Chrysanthemum extract against vegf and uses thereof

Abstract

The present application provides a kind of anti-vascular endothelial growth factor (VEGF) chrysanthemum extract, the extract uses chrysanthemum medicinal material as raw material, by specific extraction, refining and effective component separation and purification method, so that anti-VEGF effective component content is significantly improved, wherein total flavone content is greater than 50%, total isochlorogenic acid content is greater than 15%.The chrysanthemum extract reduces neovascularization from inhibiting retinal pigment epithelial cell proliferation, reducing VEGF content, and achieves the relief or treatment of neovascular disease symptoms, thereby significantly improving the efficacy, reducing the amount of drug and ensuring the stability of drug quality.The present application also provides the use of the extract in the preparation of drugs for treating or preventing neovascular eye diseases, including age-related macular degeneration, diabetic retinopathy, pathological myopia, neovascular glaucoma.The preparation process of the present application uses ethanol as solvent and eluent, which is suitable for industrialization and realizes green production.

Description

Technical Field

[0001] This invention belongs to the field of plant extracts, and specifically relates to a chrysanthemum extract that resists VEGF and its uses. Background Technology

[0002] Chrysanthemum, the dried capitulum of the chrysanthemum plant *Chrysanthemum morifolium* Ramat. (family Asteraceae), has the effects of 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, polysaccharides, nucleosides, and amino acids, and has medicinal activities such as antioxidation, anti-inflammation, antibacterial, hypoglycemic, liver protection, and immune regulation.

[0003] Neovascular eye diseases are a common group of blinding eye diseases based on pathological neovascularization. Depending on the anatomical location, they can be classified into corneal neovascular diseases, iris neovascular diseases, retinal neovascular diseases, and choroidal neovascular diseases, specifically including age-related macular degeneration (AMD), diabetic retinopathy, pathological myopia, and neovascular glaucoma. Age-related macular degeneration (AMD) is an age-related, chronically progressive, blinding eye disease. Clinically, AMD is divided into atrophic AMD and wet AMD, with wet AMD, also known as neovascular AMD, being the main clinical type causing visual impairment. The pathogenesis of wet AMD is a complex process, mainly involving the complementary effects of multiple growth factors, activation of the complement system, participation of inflammatory factors, and regulation of the autophagy system.

[0004] Currently, intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs have shown some effectiveness in treating neovascular eye diseases. However, patients exhibit varying sensitivities to VEGF drugs, and long-term use may lead to drug resistance. Some patients may also experience complications of varying degrees. Furthermore, these drugs present challenges such as frequent injections and low patient compliance. Antioxidant vitamins, mineral supplements, and traditional Chinese medicine (such as Zhixue Quyu Mingmu Pian and Hexue Mingmu Pian) can all help slow the progression of wet AMD, but they can currently only be used as adjunctive therapies. Finding more effective treatment methods is an urgent need.

[0005] Research on chrysanthemum in the field of AMD has been published. The study "Protective Effect of Chrysanthemum Extract on Photoretinal Light Damage in Mice" (New Advances in Ophthalmology, 2021, 41(02):110-115) indicates that photoretinal damage causes abnormalities in the morphology and electrophysiological function of the mouse retina, resulting in retinal changes similar to age-related macular degeneration. Pre-treatment with a certain concentration of chrysanthemum aqueous extract has a protective effect against photoretinopathy caused by photoretinopathy. This chrysanthemum aqueous extract was prepared by water extraction, concentration, and drying. Through its antioxidant effect, this extract alleviates the oxidative stress level of photoreceptor cells, thus achieving a protective effect against photoretinal light damage in mice and has a preventative effect against AMD. It does not treat AMD by inhibiting VEGF. Therefore, the treatment of AMD with chrysanthemum aqueous extract lacks specificity and tissue targeting, resulting in poor therapeutic effects.

[0006] CN112220817A, "Chrysanthemum and Chrysanthemum Stem and Leaf Extract with Therapeutic Effect on Age-Related Macular Degeneration and Its Application" (Publication Date: 2021.01.15), provides a chrysanthemum and chrysanthemum stem and leaf extract prepared by the following method: chrysanthemum stem and leaf or chrysanthemum sample is appropriately pulverized, weighed, and then water is added. The mixture is heated under reflux for extraction, filtered while hot, and the filtrates are combined. The extract is then concentrated under reduced pressure. Oxidative stress is one of the causes of age-related macular degeneration. The patent uses sodium iodate oxidative stress to establish an AMD mouse model. The chrysanthemum water extract contains antioxidant components, which can treat AMD by combating oxidative stress.

[0007] Patent CN103249422A, entitled "Tie2 Activator, Vascular Endothelial Growth Factor Inhibitor, Angiogenesis Inhibitor, Vascular Maturation Agent, Vascular Normalizing Agent and Vascular Stabilizer, and Pharmaceutical Composition" (Publication Date: August 14, 2013), discloses chrysanthemum extract as an angiogenesis inhibitor and a tyrosine kinase Tie2 activator. Angiogenesis is divided into two stages: vasculogenesis and angiogenesis. Vasculogenesis forms new blood vessels, while angiogenesis involves existing blood vessels extending and branching to form new vascular networks. The former involves the action of VEGF, which is involved in angiogenesis, encompassing a very broad range from the initial development of blood vessels (known as vascular formation) to subsequent angiogenesis. The latter involves the action of angiopoietin (Ang), which is involved in controlling the adhesion between vascular endothelial cells and vascular wall cells and stabilizing vascular structures. Activation of Tie2 expressed in vascular endothelial cells can inhibit angiogenesis, expand the vascular lumen, and inhibit vascular endothelial cell death. The patent discloses different mechanisms of action of chrysanthemum extract in treating AMD, but does not disclose the preparation method of enriching anti-VEGF components or the specific effective components.

[0008] CN116785336A, "Chrysanthemum Active Bacterial Agent for Improving Age-Related Macular Degeneration and its Preparation Method and Application" (Publication Date: 2023.09.22), describes a chrysanthemum live bacteria preparation obtained by adding Lactobacillus bacteria to chrysanthemum water extract or chrysanthemum water extract alcohol precipitation supernatant, and then compounding or co-fermenting it to improve age-related macular degeneration. In the examples, a mouse model of AMD was established using sodium iodate oxidative stress. The chrysanthemum live bacteria preparation uses the antioxidant components in chrysanthemum extract to improve AMD; simultaneously, the live bacteria preparation regulates the intestinal flora and improves the gastrointestinal function of the model mice. This technical solution requires sophisticated equipment for the production of the live bacteria preparation, and the preparation lacks specificity and tissue targeting. Summary of the Invention

[0009] This invention provides a chrysanthemum extract with anti-VEGF properties, addressing the problems of lack of specificity, tissue targeting, and poor efficacy of existing chrysanthemum extracts in treating neovascular eye diseases. The chrysanthemum extract provided by this invention utilizes specific extraction, purification, and effective component separation and purification methods to significantly increase the content of anti-VEGF active ingredients, with total flavonoid content exceeding 50% and total isochlorogenic acid content exceeding 15%. Simultaneously, this chrysanthemum extract synergistically reduces angiogenesis by inhibiting retinal pigment epithelial cell proliferation and lowering VEGF levels, thereby alleviating or treating AMD symptoms, significantly improving efficacy, reducing drug dosage, and ensuring stable drug quality. Furthermore, the preparation method of this chrysanthemum extract is suitable for industrial-scale application.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0011] A chrysanthemum extract with VEGF resistance is prepared as follows: Chrysanthemum medicinal material is extracted with 60%–90% ethanol solution or water at 90℃–100℃, concentrated to a relative density of 1.00–1.15 at 50℃, and filtered to obtain the supernatant. The supernatant is passed through a macroporous resin column, eluted sequentially with 1–5 column volumes of water and 1.5–3 column volumes of 60%–90% ethanol solution. The ethanol eluent is collected, concentrated, and dried to obtain the extract. Preferably, the macroporous resin column type includes HPD100, HPD450, or D101; the packing weight is 3–5 times the feed amount.

[0012] Preferably, the amount of ethanol solution used is 8 to 12 times the amount of feed, the extraction temperature is 75℃ to 80℃, the concentration is reduced to a relative density of 1.00 to 1.15 at 50℃, the mixture is allowed to stand, and then filtered to obtain the supernatant.

[0013] Preferably, the water dosage is 6 to 10 times the feed amount, concentrated to a relative density of 1.05 to 1.15 at 50°C, and then precipitated with 2 to 5 times the volume of the concentrated liquid in 75% to 85% ethanol solution, followed by filtration to obtain the supernatant.

[0014] Further confirmation of the effective components in the extract revealed that the effective components of the extract include total flavonoids and total isochlorogenic acid, wherein the content of total flavonoids is greater than 50% and the content of total isochlorogenic acid is greater than 15%, and the total flavonoid compounds include luteolin, apigenin, sennaol, and acaciain.

[0015] Preferably, the luteolin compounds include one or more of luteolin-7-O-neohesperidin, luteolin-7-O-glucoside, luteolin-4'-O-(6”-O-acetyl)-glucoside, and luteolin.

[0016] Preferably, the apigenin compounds include one or more of apigenin-7-O-glucoside, apigenin-7-O-glucuronide, apigenin-7-O-(6”-O-malonyl)-glucoside, apigenin-7-O-(2”-O-acetyl)-glucoside, apigenin-7-O-(6”-O-acetyl)-glucoside, and apigenin.

[0017] Preferably, the sageol compounds include sageol-7-O-glucoside.

[0018] Preferably, the farnesoids include one or more of farnesoid-7-O-glucoside, senna glycoside, farnesoid-7-O-glucuronide, farnesoid-7-O-(6”-O-malonyl)-glucoside, and farnesoid-7-O-(6”-O-acetyl)-glucoside.

[0019] Preferably, the total isochlorogenic acid includes one or more of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C.

[0020] The present invention also provides the use of the extract in the preparation of drugs for the treatment or prevention of neovascular eye diseases.

[0021] Preferably, the neovascular eye diseases include age-related macular degeneration, diabetic retinopathy, pathological myopia, and neovascular glaucoma.

[0022] Preferably, the age-related macular degeneration is wet age-related macular degeneration.

[0023] Different compounds exhibit varying anti-VEGF activities and cell proliferation inhibitory activities, and the inhibition of cell proliferation by a compound is not directly related to its anti-VEGF activity. This invention confirms through experiments that luteolin-based compounds, apigenin-based compounds, and especially isochlorogenic acids represented by isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C, possess good anti-VEGF activity. The chrysanthemum extract provided by this invention is prepared using specific extraction, purification, and effective component separation and purification methods. Its anti-VEGF effect is superior to that of simple aqueous extracts of chrysanthemum disclosed in the prior art, as well as chrysanthemum extracts obtained by polyamide column chromatography, which is conventionally used for enriching flavonoids, isochlorogenic acid, and other phenolic acids, producing unexpected technical effects.

[0024] This invention has the following advantages:

[0025] (1) The present invention obtains chrysanthemum extract through specific extraction, refining and separation and purification of effective components, which has a better anti-VEGF effect than the prior art.

[0026] The prior art CN112220817A discloses that the aqueous extracts of chrysanthemum and chrysanthemum stems and leaves have therapeutic effects on AMD. Therefore, this invention sets extract 8, chrysanthemum aqueous extract, as a control of the prior art. On the one hand, through tracking the effective components and detecting the content of total flavonoids and total isochlorogenic acid, the inventors found that the content of major flavonoids such as luteolin and apigenin in extract 8, as well as the content of total flavonoids and total isochlorogenic acid, were significantly lower than those of extracts 1-6 of this invention, as shown in Table 1. On the other hand, through the cobalt chloride (CoCl2) induced ARPE-19 cell proliferation assay and VEGF content detection, it was found that under the same amount of raw drug, the cell proliferation inhibition rate of extract 8 (28.54%) was reduced by 22% compared with extract 1 (50.84%), and its VEGF content inhibition rate (45.68%) was reduced by 21% compared with extract 1 (67.11%). The cell proliferation inhibition rate and VEGF inhibition rate of extracts 1-6 provided by this invention are similar. Therefore, it is evident that simple water extraction of extract 8 cannot effectively enrich anti-VEGF components, and existing technologies have poor specificity in treating AMD.

[0027] Furthermore, the antioxidant stress effect of extract 8 was lower than that of extracts 1-6. The inventors detected the antioxidant stress effect of the extracts by measuring the ROS levels in H2O2-induced ARPE-19 cells. Relative fluorescence intensity represents the level of antioxidant stress; the lower the value, the better the antioxidant stress effect. The experimental results showed that, under the same drug content, the relative fluorescence intensity of extract 8 was 78.61%, higher than that of extract 1 (68.28%); the relative fluorescence intensities of extracts 1-6 were similar. Therefore, extracts 1-6 are superior to extract 8 in multiple aspects, including antioxidant stress, anti-cell proliferation, and anti-VEGF activity, significantly enhancing the drug's efficacy.

[0028] Table 1. Component content of extracts 1-12 (unit: %)

[0029]

[0030] (2) This invention obtains a chrysanthemum extract with better anti-VEGF effect by tracking effective ingredients, specific preparation process and efficacy verification.

[0031] The inventors discovered through active ingredient tracking that isochlorogenic acid and luteolin glycosides possess good anti-VEGF activity. To enrich these components, the inventors modified the extraction solvent, chromatography column, elution solvent, and elution method to obtain extracts 7, 9-12, the contents of which are shown in Table 1. Even with a uniform amount of crude drug, the cell proliferation inhibition rate and VEGF inhibition rate of the simple ethanol extract 7 were still lower than those of extract 1. Specifically, the cell proliferation inhibition rate of 220 μg / mL extract 7 was 20% lower than that of 75 μg / mL extract 1 (with the same amount of crude drug), and the VEGF inhibition rate was 27% lower than that of extract 1, indicating that simple ethanol extraction cannot effectively enrich the target components.

[0032] Extract 9 was extracted using alcohol followed by a polyamide column chromatography; extract 10 was extracted using water followed by alcohol precipitation and then a polyamide column chromatography; extracts 11 and 12 were extracted using different concentrations of the ethanol solution and different elution steps, resulting in increased contents of luteolins, apigenins, and total isochlorogenic acid compared to extracts 1-6. In particular, the combined content of flavonoids and total isochlorogenic acid in extract 9 (1+3 total) was 17% higher than that in extract 1. The efficacy results, as shown in Tables 12 and 15, indicate that extracts 9-12 at 75 μg / mL exhibited cell proliferation inhibition rates of 22.91%–32.03%, lower than the 42.04% inhibition rate of extract 1 at the same concentration; the VEGF inhibition rates of extracts 9-12 at 75 μg / mL were 46.09%–50.53%, lower than the 66.18% inhibition rate of extract 1 at the same concentration. Unlike conventional techniques that assume higher concentrations of active ingredients equate to better efficacy, this invention achieves better efficacy with lower concentrations of the target ingredient, resulting in unexpected technical effects. This may be because the invention employs a specific technical solution to enrich the VEGF-inhibiting active ingredient and remove impurities, thereby enhancing the overall anti-VEGF effect of the extract; or, different major components may have a synergistic effect within a specific ratio range, enhancing the overall anti-VEGF effect.

[0033] The inventors further demonstrated through antioxidant stress experiments that there is no direct correlation between the anti-VEGF effect and the antioxidant stress effect. Extracts 9-12, obtained through process modification and containing higher levels of flavonoids and total isochlorogenic acid, exhibited relative fluorescence intensities of 56.31%-64.97% at 75 μg / mL; extract 1, at the same concentration, showed a relative fluorescence intensity of 70.51%, indicating that extract 1 has lower antioxidant activity than extracts 9-12.

[0034] Based on the results of cell proliferation inhibition rate, anti-VEGF effect, and oxidative stress test, it is evident that extracts 1-6 of this invention alleviate and treat AMD symptoms through anti-cell proliferation and anti-VEGF secretion, rather than by enriching antioxidant stress components to enhance anti-AMD activity. This invention, through tracking effective components, a specific preparation process, and efficacy verification, yielded chrysanthemum extracts with better anti-VEGF effects, resulting in better improvement or treatment of neovascular eye diseases and demonstrating specificity and tissue targeting in the treatment of AMD.

[0035] (3) This invention inhibits angiogenesis by inhibiting cell proliferation and resisting VEGF, and enhances drug efficacy through synergistic effect.

[0036] Senescence and dysfunction of retinal pigment epithelial cells (RPE) are significant factors contributing to the progression of AMD. Excessive RPE cell proliferation can lead to angiogenesis and fibrotic scarring, thus exacerbating AMD. Inhibiting RPE cell proliferation can alleviate or treat AMD. VEGF is a key regulator of retinal angiogenesis, and neovascular AMD in AMD is caused by abnormal angiogenesis. Higher anti-VEGF capacity leads to better improvement or treatment of AMD.

[0037] This invention establishes an active ingredient screening model for AMD (Advanced Mesothelial Plasty) using a CoCl2-simulated hypoxic environment to evaluate the proliferation and VEGF expression of ARPE-19 retinal pigment epithelial cells. The inventors first screened for active ingredients using common chrysanthemum compounds such as luteolin-7-O-glucuronide, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. Experimental results showed that different compounds inhibiting ARPE-19 cell proliferation exhibited both inhibitory and VEGF-promoting effects, and there was no direct correlation between the compounds' cell proliferation inhibition and anti-VEGF activity. Extracts 1-6 provided by this invention simultaneously possess both cell proliferation inhibition and VEGF inhibition activities, inhibiting angiogenesis from two aspects and synergistically enhancing the overall effect. Attached Figure Description

[0038] The accompanying drawings are provided to further illustrate the invention, and the illustrative embodiments and descriptions thereof are used to explain the invention and do not constitute an improper limitation thereof.

[0039] Figure 1 Effects of different concentrations of CoCl2 on the survival rate of ARPE-19 cells The CoCl2 concentration in the control group was 0, compared to the control group. ### P<0.001.

[0040] Figure 2 Effects of different compounds on VEGF content in CoCl2-induced ARPE-19 cells Among them, SG is luteolin-7-O-glucuronide, SJ is isochlorogenic acid A, SI isochlorogenic acid B, SL isochlorogenic acid C, SN is apigenin-7-O-(6”-O-malonyl)-glucoside, SO is stigmosiderin, SH is luteolin-7-O-glucoside, and SK is apigenin-7-O-glucoside. Compared with the control group... ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0041] Figure 3 Effects of different concentrations of H2O2 on the survival rate of ARPE-19 cells The H2O2 concentration in the control group was 0. Compared with the control group, ### P<0.001.

[0042] Figure 4 Effects of extracts 1–4 on intracellular ROS levels in H2O2-induced ARPE-19 cells Here, E-1 to E-4 represent extracts 1 to 4, respectively. Compared with the Control group, ### P<0.001; compared with the Model group, ***P<0.001.

[0043] Figure 5 Effects of extracts 5-8 on intracellular ROS levels induced by H2O2 in ARPE-19 cells Here, E-5 to E-8 represent extracts 5 to 8, respectively. Compared with the Control group, ### P<0.001; compared with the Model group, ***P<0.001.

[0044] Figure 6 Effects of extracts 9–12 on intracellular ROS levels in H2O2-induced ARPE-19 cells Here, E-9 to E-12 represent extracts 9 to 12, respectively. Compared with the Control group, ###P<0.001; compared with the Model group, *P<0.05, ***P<0.001.

[0045] Figure 7 Effects of different chrysanthemum extracts on ocular angiogenesis in zebrafish AMD model Here, E-1, E-4, E-7, and E-8 represent extracts 1, 4, 7, and 8, respectively. Compared with the Control group, ## P<0.01; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0046] The following examples are intended to further illustrate the present invention and are not intended to limit the invention.

[0047] Example 1: Screening of the anti-VEGF activity of chrysanthemum components

[0048] 1. Test materials

[0049] Human retinal pigment epithelial cells (ARPE-19) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. Cobalt chloride (Sigma); DMEM / F12 medium, fetal bovine serum (FBS) (Gibco), penicillin-streptomycin mixture (100×) (Sologopharm Technology Co., Ltd.); CCK-8 assay kit (Beyotime Biotechnology Co., Ltd.); trypsin-EDTA digestion solution, PBS buffer (Sologopharm Technology Co., Ltd.); VEGF ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd.).

[0050] 2. Test methods

[0051] 2.1 ARPE-19 cell culture

[0052] 2.1.1 Preparation of complete culture medium

[0053] Take 5 mL of FBS and 500 μL of penicillin-streptomycin mixture and add it to 44.5 mL of DMEM / F12 basal medium to prepare a complete medium containing 10% FBS. Store in a 4°C refrigerator and allow to equilibrate to room temperature for half an hour before use.

[0054] 2.1.2 Cell passage

[0055] When the cells cover about 80% of the culture dish, discard the old culture medium and wash twice with PBS. Add 1 mL of trypsin-EDTA digestion solution (hereinafter referred to as "trypsin"), digest at 37°C for 3 min, disperse the cells evenly and collect them in a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in complete culture medium, and then culture them in a conventional manner at a ratio of 1:3.

[0056] 2.1.3 Cell cryopreservation

[0057] When the cells are in the logarithmic growth phase, collect the cell suspension according to the cell passage method, centrifuge at 1000 rpm for 5 min, discard the supernatant, add cell cryopreservation solution (FBS:DMSO = 9:1), resuspend the cells, collect them into cryovials, then place the cryovials in a programmed cooling box, place them in an ultra-low temperature freezer at -70℃ for 1-2 days, and then transfer the cryovials to a liquid nitrogen tank for long-term storage.

[0058] 2.1.4 Cell resuscitation

[0059] Immediately place the cryovials containing cells from the liquid nitrogen container into a 37°C water bath and gently shake them to accelerate thawing. Once the liquid in the cryovials has completely thawed, transfer them to centrifuge tubes containing complete culture medium. Centrifuge at 1000 rpm for 5 minutes. Wipe the surface and seal of the cryovials with a cotton ball containing 75% alcohol, discard the supernatant, add 2 mL of complete culture medium, and gently pipette the culture medium to agitate the cell clusters. After the cells are resuspended, transfer them to a culture dish, add an appropriate amount of complete culture medium, mix well, and incubate overnight. The next day, remove the cells, discard the culture medium, rinse twice with PBS, and replace with fresh complete culture medium for routine culture.

[0060] 2.2 Drugs and Preparation

[0061] Preparation of medium containing 1% FBS: Take 500 μL of FBS and 500 μL of penicillin-streptomycin mixture and add it to 49 mL of DMEM / F12 basal medium. Store in a 4°C refrigerator and allow to equilibrate to room temperature for half an hour before use.

[0062] Anhydrous cobalt chloride was purchased from Sigma-Aldrich. It was dissolved in an appropriate amount of distilled water to prepare a 50 mM stock solution. Certain amounts of luteolin-7-O-glucuronide, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, apigenin-7-O-(6”-O-malonyl)-glucosinolate, budesonin, luteolin-7-O-glucosinolate, and apigenin-7-O-glucosinolate were weighed and dissolved in DMSO to prepare a 200 mg / mL stock solution. The stock solution was diluted with 1% FBS medium to the final concentration required for the experiment.

[0063] 2.3 Concentration Screening of CoCl2 as a Modeling Drug

[0064] Digestion was performed according to the method described in section 2.1.2, and the ARPE-19 cell suspension was collected. The solution was then diluted with 6 × 10⁻⁶ cells. 3 Cells were seeded per well in 96-well plates and cultured overnight to allow them to adhere. The culture medium was discarded. The control group was treated with 100 μL of medium containing 1% FBS, while the other groups were treated with 1% FBS medium containing 25, 50, 100, 200, 400, and 800 μM CoCl2, respectively, for 24 h and 48 h. The solution in each well was then discarded, and 100 μL of CCK-8 working solution (a 1:10 mixture of CCK-8 solution and DMEM / F12 medium) was added to each well. The plates were incubated at 37°C in the dark for 2 h, and the absorbance (A) at 450 nm was measured using a microplate reader to calculate cell viability.

[0065]

[0066] 2.4 Screening of safe and effective concentrations of compounds

[0067] ARPE-19 cells in the logarithmic growth phase were harvested at a concentration of 6 × 10⁻⁶. 3 Each sample was seeded per well in a 96-well plate and incubated overnight to allow adhesion. The culture medium was discarded. The control group was treated with 100 μL of culture medium containing 1% serum, while the other groups were treated with 100 μL of each monomeric drug concentration for 24 h. Luteolin-7-O-glucuronide, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, apigenin-7-O-(6”-O-malonyl)-glucosidase, and scutellarin were prepared at 50, 150, and 200 μM, respectively. Luteolin-7-O-glucosidase and apigenin-7-O-glucosidase were prepared at 30, 40, and 50 μM, respectively. After incubation in a cell culture incubator for 24 h, the solution in the wells was discarded, and 100 μL of CCK-8 working solution (a mixture of CCK-8 solution and DMEM / F12 medium at a ratio of 1:10) was added to each well. The cells were incubated at 37°C in the dark for 2 h, and the absorbance (A) at 450 nm was measured using a microplate reader to calculate the cell viability.

[0068]

[0069] 2.5 CCK-8 assay to detect the effect of compounds on CoCl2-induced ARPE-19 cell proliferation

[0070] ARPE-19 cells in the logarithmic growth phase were harvested at a concentration of 6 × 10⁻⁶. 3Cells were seeded per well in 96-well plates and incubated overnight to allow adherence. The culture medium was discarded. The control group received 100 μL of medium containing 1% FBS, while the model and experimental groups received 200 μM CoCl2. The drug concentrations in the experimental groups were set at 18.8, 37.5, and 75 μM. After 24 h, the solutions in the wells were discarded, and 100 μL of LCK-8 working solution was added to each well. The plates were incubated at 37°C in the dark for 2 h. The absorbance (A) at 450 nm was measured using a microplate reader, and cell viability and inhibition rate were calculated. Inhibition rate = (Model group survival rate - Experimental group survival rate) / Model group survival rate * 100%.

[0071]

[0072] 2.6 ELISA method for determining the protein concentration of VEGF in cell supernatant

[0073] ARPE-19 cells in the logarithmic growth phase were harvested at a concentration of 6 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates and incubated overnight to allow adhesion. The culture medium was discarded. The control group received 100 μL of medium containing 1% FBS, while the model group and other experimental groups received 200 μM CoCl2. Drug concentrations for the drug treatment groups were set at 18.8, 37.5, and 75 μM. The cell culture supernatant from each group was collected, and VEGF content and inhibition rate were measured according to the ELISA kit instructions. Inhibition rate = (model group - experimental group) / model group * 100%.

[0074] 3. Test Results

[0075] 3.1 Effect of CoCl2 on ARPE-19 cell viability

[0076] The CCK-8 assay was used to investigate the effect of different concentrations of CoCl2 on the survival rate of ARPE-19 cells at different treatment times. The experimental results are as follows: Figure 1 As shown, under the same treatment time, cell viability initially increased and then decreased with increasing CoCl2 concentration, reaching its highest level at a CoCl2 concentration of 200 μM. When the CoCl2 concentration remained constant, cells treated with CoCl2 for 24 h had a higher viability than those treated for 48 h. Therefore, 200 μM CoCl2 treatment for 24 h was chosen as the modeling condition for the ARPE-19 cell hypoxia model.

[0077] 3.2 Effects of the compound on ARPE-19 cell survival

[0078] As shown in Table 2, cell viability was not affected when isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and apigenin-7-O-(6”-O-malonyl)-glucoside were administered at concentrations ranging from 0 to 200 μM; cell viability was not affected when budesonide was administered at a concentration of 50 μM; cytotoxicity was observed when luteolin-7-O-glucuronide was administered at a concentration of 50 μM; and cytotoxicity was observed when luteolin-7-O-glucoside and apigenin-7-O-glucoside were administered at concentrations of 30 μM. Setting the administration concentrations to 18.8, 30, and 75 μM met the cytotoxicity requirements for most compounds.

[0079] Table 2. Cell viability results for different compounds

[0080]

[0081] Note: Compared to the Control group, # P<0.05, ## P<0.01, ### P<0.001.

[0082] 3.3 Effects of compounds on CoCl2-induced proliferation of ARPE-19 cells

[0083] Excessive proliferation of retinal pigment epithelial cells (ARPE-19) can lead to angiogenesis and fibrotic scarring, thus exacerbating AMD. CoCl2, as a chemical hypoxia inducer, can mimic a hypoxic environment and promote the proliferation of ARPE-19 cells. Inhibiting ARPE-19 cell proliferation can help screen for active ingredients that can alleviate or treat AMD symptoms.

[0084] As shown in Tables 3 and 4, intervention with 200 μM CoCl2 for 24 h significantly increased cell number, with the survival rate of the model group increasing by 42%–53% compared to the control group, indicating successful model establishment. Compared to the model group, apigenin-7-O-(6”-O-malonyl)-glucoside and apigenin-7-O-glucoside both produced cell proliferation inhibition rates of 14%–43% within the concentration range of 18.8–75 μM; the remaining compounds did not have a significant effect on cell proliferation.

[0085] Table 3. Inhibition of CoCl2-induced ARPE-19 cell proliferation by monomeric compounds such as isochlorogenic acid A.

[0086]

[0087]

[0088] Note: Compared to the Control group, ###P<0.001; Compared with the Model group, **P<0.01, ***P<0.001.

[0089] Table 4. Inhibition of CoCl2-induced ARPE-19 cell proliferation by monomeric compounds such as buddlejasin.

[0090]

[0091] Note: Compared to the Control group, ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0092] 3.4 Effects of the compound on VEGF content in the supernatant of CoCl2-induced ARPE-19 cells

[0093] Under CoCl2-simulated hypoxic conditions, ARPE-19 cells increase VEGF expression, thereby promoting angiogenesis. Inhibiting VEGF expression can suppress angiogenesis, thus alleviating or treating AMD.

[0094] From Table 5 and Figure 2 It was found that the VEGF content in the model group increased by 66.5% compared with the control group, indicating successful model establishment. Compared with the model group, neither apigenin-7-O-(6”-O-malonyl)-glucoside nor luteolin at concentrations of 18.8–75 μM inhibited VEGF production; the order of anti-VEGF activity of the remaining compounds was isochlorogenic acid A > isochlorogenic acid C > isochlorogenic acid B > luteolin-7-O-glucuronide > luteolin-7-O-glucoside > apigenin-7-O-glucoside. The experimental results indicate that isochlorogenic acids and luteolin glycosides have good anti-VEGF activity. Combined with the cell proliferation inhibition in Tables 3–4, it can be seen that the inhibition of cell proliferation by the compounds is not directly related to their anti-VEGF activity. Chrysanthemum is rich in isochlorogenic acids and luteolin glycosides; enriching these major compounds through the preparation process is beneficial for obtaining chrysanthemum extracts with good anti-VEGF effects.

[0095] Table 5. Inhibition of VEGF in CoCl2-induced ARPE-19 cell supernatant by different compounds.

[0096]

[0097]

[0098] Note: Compared to the Control group, ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0099] Example 2: Preparation of Chrysanthemum Extract 1

[0100] Take 35 kg of chrysanthemum medicinal material and extract it three times with 75% ethanol solution at 10 times the feed amount at an extraction temperature of 75℃. Combine the extracts and concentrate the filtrate to a relative density of 1.00 at 50℃. Filter to obtain the supernatant. Pass the supernatant through an HPD100 macroporous resin column with a packing volume of 3 times the feed amount. Elute with 4 BV water and 2 BV 70% ethanol solution in sequence. Collect the ethanol eluent, concentrate, and dry to obtain 1.57 kg of extract 1, with a yield of 4.49%.

[0101] Example 3: Preparation of Chrysanthemum Extract 2

[0102] Take 300g of chrysanthemum medicinal material and extract it three times with 12 times the amount of 60% ethanol solution at an extraction temperature of 80℃. Combine the extracts, concentrate the filtrate to a relative density of 1.10 at 50℃, and filter to obtain the supernatant. Pass the supernatant through an HPD450 macroporous resin column with a packing volume of 4 times the amount of raw material. Elute with 2 BV of water and 1.5 BV of 60% ethanol solution in sequence. Collect the ethanol eluent, concentrate, and dry to obtain 17.46g of extract 2, with a yield of 5.82%.

[0103] Example 4: Preparation of Chrysanthemum Extract 3

[0104] Take 300g of chrysanthemum medicinal material and extract it twice with 8 times the amount of 90% ethanol solution at an extraction temperature of 78℃. Combine the extracts and concentrate the filtrate to a relative density of 1.15 at 50℃. Filter to obtain the supernatant. Pass the supernatant through a D101 macroporous resin column with a packing volume of 5 times the amount of raw material. Elute with 2BV of water and 4BV of 90% ethanol solution in sequence. Collect the ethanol eluent, concentrate, and dry to obtain 11.82g of extract 3, with a yield of 3.94%.

[0105] Example 5: Preparation of Chrysanthemum Extract 4

[0106] Take 1 kg of chrysanthemum medicinal material and extract it three times with 10 times the amount of water at an extraction temperature of 95℃. Combine the extracts and concentrate the filtrate to a relative density of 1.05 at 50℃. Add 5 times the volume of 80% ethanol solution for alcohol precipitation and filter to obtain the supernatant. Pass the supernatant through an HPD100 macroporous resin column with a packing volume of 4 times the amount of raw material. Elute with 3 BV of water and 3 BV of 60% ethanol solution in sequence. Collect the ethanol eluent, concentrate and dry to obtain 55.11 g of extract 4, with a yield of 5.51%.

[0107] Example 6: Preparation of Chrysanthemum Extract 5

[0108] Take 300g of chrysanthemum medicinal material and extract it twice with 12 times the amount of water at an extraction temperature of 90℃. Combine the extracts and concentrate the filtrate to a relative density of 1.10 at 50℃. Add 4 times the volume of 85% ethanol solution for alcohol precipitation and filter to obtain the supernatant. Pass the supernatant through an HPD450 macroporous resin column with a packing volume of 3 times the amount of raw material. Elute with 4 BV of water and 2 BV of 75% ethanol solution in sequence. Collect the ethanol eluent, concentrate and dry to obtain 16.89g of extract 5, with a yield of 5.63%.

[0109] Example 7: Preparation of Chrysanthemum Extract 6

[0110] Take 300g of chrysanthemum medicinal material, extract it twice with 8 times the amount of water, and extract it at 100℃. Combine the extracts, concentrate the filtrate to a relative density of 1.15 at 50℃, add 3 times the volume of 95% ethanol solution for alcohol precipitation, and filter to obtain the supernatant. Pass the supernatant through an HPD100 macroporous resin column with a packing volume of 5 times the amount of material. Elute with 3 BV of water and 4 BV of 90% ethanol solution in sequence. Collect the ethanol eluent, concentrate it, and dry it to obtain 14.95g of extract 6, with a yield of 4.98%.

[0111] Example 8: Preparation of Chrysanthemum Extract 7

[0112] Based on Example 2, a simple alcohol extract was obtained.

[0113] Take 300g of chrysanthemum medicinal material and extract it three times with 75% ethanol solution at 10 times the amount of raw material at an extraction temperature of 75℃. Combine the extracts, concentrate the filtrate, and dry it to obtain 112.2g of extract 7, with a yield of 37.4%.

[0114] Example 9: Preparation of Chrysanthemum Extract 8

[0115] Based on Example 5, a simple water extract was obtained.

[0116] Take 300g of chrysanthemum medicinal material, extract it three times with 10 times the amount of water, at an extraction temperature of 95℃, combine the extracts, concentrate the filtrate, and dry it to obtain 101.67g of extract 8, with a yield of 33.89%.

[0117] Example 10: Preparation of Chrysanthemum Extract 9

[0118] Based on Example 3, a polyamide column was used to replace the macroporous resin column, and the eluent concentration was modified to 75% ethanol solution to obtain chrysanthemum extract 9.

[0119] Take 300g of chrysanthemum medicinal material and extract it three times with 12 times the amount of 60% ethanol solution at an extraction temperature of 80℃. Combine the extracts and concentrate the filtrate to a relative density of 1.10 at 50℃. Filter to obtain the supernatant. Pass the supernatant through a polyamide column with a packing volume of 3 times the amount of raw material. Elute with 2 BV of water and 3 BV of 75% ethanol solution in sequence. Collect the ethanol eluent, concentrate, and dry to obtain 10.71g of extract 9, with a yield of 3.57%.

[0120] Example 11: Preparation of Chrysanthemum Extract 10

[0121] Based on Example 6, a polyamide column was used to replace the macroporous resin column, the alcohol precipitation concentration was modified to 80% ethanol solution, and the eluent concentration was modified to 75% ethanol solution to obtain 10g of chrysanthemum extract.

[0122] Take 300g of chrysanthemum medicinal material and extract it twice with 12 times the amount of water at an extraction temperature of 90℃. Combine the extracts and concentrate the filtrate to a relative density of 1.10 at 50℃. Add 5 times the volume of 80% ethanol solution for alcohol precipitation and filter to obtain the supernatant. Pass the supernatant through a polyamide column with a packing volume of 3 times the amount of feed material. Elute with 2 BV of water and 3 BV of 75% ethanol solution in sequence. Collect the ethanol eluent, concentrate and dry to obtain 12.36g of extract 10, with a yield of 4.12%.

[0123] Example 12: Preparation of Chrysanthemum Extract 11

[0124] Based on Example 2, the concentration of the elution solution was changed to enrich isochlorogenic acid and luteolin glycoside components.

[0125] Take 300g of chrysanthemum medicinal material and extract it three times with 75% ethanol solution at 10 times the feed amount at 75℃. Combine the extracts and concentrate the filtrate to a relative density of 1.00 at 50℃. Filter to obtain the supernatant. Pass the supernatant through an HPD100 macroporous resin column with a packing volume of 4 times the feed amount. Elute with 3BV 50% ethanol solution. Collect the ethanol eluent, concentrate, and dry to obtain 10.08g of extract 11, with a yield of 3.36%.

[0126] Example 13: Preparation of Chrysanthemum Extract 12

[0127] Based on Example 2, the elution method was changed to enrich isochlorogenic acid and luteolin glycoside components.

[0128] Take 300g of chrysanthemum medicinal material and extract it three times with 10 times the amount of 75% ethanol solution at 75℃. Combine the extracts and concentrate the filtrate to a relative density of 1.00 at 50℃. Filter to obtain the supernatant. Pass the supernatant through an HPD450 macroporous resin column with a packing volume of 4 times the amount of raw material. Elute with 2BV of 30% ethanol solution and 2BV of 75% ethanol solution successively. Collect the 75% ethanol eluent, concentrate, and dry to obtain 8.61g of extract 12, with a yield of 2.87%.

[0129] Example 14: Determination of Major Component Categories

[0130] 1. Detection Method

[0131] 1.1 Detection of total flavonoid content

[0132] Preparation of reference solution: Take an appropriate amount of luteolin reference standard, accurately weigh it, and add 60% ethanol to prepare a solution containing 0.10 mg per 1 mL.

[0133] Preparation of the standard curve: Accurately measure 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mL of the reference solution and place them in separate 25 mL volumetric flasks. Add 60% ethanol to each flask to a final volume of 8.0 mL. Add 1 mL of 5% sodium nitrite solution to each flask sequentially, shake well, and let stand for 6 minutes. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 mL of 4% NaOH solution, then add water to the mark and shake well. Using the corresponding reagents as blanks, immediately measure the absorbance at 512 nm using UV-Vis spectrophotometry (General Rule 0401). Plot the standard curve with absorbance as the ordinate and the reference concentration as the abscissa.

[0134] Assay: Accurately weigh 30 mg of extract 1–12 mg, place them in a 25 mL volumetric flask, dissolve in 60% ethanol and dilute to the mark, shake well to obtain the test solution. Accurately measure 1 mL of the test solution and place it in a 25 mL volumetric flask. Following the method under the standard curve preparation section, starting from "add 60% ethanol to 8.0 mL", determine the absorbance according to the method. Read the amount of luteolin in the test solution from the standard curve. Calculate the content using luteolin reference standard and multiply by a correction factor of 1.57 to convert it to luteolin glycoside content, thus obtaining the total flavonoid content.

[0135] 1.2 Detection of total isochlorogenic acid content

[0136] Chromatographic conditions and system suitability test: Octylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with the elution gradient shown in Table 6; the column temperature was 30℃; the detection wavelength was 328nm; the flow rate was 1.0mL per minute; the theoretical plate number calculated based on the isochlorogenic acid C peak should not be less than 5000.

[0137] Table 6 Elution gradient

[0138]

[0139] Preparation of reference solution: Take appropriate amounts of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C reference standards, accurately weigh them, and add 70% methanol to prepare a reference solution containing 50 μg of isochlorogenic acid A, 10 μg of isochlorogenic acid B, and 60 μg of isochlorogenic acid C per 1 ml.

[0140] Preparation of the test solution: Weigh 25 mg of extract 1-12 respectively, place them in a 25 mL volumetric flask, add 70% methanol, sonicate to dissolve and dilute to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0141] Assay: Accurately pipette 5 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0142] 2. Results of major component analysis

[0143] The total flavonoid content and total isochlorogenic acid percentage of extracts 1–12 were determined, with the total isochlorogenic acid content calculated as the sum of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C. The experimental results are shown in Table 7. The total flavonoid and total isochlorogenic acid contents of extracts 1–6 were similar. Extracts 7 and 8 were extracted with simple alcohol and water, respectively, and no active ingredients were enriched. Extracts 9 and 10 were better enriched with isochlorogenic acid using a polyamide column, and the total isochlorogenic acid content was higher than that of extracts 1–6. The isochlorogenic acid content of extracts 11 and 12 was enriched by adjusting the concentration of the ethanol elution solvent and the elution order.

[0144] Table 7. Total flavonoids and total isochlorogenic acid content of extracts 1-12

[0145] Extract number Total flavonoid content (%) Total isochlorogenic acid content (%) total(%) 1 56.8 19.2 76.0 2 52.3 16.8 69.1 3 62.0 15.2 77.2 4 59.3 15.8 75.1 5 53.8 17.3 71.1 6 51.9 16.9 68.8 7 8.9 3.2 12.1 8 9.1 4.1 13.2 9 57.0 26.8 83.8 10 46.3 22.5 68.8 11 49.2 26.6 75.8 12 48.0 27.7 75.7

[0146] Example 15: Detection of Flavonoid Content

[0147] 1. Test Methods

[0148] 1.1 Preparation of reference solution

[0149] Accurately weigh the standards of luteolin, apigenin, sennaol, and farnesin, dissolve them in methanol to prepare a mixed solution containing 50 μg of each per mL.

[0150] Luteolin compounds include luteolin-7-O-neohesperidin, luteolin-7-O-glucoside, luteolin-4'-O-(6”-O-acetyl)-glucoside, and luteolin;

[0151] Apigenin compounds include apigenin-7-O-glucoside, apigenin-7-O-glucuronide, apigenin-7-O-(6”-O-malonyl)-glucoside, apigenin-7-O-(2”-O-acetyl)-glucoside, apigenin-7-O-(6”-O-acetyl)-glucoside, and apigenin;

[0152] The sage-derived phenolic compound is sage-derived 7-O-glucoside;

[0153] The acacia compounds include acacia-7-O-glucoside, safflower glycoside, acacia-7-O-glucuronide, acacia-7-O-(6”-O-malonyl)-glucoside, and acacia-7-O-(6”-O-acetyl)-glucoside.

[0154] 1.2 Preparation of test solution

[0155] Take 25 mg of extract 1-12 respectively, accurately weigh them, place them in a stoppered conical flask, accurately add 25 mL of 70% methanol solution, stopper tightly, sonicate to dissolve, shake well, filter, and collect the filtrate to obtain the extract.

[0156] 1.3 Detection Methods

[0157] Liquid phase conditions: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; the detection wavelength was 348 nm, the flow rate was 1.0 mL / min, and the column temperature was 31 °C.

[0158] Table 8. Mobile Phase Gradient

[0159]

[0160] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0161] 2. Test Results

[0162] The contents of luteolin, apigenin, sennaol, and acacia compounds in chrysanthemum extract were detected and classified and statistically analyzed in conjunction with the total isochlorogenic acid content in Example 14, as shown in Table 9.

[0163] Table 9 shows the results of component content detection for extracts 1–12 (unit: %).

[0164] Extract number Luteolins Celery sage phenols acacia compounds Total isochlorogenic acid total 1 13.3 10.8 10.6 2.8 19.2 56.7 2 10.5 8.1 7.9 2.2 16.8 45.5 3 10.1 8.3 7.9 2.4 15.2 43.9 4 10.3 7.6 7.3 2.1 15.8 43.1 5 13.4 7.1 7.0 1.9 17.3 46.7 6 10.5 8.8 7.6 2.2 16.9 46.0 7 1.7 1.3 1.2 0.4 3.2 7.8 8 1.7 1.4 1.2 0.3 4.1 8.6 9 16.8 13.5 12.9 3.7 26.8 73.7 10 14.6 12.1 10.6 2.7 22.5 62.5 11 15.7 12.5 8.7 2.5 26.6 66.0 12 16.5 13.5 9.6 2.9 27.7 70.2

[0165] The elution results of column chromatography depend on various factors, including the type and concentration of the elution solvent and the material characteristics of the packing material. Selecting appropriate elution conditions can significantly improve the separation and purification efficiency. Experimental results show that extracts 9–12 have higher contents of luteolin and isochlorogenic acid compounds. Specifically, extracts 9 and 10 were enriched by replacing the macroporous resin column with a polyamide column; extracts 11 and 12 were enriched by using a macroporous resin column and varying the concentration of the ethanol solution in the elution solution.

[0166] Example 16: Screening for the anti-AMD activity of chrysanthemum extract

[0167] Take 1-12 extracts and conduct efficacy experiments according to the experimental steps of Example 1.

[0168] 1. Test materials

[0169] As shown in Example 1.

[0170] 2. Test methods

[0171] 2.1 ARPE-19 cell culture

[0172] As shown in Example 1.

[0173] 2.2 Drugs and Preparation

[0174] Preparation of medium containing 1% FBS: Take 500 μL of FBS and 500 μL of penicillin-streptomycin mixture and add it to 49 mL of DMEM / F12 basal medium. Store in a 4°C refrigerator and allow to equilibrate to room temperature for half an hour before use.

[0175] Anhydrous cobalt chloride was purchased from Sigma-Aldrich. It was dissolved in an appropriate amount of distilled water to prepare a 50 mM stock solution. A certain amount of extract 1–12 was weighed and dissolved in DMSO to prepare a 200 mg / mL stock solution. The stock solution was diluted with 1% FBS medium to the final concentration required for the experiment.

[0176] 2.3 Experimental grouping and intervention

[0177] Control group: ARPE-19+ medium containing 1% FBS.

[0178] Model group: ARPE-19 + 200μM CoCl2.

[0179] High-dose group: ARPE-19 + 200μM CoCl2 + 75μg / mL extract 1-6, 9-12.

[0180] Medium-dose group: ARPE-19 + 200μM CoCl2 + 37.5μg / mL extract 1-6, 9-12.

[0181] Low-dose group: ARPE-19 + 200μM CoCl2 + 18.8μg / mL extract 1-6, 9-12.

[0182] Extracts 7 and 8 are alcohol extract and water extract, respectively, with low content of active ingredients. To unify the amount of raw medicinal material, the dosage concentration was increased: the dosage concentration for the high-dose group was set at 220 μg / mL; the dosage concentration for the medium-dose group was set at 110 μg / mL; and the dosage concentration for the low-dose group was set at 55 μg / mL.

[0183] 2.4 Effect of chrysanthemum extract on CoCl2-induced proliferation of ARPE-19 cells as determined by CCK-8 assay

[0184] ARPE-19 cells in the logarithmic growth phase were harvested at a concentration of 6 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates and cultured overnight to allow them to adhere. The culture medium was discarded, and cells were treated according to the method described in section "2.3". After 24 hours, the solution in each well was discarded, and 100 μL of CCK-8 working solution was added to each well. The plates were incubated at 37°C in the dark for 2 hours. The absorbance (A) at 450 nm was measured using a microplate reader, and cell viability and inhibition rate were calculated. Inhibition rate = (Model group - Experimental group) / Model group * 100%.

[0185] 2.5 ELISA method was used to determine the protein concentration of VEGF in cell supernatant.

[0186] ARPE-19 cells in the logarithmic growth phase were seeded in 96-well plates. After processing the cells according to the method in section "2.3", the cell culture supernatant of each group was collected. The VEGF content was detected and the inhibition rate was calculated according to the ELISA kit instructions. Inhibition rate = (model group - experimental group) / model group * 100%.

[0187] 3. Test Results

[0188] 3.1 Effects of Chrysanthemum Extract on CoCl2-Induced Proliferation of ARPE-19 Cells

[0189] The inhibitory effects of chrysanthemum extracts 1–12 on CoCl2-induced ARPE-19 cell proliferation are shown in Tables 10–12. Since the cell viability differed among the model groups, extract 1 at 75 μg / mL was used as a reference.

[0190] Compared with the control group, the cell survival rate of the model groups in the three experiments increased by 72% to 117%, and the model was successfully established.

[0191] At a concentration of 75 μg / mL, the cell proliferation inhibition rates of extracts 1–4 in experimental group 1 ranged from 35% to 41%, indicating similar efficacy.

[0192] In experimental group 2, the survival rate of cell proliferation in the model group was higher, and the cell proliferation inhibition rate of extract 1 was 51%, similar to that of extracts 5-6; the inhibition rate of extracts 7-8 was 29%-31%, which was 20%-22% lower than that of extract 1.

[0193] In experimental group 3, the cell proliferation inhibition rate of extract 1 was 42%, while the cell proliferation inhibition rate of extracts 9-12 was reduced by 10%-19% compared with extract 1.

[0194] The results of the three sets of experiments show that the cell proliferation inhibition rates of extracts 1-6 are similar, but significantly higher than those of extracts 7-12.

[0195] Table 10. Inhibition of CoCl2-induced ARPE-19 cell proliferation by extracts 1–4

[0196]

[0197] Note: Compared to the Control group, ### P<0.001; Compared with the Model group, **P<0.01, ***P<0.001.

[0198] Table 11. Inhibition of CoCl2-induced ARPE-19 cell proliferation by extracts 5–8

[0199]

[0200]

[0201] Note: Compared to the Control group, ### P<0.001; Compared with the Model group, **P<0.01, ***P<0.001.

[0202] Table 12 Inhibition of extracts 9–12 on CoCl2-induced ARPE-19 cell proliferation

[0203]

[0204] Note: Compared to the Control group, ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0205] 3.2 Effect of Chrysanthemum Extract on VEGF Content in CoCl2-Induced ARPE-19 Cell Supernatant

[0206] The VEGF content in the cell supernatant of each group was detected by ELISA. The experimental results of chrysanthemum extracts 1-12 are shown in Tables 13-15. The VEGF content of the model groups in different experiments was different, so 75 μg / mL extract 1 was used as a reference.

[0207] Compared with the control group, the VEGF content in the model group cells increased by about 1 times, which was statistically significant, indicating that the model was successfully established.

[0208] At a concentration of 75 μg / mL, the cell inhibition rates of extracts 1-4 in experimental group 1 were between 55% and 60%, indicating that extracts 1-4 had similar efficacy.

[0209] In experimental groups 2 and 3, the model group cells secreted more VEGF than those in experimental group 1. Compared to the control group, the VEGF increase was similar in each model group, corresponding to similar VEGF inhibition rates of extract 1 (66%–67%). Therefore, experimental groups 2 and 3 could be compared simultaneously. The VEGF inhibition rates of extracts 5–6 were 63%–65%, similar to the efficacy of extract 1; the VEGF inhibition rates of extracts 7–12 were 40%–51%, a decrease of 16%–27% compared to extract 1.

[0210] VEGF is a key regulator of retinal angiogenesis and a key indicator for detecting pathological retinal vascular proliferation. Higher anti-VEGF activity leads to better improvement or treatment of pathological retinal vascular proliferation. Combining the efficacy results of monomeric compounds in Tables 4-5 with the content detection results of extracts 1-12 in Table 9, it is evident that extracts 1-6 have lower levels of isochlorogenic acid and luteolin compared to extracts 9-12. Since isochlorogenic acid and luteolin are active anti-VEGF components, the anti-VEGF effect of extracts 9-12 should be greater than that of extracts 1-6. The anti-VEGF test results show that extracts 1-6 have similar and significantly higher anti-VEGF effects than extracts 9-12. This contradicts the conventional assumption in the technical optimization process that higher levels of enriched active ingredients generally equate to better extract efficacy, resulting in an unexpected technical effect.

[0211] Existing technologies CN112220817A and "Protective Effect of Chrysanthemum Extract on Retinal Photodamage in Mice" disclose that chrysanthemum water extract has anti-AMD effects. In this experiment, extract 8 was obtained by water extraction at 95℃, serving as a control for the prior art. The results showed that at a concentration of 220 μg / mL, extract 8 exhibited a VEGF inhibition rate of 45.68%, which was 22% lower than that of extract 1 at a concentration of 75 μg / mL. This demonstrates that simple water extraction cannot effectively enrich the anti-VEGF active components in chrysanthemum.

[0212] Extract 7, obtained by extraction with 75% ethanol solution, served as a simple ethanol extraction control. Extracts 11–12, obtained by 75% ethanol extraction + HPD macroporous resin column + different elution gradients, served as controls using the same technical approach. The results showed that at a concentration of 220 μg / mL, extract 7 exhibited a VEGF inhibition rate of 40.16%, a 27% decrease compared to extract 1 at a concentration of 75 μg / mL (with the same amount of crude drug). Extracts 11–12, enriched with isochlorogenic acid and luteolin compounds by varying the elution gradient, showed anti-VEGF inhibition rates of 47.00% and 50.53%, respectively, a 19% and 16% decrease compared to extract 1. The results indicate that extracts 1–6 require specific extraction methods, chromatographic columns, and elution conditions to achieve better VEGF inhibition. These specific technical approaches may have enriched the VEGF-inhibiting active components and removed impurities, thereby improving the overall anti-VEGF effect of the extracts; or, different major components may have a synergistic effect within a specific ratio range, enhancing the overall anti-VEGF effect.

[0213] Furthermore, the pharmacodynamic experiments of the compounds showed that there was no direct correlation between the VEGF inhibition rate and the cell proliferation inhibition rate. Extracts 1-6 simultaneously exhibited good cell proliferation inhibition rate and VEGF inhibition rate, reducing the formation of new blood vessels at different levels and treating or improving the symptoms of AMD.

[0214] Table 13 shows the inhibitory effect of extracts 1-4 on VEGF in the supernatant of CoCl2-induced ARPE-19 cells.

[0215]

[0216] Note: Compared to the Control group, ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0217] Table 14. Inhibition of VEGF in CoCl2-induced ARPE-19 cell supernatant by extracts 5-8.

[0218]

[0219] Note: Compared to the Control group, ### P<0.001; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

[0220] Table 15. Inhibition of VEGF in CoCl2-induced ARPE-19 cell supernatant by extracts 9–12.

[0221]

[0222] Note: Compared to the Control group, ### P<0.001; Compared with the Model group, **P<0.01, ***P<0.001.

[0223] Example 17: Study on the protective effect and mechanism of ARPE-19 cells against oxidative damage

[0224] 1. Test materials

[0225] ARPE-19 cells are detailed in Example 1. Reactive oxygen species fluorescence assay kit (Wuhan Elite Biotechnology Co., Ltd.)

[0226] 2. Test methods

[0227] 2.1 Cultivation of ARPE-19 cells

[0228] As shown in Example 1.

[0229] 2.2 Drugs and Preparation

[0230] As shown in Example 1.

[0231] 2.3 Concentration screening of the modeling drug H2O2

[0232] ARPE-19 cells were digested according to the passage method, and the ARPE-19 cell suspension was collected at 6 × 10⁶ cells / mL. 3 Cells were seeded per well in 96-well plates and cultured overnight to allow adhesion. The culture medium was discarded, and each group was incubated with 100 μL of medium containing 1% FBS for 24 h. The control group was incubated with 100 μL of medium containing 1% FBS, while the other groups were incubated with 100 μL of medium containing 1% FBS (100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM H2O2, respectively) for 2 h. The solutions in the wells were then discarded, and 100 μL of CCK-8 working solution was added to each well. The plates were incubated at 37°C in the dark for 2 h, and the absorbance (A) at 450 nm was measured using a microplate reader to calculate cell viability.

[0233]

[0234] 2.4 Experimental grouping and intervention

[0235] Control group: ARPE-19 + medium containing 1% FBS;

[0236] Model group: ARPE-19 + 400 μM H2O2;

[0237] High-dose group: ARPE-19 + 400 μM H2O2 + 75 μg / mL extract 1-6, 9-12;

[0238] Medium-dose group: ARPE-19 + 400μM H2O2 + 37.5μg / mL extract 1-6, 9-12;

[0239] Low-dose group: ARPE-19 + 400 μM H2O2 + 18.8 μg / mL extract 1-6, 9-12.

[0240] Extracts 7 and 8 are alcohol extract and water extract, respectively. To maintain a uniform amount of raw drug, the drug concentration was increased: the high-dose group had a concentration of 220 μg / mL; the medium-dose group had a concentration of 110 μg / mL; and the low-dose group had a concentration of 55 μg / mL.

[0241] 2.5 Detection of ROS content in cells using enzyme-linked immunosorbent assay (ELISA)

[0242] ARPE-19 cells in the logarithmic growth phase were collected and processed at a rate of 6 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates and cultured overnight to allow adhesion. The culture medium was discarded, and each group was incubated with 100 μL of medium containing 1% FBS for 24 h. Following the intervention steps in section "2.4", the cells were incubated for 2 h, then the solution in the wells was discarded. The cells were washed once with PBS, and 100 μL of 1 μM DCFH-DA probe was added to each well. The cells were incubated at 37°C in the dark for 30 min. The probe was carefully discarded, and the cells were washed three times with PBS buffer to remove any probe that had not yet entered the cells. Fluorescence (B) values ​​were measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The PBS was discarded, and 100 μL of LCK-8 working solution was added to each well. The cells were incubated at 37°C in the dark for 2 h, and the absorbance (A) value at 450 nm was measured using a microplate reader. The relative fluorescence intensity of each group was calculated based on the fluorescence and optical density values.

[0243]

[0244] 3. Test Results

[0245] 3.1 Effect of H2O2 on ARPE-19 cell viability

[0246] The effect of different concentrations of H2O2 on the survival rate of ARPE-19 cells was investigated using the CCK-8 assay. Figure 3 It was found that cell viability decreased with increasing H2O2 concentration, and the cell viability was in the range of 50%–60% at an H2O2 concentration of 400 μM. Therefore, this study selected 24 h of pre-drug administration followed by 2 h of intervention with 400 μM H2O2 as the modeling conditions for ARPE-19 cell oxidative damage.

[0247] 3.2 Effects of Chrysanthemum Extract on ROS Levels in H2O2-Induced ARPE-19 Cells

[0248] Extract 1 at 75 μg / mL was used as a control group between different experimental groups. The experimental results are shown in Tables 16-18. Figures 4-6 As shown.

[0249] Compared with the control group, the relative fluorescence intensity of the model groups in the three experiments ranged from 166% to 184%, which was 66% to 84% higher than that of the control group, indicating that the model was successfully established. Relative fluorescence intensity represents the level of antioxidant stress; the lower the relative fluorescence intensity, the better the antioxidant stress effect.

[0250] At a concentration of 75 μg / mL, the relative fluorescence intensities of extracts 1–4 in experimental group 1 were 65%–73%; those of extracts 5–8 in experimental group 2 were 67%–79%; and those of extracts 9–12 in experimental group 3 were 56%–65%. The results indicate that extracts 1–8 had similar antioxidant stress effects, while extracts 9–12 showed better antioxidant stress effects than extract 1.

[0251] Screening tests on the anti-AMD activity of chrysanthemum extract showed that the cell proliferation inhibition rate and anti-VEGF effect of extracts 9-12 were lower than those of extracts 1-6. It can be seen that extracts 1-6 of the present invention achieve the relief and treatment of AMD symptoms by inhibiting cell proliferation and inhibiting VEGF secretion, rather than by enriching antioxidant stress components to enhance anti-AMD activity.

[0252] Table 16 Inhibition rates of extracts 1–4 on H2O2-induced intracellular ROS levels in ARPE-19 cells

[0253]

[0254] Note: Compared to the Control group, ### P<0.001; compared with the Model group, ***P<0.001.

[0255] Table 17 Inhibition rate of extracts 5-8 on H2O2-induced intracellular ROS levels in ARPE-19 cells

[0256]

[0257]

[0258] Note: Compared to the Control group, ### P<0.001; compared with the Model group, ***P<0.001.

[0259] Table 18. Inhibition rate of extracts 9-12 on H2O2-induced intracellular ROS levels in ARPE-19 cells

[0260]

[0261] Note: Compared with the Control group, ### P<0.001; compared with the Model group, *P<0.05, ***P<0.001.

[0262] Example 18: Effect of chrysanthemum extract on ocular angiogenesis in zebrafish AMD model

[0263] 1. Test materials

[0264] Transgenic vascular green fluorescent zebrafish (Fli-1 strain) were reproduced by natural pairwise mating. The age of zebrafish used in the experiment was 1 day after fertilization (1 dpf), and all were raised in fish water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3). The license number for the use of experimental animals is: SYXK (Zhejiang) 2012-0171. Breeding management meets the requirements of international AAALAC certification (certification number: 001458).

[0265] 2. Test methods

[0266] 2.1 Establishment of zebrafish AMD model

[0267] 1 dpf transgenic vascular green fluorescent strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. 1 dpf transgenic vascular fluorescent zebrafish were first treated with 5 mg / mL CoCl2 for 10 min. After the liquid was washed off, the zebrafish were treated with 0.1 mg / mL CoCl2 until 4 dpf, to establish the zebrafish AMD model.

[0268] 2.2 Drug and preparation

[0269] CoCl2 was prepared into a 10.0 mg / mL stock solution with sterilized water for later use. Extract 1 and 4 were prepared into 2.5 mg / mL stock solutions with DMSO, and extracts 7 and 8 were prepared into 10.0 mg / mL stock solutions with DMSO, which were stored at -20°C for later use.

[0270] 2.3 Maximum tolerated concentration (MTC) of chrysanthemum extract

[0271] 1-day-fiber transgenic vascularized green fluorescent zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. A control group, a model group, and treatment groups for extracts 1, 4, 7, and 8 were established. Except for the control group, all experimental groups were treated with water-soluble CoCl2 to establish a zebrafish AMD model. The concentrations of extracts 1, 4, and 7 were set at 6.25, 12.5, 25.0, 50.0, and 100 μg / mL; the concentration of extract 8 was set at 1.56, 3.13, 6.25, 12.5, and 25.0 μg / mL. During the treatment period, the number of dead zebrafish in each experimental group was counted daily and removed promptly. After treatment at 28℃ for 3 days, the mean toxicity (MTC) of each extract for the model zebrafish was calculated and determined.

[0272] 2.4 Experimental grouping and intervention

[0273] Control group: Water used for fish farming.

[0274] Model group: CoCl2.

[0275] Extract 1 group: CoCl2+ 12.5μg / mL, 6.25μg / mL, 3.13μg / mL Extract 1.

[0276] Extract group 4: CoCl2+ 12.5μg / mL, 6.25μg / mL, 3.13μg / mL extract 4.

[0277] Extract group 7: CoCl2+ 6.25μg / mL, 3.13μg / mL, 1.56μg / mL extract 7.

[0278] Extract group 8: CoCl2+ 25.0 μg / mL, 12.5 μg / mL, 6.25 μg / mL extract 8.

[0279] 2.5 Effects of Chrysanthemum Extract on Ocular Angiogenesis in Zebrafish AMD Model

[0280] 1 dpf transgenic zebrafish with green fluorescent blood vessels were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Experimental grouping and intervention are detailed in Section 2.4. After 3 days of treatment at 28℃, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the ocular blood vessel area and evaluate the effects of extracts 1, 4, 7, and 8 on ocular angiogenesis in the zebrafish AMD model.

[0281] 3. Test Results

[0282] 3.1 MTC Measurement

[0283] Extract 1 did not cause mortality or show any obvious abnormalities in zebrafish at concentrations of 6.25 and 12.5 μg / mL; however, at concentrations of 25.0 and 100 μg / mL, the condition was worse than that of the model group; at a concentration of 50.0 μg / mL, it induced mortality in 10% (3 / 30) of the zebrafish, and the remaining zebrafish were in worse condition than the model group. Therefore, the MTC of extract 1 for model zebrafish was determined to be 12.5 μg / mL.

[0284] Extract 4 did not induce mortality or show any obvious abnormalities in zebrafish at concentrations of 6.25 and 12.5 μg / mL. At a concentration of 25 μg / mL, the condition was worse than that of the model group. At concentrations of 50 and 100 μg / mL, zebrafish mortality occurred, with 3.3% (1 / 30 fish) of zebrafish dying at 50.0 μg / mL and 17% (5 / 30 fish) dying at 100 μg / mL. The MTC of extract 4 for model zebrafish was determined to be 12.5 μg / mL.

[0285] Extract 7 did not cause death or obvious abnormalities in zebrafish at a concentration of 6.25 μg / mL; however, at concentrations of 12.5 and 25.0 μg / mL, the condition was worse than that of the model group. At a concentration of 50.0 μg / mL, 30% (9 / 30) of zebrafish died, and at a concentration of 100 μg / mL, 17% (5 / 30) of zebrafish died. Therefore, the MTC of extract 7 for model zebrafish was determined to be 6.25 μg / mL.

[0286] Extract 8 did not cause mortality or show any obvious abnormalities in zebrafish at concentrations ranging from 1.56 to 25.0 μg / mL. Therefore, the MTC of extract 8 for model zebrafish was determined to be 25.0 μg / mL. Based on the above results, the dosage concentrations of extracts 1 and 4 were set at 3.13, 6.25, and 12.5 μg / mL, respectively; the dosage concentrations of extract 7 were set at 1.56, 3.13, and 6.25 μg / mL; and the dosage concentrations of extract 8 were set at 6.25, 12.5, and 25 μg / mL.

[0287] 3.2 Effects of Chrysanthemum Extract on Ocular Angiogenesis in Zebrafish AMD Model

[0288] As shown in Table 19 and Figure 7As shown, compared with the control group, the ocular vascular area of ​​zebrafish in the model group was significantly increased (P<0.01), indicating that the model was successfully established. Compared with the model group, the ocular vascular area of ​​zebrafish treated with different concentrations of extracts 1, 4, 7, and 8 was significantly decreased (P<0.01, P<0.05, P<0.001), indicating that all four extracts can effectively inhibit ocular angiogenesis. The vascular area of ​​extract 8 at 12.5 μg / mL was greater than that of extracts 1 and 4 at 3.13 μg / mL, indicating that extract 8 corresponds to a larger vascular area at a higher drug concentration. Extracts 1 and 4 were more effective in treating AMD than extract 8 obtained by simple water extraction. Extract 7 had better anti-AMD activity than extract 8, but its activity and safety were lower than those of extracts 1 and 4.

[0289] Table 19 Effects of extracts 1, 4, 7, and 8 on ocular angiogenesis in a zebrafish AMD model

[0290]

[0291] Note: Compared to the Control group, ## P<0.01; compared with the Model group, *P<0.05, **P<0.01, ***P<0.001.

Claims

1. A chrysanthemum extract with anti-VEGF properties, wherein the preparation method of the extract is as follows: Chrysanthemum medicinal material is extracted with 60%–90% ethanol solution or water at 90℃–100℃, concentrated to a relative density of 1.00–1.15 at 50℃, and filtered to obtain the supernatant. The supernatant is passed through a macroporous resin column and eluted sequentially with 1–5 column volumes of water and 1.5–3 column volumes of 60%–90% ethanol solution. The ethanol eluent is collected, concentrated, and dried to obtain the extract.

2. The extract according to claim 1, characterized in that, The amount of ethanol solution used is 8 to 12 times the amount of feed, the extraction temperature is 75℃ to 80℃, the concentration is reduced to 1.00 to 1.15 at 50℃, the mixture is allowed to stand, and the supernatant is obtained by filtration.

3. The extract according to claim 1, characterized in that, The amount of water used is 6 to 10 times the amount of feed, concentrated to a relative density of 1.05 to 1.15 at 50°C, and then precipitated with 75% to 85% ethanol solution at a volume of 2 to 5 times the volume of the concentrated liquid. The supernatant is then obtained by filtration.

4. The extract according to claim 1, characterized in that, The macroporous resin column models include HPD100, HPD450, or D101.

5. The extract according to claim 1 or 4, characterized in that, The packing material of the macroporous resin chromatography column is 3 to 5 times the amount of raw material fed in.

6. The extract according to claim 1, characterized in that, The active ingredients of the extract include total flavonoids and total isochlorogenic acid, wherein the total flavonoid content is greater than 50% and the total isochlorogenic acid content is greater than 15%, and the total flavonoid compounds include luteolin, apigenin, sennaol, and acaciain.

7. The extract according to claim 6, characterized in that, The luteolin compounds include one or more of luteolin-7-O-neohesperidin, luteolin-7-O-glucoside, luteolin-4'-O-(6”-O-acetyl)-glucoside, and luteolin.

8. The extract according to claim 6, characterized in that, The apigenin compounds include one or more of the following: apigenin-7-O-glucoside, apigenin-7-O-glucuronide, apigenin-7-O-(6”-O-malonyl)-glucoside, apigenin-7-O-(2”-O-acetyl)-glucoside, apigenin-7-O-(6”-O-acetyl)-glucoside, and apigenin.

9. The extract according to claim 6, characterized in that, The sageol compounds include sageol-7-O-glucoside.

10. The extract according to claim 6, characterized in that, The acacia compounds include one or more of acacia-7-O-glucoside, safflower glycoside, acacia-7-O-glucuronide, acacia-7-O-(6”-O-malonyl)-glucoside, and acacia-7-O-(6”-O-acetyl)-glucoside.

11. The extract according to claim 6, characterized in that, The total isochlorogenic acid includes one or more of isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C.

12. Use of the extract according to claim 1 in the preparation of a medicament for the treatment or prevention of neovascular eye diseases.

13. The use according to claim 12, characterized in that, The neovascular eye diseases mentioned include age-related macular degeneration, diabetic retinopathy, pathological myopia, and neovascular glaucoma.

14. The use according to claim 12, characterized in that, The age-related macular degeneration disease mentioned is wet age-related macular degeneration disease.

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