group of mice were detected. Figure 18-19 As shown, compared with the normal group mice, the expression levels of HIF-1α and VEGF proteins in the retinal tissue of the model group mice were significantly increased (p < 0.001). Compared with the model group mice, the expression levels of HIF-1α and VEGF proteins in the retinal tissue of the herbal monomer composition group were significantly decreased (p < 0.001), and the degree of decrease was greater than that in the 70% ethanol extract of chrysanthemum group. These results suggest that the herbal monomer composition can effectively inhibit the increase in the expression levels of HIF-1α and VEGF proteins in the retinal tissue of hyperglycemic DR mice.

Claims

1. A traditional Chinese medicine monomer composition derived from chrysanthemum plant for treating diabetic retinopathy, characterized in that, The herbal monomer composition comprises herbs in the following mass ratios: 3.88~6.99: 78.77~154.32: 73.54~125.46: 53.94~90.27: 49.93~80.96: 264.90~450.42: 53.37~107.93: 50.61~89.82: 4.52~8.15: 2.56~4.47: 25.45~44.12: 9.69~17 .25∶29.82~64.36∶8.43~13.55∶5.80~14.40∶0.95~3.27∶3.45~5.71∶269.42~414.94∶3170.57~5685.61∶1 chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, Tuberonic acid Composed of apigenin glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

2. The traditional Chinese medicine monomer composition according to claim 1, characterized in that, The herbal monomer composition comprises herbs in the following mass ratios: 4.4~5.9: 85.6~105.7: 80.4~104.3: 58.3~79.5: 52.1~66.4: 280.7~312.9: 79.5~88.1: 62.9~78.3: 4.9~6.7: 2.8~3.7: 30.1~36.4: 10.9~15.7: 3 3.7~45.9∶8.8~10.6∶7.4~11.4∶1.1~2.6∶3.6~4.8∶279.8~318.7∶3495.2~3583.3∶1 chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, Tuberonic acid Composed of apigenin glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

3. The traditional Chinese medicine monomer composition according to claim 2, characterized in that, The herbal monomer composition comprises chlorogenic acid, cryptochlorogenic acid, quercetin-3,7'-di-O-β-D-glucoside, luteolin-7,4'-di-O-β-D-glucoside, and tuberonic acid in a mass ratio of 5.1:98.8:89.3:68.4:57.7:291.2:81.6:70.5:5.3:3.3:33.7:12.2:39.3:9.2:8.8:1.7:4.1:293.4:3512.1:

1. It is composed of glucoside, apigenin-6,8-bis-C-glucoside, 1,3-O-dicaffeoylquinic acid, apigenin-6-C-glucoside-8-C-arabinoside, senna-7-O-β-D-glucoside, isoquercitrin, luteolin, isochlorogenic acid B, 1,5-O-dicaffeoylquinic acid, isochlorogenic acid A, apigenin-7-O-β-D-glucoside, isochlorogenic acid C, geraniol-7-O-β-D-glucoside, geraniol-7-O-6″-malonylglucoside, farnesin-7-O-β-D-glucoside, and genkwain.

4. A method for preparing a traditional Chinese medicine monomer composition as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Add the chrysanthemum plant raw material to the extraction solvent and heat and reflux to extract. Filter and concentrate the extract, add an appropriate amount of distilled water, filter under vacuum to obtain the sample solution; (2) The sample solution was adsorbed through macroporous resin, eluted with distilled water to remove impurities, and eluted with elution solvent. The eluent was collected, concentrated, and dried to obtain the total flavonoid chemical fraction. (3) The lens was isolated from the mouse eyeball, added to PBS buffer solution, homogenized, and the supernatant was collected. The crude enzyme extract was obtained by adjusting the concentration of ammonium sulfate solution in the supernatant in turn. The purified enzyme solution was obtained by dialysis. (4) Mix the total flavonoid chemical fraction solution with the purified enzyme solution, incubate in a water bath at 37 °C for 20-40 min, centrifuge, add PBS buffer solution to remove compounds that have not been bound to the enzyme, add 50%-80% methanol solution, incubate at room temperature and centrifuge to obtain the traditional Chinese medicine monomer composition.

5. The method for preparing the traditional Chinese medicine monomer composition according to claim 4, characterized in that, In step (1), the chrysanthemum plant raw materials include chrysanthemum inflorescences, chrysanthemum stems and leaves or chrysanthemum roots; the extraction solvent is distilled water or 30%~85% ethanol solution; the material-to-liquid ratio is 1:10~20 g / mL, the extraction time is 1~3 h, and the extraction is performed 1~2 times.

6. The method for preparing the traditional Chinese medicine monomer composition according to claim 4, characterized in that, In step (2), The mass concentration of the loading solution is 25~125 mg / mL; and / or The volume of the sample loading solution is 10-110 mL; and / or The macroporous resin is any one of AB-8, NKA-9, HPD-500, DM-130, or DM-301; and / or The volumetric flow rate of the sample loading solution is 0.5~2.0 mL / min; and / or The column volume of the distilled water is 1~2 BV; and / or The elution solvent is a 60%~80% ethanol solution; and / or The volume of the elution solvent is 2-4 BV; and / or In step (4), the volume ratio of the total flavonoid chemical fraction solution to the purified enzyme solution is 1:1~3; and / or The concentration of the total flavonoid chemical fraction solution is 0.5~2 mg / mL; and / or The concentration of the purified enzyme solution is 1~3.5 mg / mL.

7. The use of a traditional Chinese medicine monomer composition as described in any one of claims 1 to 3 in the preparation of a medicament for treating diabetic retinopathy.

8. The application according to claim 7, characterized in that, The herbal monomer composition plays a role in the preparation of drugs for treating diabetic retinopathy by inhibiting body weight gain caused by high blood sugar, reducing blood sugar levels, improving retinal tissue structure damage, restoring retinal tissue thickness, inhibiting retinal cell apoptosis and inflammatory response, and reducing the expression levels of HIF-1α and VEGF proteins in retinal tissue.

9. A drug for treating diabetic retinopathy, characterized in that, The drug comprises an effective amount of the traditional Chinese medicine monomer composition as described in any one of claims 1 to 3 and pharmaceutically acceptable excipients.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes any one of the following: decoction, oral liquid, patch, tablet, granule, capsule, ointment, pill, powder, elixir, injection, or sustained-release preparation.

Images