Application of lonicera confusa and lonicera confusa extract in preparation of medicine for treating glaucoma
By inhibiting trabecular meshwork cell fibrosis through honeysuckle extract and its active ingredients, the problem of lacking effective drug treatment for trabecular meshwork fibrosis in existing technologies has been solved, thus achieving effective prevention and treatment of glaucoma.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUIZHOU MEDICAL UNIV
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-12
AI Technical Summary
Current technology lacks effective drugs that can specifically treat trabecular meshwork cell fibrosis, which increases the probability of developing diseases such as glaucoma. The pathological mechanism of fibrosis is complex and not fully understood.
The extract of honeysuckle and its active ingredients chlorogenic acid, isochlorogenic acid A, Dipsacus saponin B and Lonicera japonica saponin B were used to prepare an anti-trabecular meshwork cell fibrosis drug, which inhibited the upregulation of transcription levels of cell fibronectin, α-smooth muscle actin gene, type I collagen gene and type IV collagen gene.
It effectively inhibits fibrosis of trabecular meshwork cells, reduces aqueous humor outflow resistance, and decreases the probability of glaucoma, showing good potential for drug development and clinical application value.
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Figure CN122005643A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically the application of honeysuckle and its extracts in the preparation of drugs for treating glaucoma. Background Technology
[0002] The trabecular meshwork, located between the scleral venous sinus and the anterior chamber angle, is the essential pathway for aqueous humor to return to the scleral venous sinus and the primary channel for aqueous humor outflow, accounting for approximately 70% of aqueous humor circulation. Therefore, pathological changes in the trabecular meshwork tissue can severely affect the normal maintenance of intraocular pressure. According to literature reports, abnormal accumulation of extracellular matrix proteins (ECM), such as fibronectin and collagen, can lead to fibrosis and hardening of the trabecular meshwork tissue, thereby increasing resistance to aqueous humor outflow, causing pathological increases in intraocular pressure, and consequently increasing the probability of glaucoma.
[0003] However, due to the complexity of the pathological mechanism of fibrosis, which has not yet been fully elucidated, there are very few drugs that can specifically treat fibrosis. Therefore, the development of novel anti-fibrotic drugs has broad application prospects and important social significance. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides the application of honeysuckle extract and chlorogenic acid, isochlorogenic acid A, dipsacus saponin B, and honeysuckle saponin B in the preparation of drugs against trabecular meshwork cell fibrosis, as detailed below: Application of honeysuckle and its extracts in the preparation of drugs for treating glaucoma.
[0005] Furthermore, the glaucoma treatment drug is an anti-trabecular meshwork cell fibrosis drug. The anti-trabecular meshwork cell fibrosis includes: inhibiting the upregulation of fibronectin expression in cells and / or inhibiting the upregulation of transcriptional levels of at least one of the following genes: fibronectin gene, α-smooth muscle actin gene, type I collagen gene, and type IV collagen gene.
[0006] Furthermore, the glaucoma treatment drug is a drug for treating iridocorneal endothelial syndrome.
[0007] Furthermore, the honeysuckle extract includes honeysuckle extract or its target compound.
[0008] Furthermore, the honeysuckle extract or its target compound includes the active ingredient of the honeysuckle extract, and at least one of a pharmaceutically acceptable salt, ester, solvate, stereoisomer, and tautomer of the active ingredient.
[0009] Furthermore, the active ingredient includes at least one of organic acids and triterpenoid saponins.
[0010] Furthermore, the active ingredient includes at least one of chlorogenic acid, isochlorogenic acid A, Dipsacus saponin B, and Lonicera japonica saponin B.
[0011] Furthermore, the structural formulas of chlorogenic acid, isochlorogenic acid A, dipsacus saponin B, and honeysuckle saponin B are shown in Formulas 1 to 4, respectively: Formula 1: Formula 2: ; Formula 3: ; Formula 4: .
[0012] Furthermore, the honeysuckle extract is a honeysuckle alcohol extract.
[0013] Furthermore, the honeysuckle extract is a water extract of honeysuckle.
[0014] Compared with the prior art, the technical effects of this invention are reflected in: This application innovatively discovers the anti-trabecular meshwork cell fibrosis efficacy of honeysuckle extract and its active ingredients, which can be applied to the preparation of drugs for anti-trabecular meshwork cell fibrosis and prevention and treatment of trabecular meshwork cell fibrosis-related diseases such as glaucoma. Specifically, honeysuckle extract, along with chlorogenic acid, isochlorogenic acid A, dipsacusin B, and honeysuckle saponin B, can be used in the preparation of drugs for anti-trabecular meshwork cell fibrosis. They can also be used to prepare drugs that inhibit the upregulation of FN, α-SMA, and COL-1 protein levels, as well as drugs that inhibit the upregulation of FN1, ACTA2, COL1A1, and COL4A1 transcriptional levels. This has good drug development potential and broad value and prospects for development, translation, and clinical application. Attached Figure Description
[0015] Figure 1 The following figures illustrate the cytotoxicity results of honeysuckle aqueous extract (WELF) and honeysuckle alcohol extract (EELF) as a model in Example 1: a) Effect of different concentrations of WELF on the viability of human trabecular meshwork cells; b) Effect of different concentrations of EELF on the viability of human trabecular meshwork cells. Figure 2 The figure shows the effect of WELF and EELF on the expression levels of fibrosis markers FN and α-SMA in a TGF-β1-induced human trabecular meshwork cell fibrosis model in Example 2. a is the SDS-PAGE electrophoresis result, and b is the quantitative analysis statistical graph. Figure 3The figure shows the effect of EELF on the expression levels of fibrosis markers FN and α-SMA in a TGF-β1-induced human trabecular meshwork cell fibrosis model in Example 3. a is the SDS-PAGE electrophoresis result, and b is the quantitative analysis statistical graph. Figure 4 Figure 4 shows the effect of EELF on the transcriptional levels of FN1 (a), ACTA2 (b), COL4A1 (c), and COL1A1 (d) in a TGF-β1-induced human trabecular meshwork cell fibrosis model. Figure 5 In Example 5, using human trabecular meshwork cells (HTMC) as a model, the cytotoxicity results of the active components of Lonicera japonica, including chlorogenic acid, isochlorogenic acid A, dipsacoside B, and macronthoidin B, were detected. Figure a shows the results for chlorogenic acid; figure b shows the results for isochlorogenic acid A; figure c shows the results for dipsacoside B; and figure d shows the results for macronthoidin B. Figure 6 The figure shows the effect of the active components of honeysuckle (including chlorogenic acid, isochlorogenic acid A, dipsacoside B, and macramthoidin B) on the expression levels of fibrosis markers FN, α-SMA, and COL-1 in a TGF-β1-induced human trabecular meshwork cell fibrosis model in Example 6. Figure 7 The figure shows the effect of dipsacoside B, an active ingredient of honeysuckle in Example 7, on the expression levels of fibrosis markers FN, α-SMA, and COL-1 in a TGF-β1-induced human trabecular meshwork cell fibrosis model. a is the SDS-PAGE electrophoresis result, and b is the quantitative analysis statistical graph. Figure 8 Figure 8 shows the effect of the active ingredient Dipsacus asperoides B from Lonicera japonica on the transcriptional levels of FN1 (a), COL4A1 (b), COL1A1 (c), and ACTA2 (d) in a TGF-β1-induced human trabecular meshwork cell fibrosis model. Figure 9Example 9 shows the results of hematoxylin and eosin staining (HE) and Masson trichrome staining of the trabecular meshwork structure in a rat model of dexamethasone-induced glaucoma induced by chlorogenic acid, an active ingredient of honeysuckle, as well as the results of intraocular pressure changes in rats. a) shows the results of HE and Masson staining; b) shows the results of intraocular pressure changes in rats. Detailed Implementation
[0016] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.
[0017] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0018] Definition of noun The term "honeysuckle extract" in this article refers to the chemical components obtained from the traditional Chinese medicine honeysuckle through processes such as extraction with a specific solvent (commonly water, ethanol, or a water-alcohol mixture), filtration, concentration, purification, and drying.
[0019] The term “treatment” in this article includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.
[0020] In this article, the term "prevention" refers to preventing the onset, development, or worsening of a disease or health problem before or at an early stage, through proactive interventions (such as medication). Its core objective is to reduce the probability of disease occurrence, delay the onset time, and / or reduce the negative impact of disease on an individual.
[0021] The term "pharmaceutical acceptable" in this article means that the form of the compound must meet requirements such as safety, stability, and suitability for formulation.
[0022] The term "stereoisomers" in this article refers to molecules with the same molecular formula and the same atomic connection order, but different spatial arrangements.
[0023] The term "tautomer" in this article refers to a dynamic equilibrium isomer of the same compound formed by proton transfer and double bond position change, commonly found in keto-enol and imine-enamine structures.
[0024] This application does not impose any special limitations on the preparation process of honeysuckle extract and its active ingredients, and extraction and preparation can be carried out based on the existing publicly disclosed related processes for honeysuckle extract and its active ingredients.
[0025] In some embodiments, the method for preparing the honeysuckle extract includes: The dried buds or newly opened flowers of *Lonicera japonica* (a traditional Chinese medicine) were extracted by reflux at 55–95°C using 80% (v / v) ethanol or distilled water. The solvent was added at a liquid-to-solid ratio of 1:10–1:15. Before the first extraction, the flowers were pre-soaked in cold water for 0.5 hours to promote cell swelling. Subsequent reflux extractions were performed 2–3 times at 55–95°C, each extraction lasting 1–3 hours. The extracts were filtered, combined, and then concentrated under reduced pressure (≤40°C) to recover the solvent. The concentrate was then concentrated to form a thick paste-like liquid.
[0026] In some embodiments, the active ingredient includes at least one of organic acids and triterpenoid saponins. Examples of organic acids include chlorogenic acid and isochlorogenic acid A. Examples of triterpenoid saponins include dipsacusin B and honeysuckle saponin B.
[0027] In some embodiments, the active ingredient includes at least one of organic acids and triterpenoid saponins. Examples of organic acids include chlorogenic acid (CGA) and isochlorogenic acid A; examples of triterpenoid saponins include dipsacoside B and macronthoidin B.
[0028] In some embodiments, the structural formulas of chlorogenic acid, isochlorogenic acid A, Dipsacus saponin B, and Lonicera japonica saponin B are shown in Formulas 1 to 4, respectively. Formula 1: ; Formula 2: ; Formula 3: ; Formula 4: .
[0029] In some embodiments, the pharmaceutically acceptable salt includes hydrochloride, sulfate, sodium salt, or potassium salt. The pharmaceutically acceptable alcohol includes ethanol or methanol. The pharmaceutically acceptable solvate includes hydrates or ethanolates.
[0030] In some embodiments, the product includes: a drug.
[0031] In some embodiments, the medicament may include at least one of pharmaceutically acceptable conventional carriers and / or excipients, such as diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbent carriers, and lubricants.
[0032] In some embodiments, the anti-trabecular meshwork cell fibrosis includes: inhibiting the upregulation of fibronectin expression in cells and / or inhibiting the upregulation of transcriptional levels of at least one of the following genes: fibronectin gene, α-smooth muscle actin gene, type I collagen gene, and type IV collagen gene.
[0033] In some embodiments, the inhibition of fibronectin expression upregulation in cells includes: reducing or terminating the abnormal upregulation of protein expression levels, so that the protein expression levels approach normal.
[0034] In some embodiments, the upregulation of transcriptional levels of at least one of the fibronectin gene, α-smooth muscle actin gene, and type I collagen gene of the inhibiting cell includes: reducing or terminating the abnormal upregulation of the transcriptional level of the target gene, so that the gene transcriptional level approaches normal.
[0035] In some embodiments, the cells comprise trabecular meshwork cells.
[0036] On the other hand, embodiments of the present invention provide the use of the target compound as described in any of the foregoing embodiments in the preparation of products for preventing diseases related to trabecular meshwork cell fibrosis.
[0037] On the other hand, embodiments of the present invention provide the use of the target compound as described in any of the foregoing embodiments in the preparation of products for treating trabecular meshwork cell fibrosis-related diseases.
[0038] In some embodiments, the trabecular meshwork cell fibrosis-related diseases include glaucoma and / or iridocorneal endothelial syndrome.
[0039] In some embodiments, the product includes a drug.
[0040] In some embodiments, the glaucoma includes at least one of primary open-angle glaucoma, secondary glaucoma, and angle-regressing glaucoma.
[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0042] Material: The dried buds or newly opened flowers of *Lonicera japonica* (a traditional Chinese medicine) were extracted by reflux at 55–95°C using 80% (v / v) ethanol or distilled water. The solvent was added at a liquid-to-solid ratio of 1:10–1:15. Before the first extraction, the flowers were pre-soaked in cold water for 0.5 hours to promote cell swelling. Subsequent reflux extractions were performed 2–3 times at 55–95°C, each extraction lasting 1–3 hours. The extracts were filtered, combined, and then concentrated under reduced pressure (≤40°C) to recover the solvent. The concentrate was then concentrated to form a thick paste-like liquid.
[0043] Example 1 The effects of WELF and EELF on the viability of human trabecular meshwork cells were tested using the CCK-8 assay kit.
[0044] (1) Experimental methods 1. Passage the trabecular meshwork cells one day in advance. After the cells adhere to the culture plate, take the target cells in the logarithmic growth phase, digest them with trypsin, resuspend them in culture medium and adjust the cell density. Seed 100 μL per well in a 96-well cell culture plate (fill the edge wells with an equal volume of PBS), and incubate them in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere and grow.
[0045] 2. Discard the old culture medium in the 96-well plate. Add 100 μL / well of WELF and EELF culture medium containing gradient concentrations (0, 25, 50, 100, 200, 400, 500 μg / mL) to the experimental group to treat trabecular meshwork cells. Add an equal volume of culture medium without the test sample to the control group. Set up 3-5 replicates for each group and continue to incubate under the above culture conditions for 24 h. 3. Add 10 μL of CCK-8 reagent to each well for incubation. After gentle shaking to mix, incubate in the dark for 1–4 h. Then, perform absorbance measurement. Remove the 96-well plate after incubation and measure the absorbance at 450 nm using a microplate reader. Record the experimental data.
[0046] 4. Finally, cell viability was calculated. The absorbance of the blank wells was used as a baseline, and the net absorbance values of the experimental and control groups were subtracted. Quantitative results were obtained using the formulas: "Cell viability (%) = (Net absorbance of experimental group - Net absorbance of blank group) / (Net absorbance of control group - Net absorbance of blank group) × 100%" and "Cell inhibition rate (%) = 100% - Cell viability". The above results, such as Figure 1 As shown.
[0047] (2) Experimental results The effect of honeysuckle extract on the toxicity of human trabecular meshwork cells was detected by CCK-8 assay. The results were obtained from... Figure 1It can be seen that as the concentration of honeysuckle extract increases, the cell survival rate decreases, while the effect of doses of 100 μg / mL and below on cell viability is small. Therefore, subsequent experiments determined 100 μg / mL as the maximum dose (n=3, *P<0.05, **P<0.01, ns, no statistical significance).
[0048] Example 2 Effects of WELF and EELF on the expression levels of fibrosis markers FN and α-SMA in a TGF-β1-induced human trabecular meshwork cell fibrosis model.
[0049] (1) Experimental methods 1. Passage trabecular meshwork cells one day in advance. After cell adhesion, add 10 ng / mL TGF-β1 (prepared in 10% complete medium) to construct a trabecular meshwork cell fibrosis model and administer the drug simultaneously. Culture the cells in an incubator (37℃, 5% CO2) according to the following protocols for at least 24 h: ① Blank group (10% complete medium) ② Model group (10 ng / mL TGF-β1) ③ Positive drug group (5 μM Y-27632) ④ Drug administration groups (low (25 μg / mL), medium (50 μg / mL), and high (100 μg / mL) doses of WELF and EELF) for 24 h. 2. Remove cells, add an appropriate amount of RIPA lysis buffer, and lyse on ice for 30 min, shaking every 5 min during lysis. Centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and determine protein concentration using the BCA method. Take an equal volume of protein sample, add 5×SDS loading buffer, and boil for 5 min to denature the protein. Perform SDS-PAGE electrophoresis to transfer the protein to a PVDF membrane. Block the PVDF membrane with 5% skim milk for 2 h, wash the membrane 3 times with TBST, add primary antibody, and incubate overnight at 4℃. The next day, wash the membrane 3 times with TBST for 10 min each time, add secondary antibody, and incubate at room temperature for 2 h. After washing the membrane 3 times with TBST, develop the color using ECL chemiluminescence, analyze the band gray values using ImageJ software, use GAPDH as an internal control, calculate the relative expression level of the target protein, and then create a quantitative analysis statistical graph.
[0050] The above results, such as Figure 2 As shown.
[0051] (2) Experimental results Depend on Figure 2 It can be seen that WELF and EELF can reduce the expression levels of FN and α-SMA proteins in the model group in a dose-dependent manner, but the inhibitory effect of EELF is more obvious, indicating that both WELF and EELF have the activity of improving trabecular meshwork cell fibrosis.
[0052] Example 3 Effects of EELF on the expression levels of fibrosis markers FN and α-SMA in a TGF-β1-induced human trabecular meshwork cell fibrosis model.
[0053] (1) Experimental methods 1. Passage trabecular meshwork cells one day in advance. After cell adhesion, add 10 ng / mL TGF-β1 (prepared in 10% complete medium) to construct a trabecular meshwork cell fibrosis model and administer the drug simultaneously. Culture the cells in an incubator (37℃, 5% CO2) according to the following protocols for at least 24 h: ① Blank group (10% complete medium) ② Model group (10 ng / mL TGF-β1) ③ Positive drug group (20 μM Y-27632) ④ Drug administration groups (low (25 μg / mL), medium (50 μg / mL), and high (100 μg / mL) doses of EELF) for 24 h. 2. Remove cells, add an appropriate amount of RIPA lysis buffer, and lyse on ice for 30 min, shaking every 5 min during lysis. Centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and determine protein concentration using the BCA method. Take an equal volume of protein sample, add 5×SDS loading buffer, and boil for 5 min to denature the protein. Perform SDS-PAGE electrophoresis to transfer the protein to a PVDF membrane. Block the PVDF membrane with 5% skim milk for 2 h, wash the membrane 3 times with TBST, add primary antibody, and incubate overnight at 4℃. The next day, wash the membrane 3 times with TBST for 10 min each time, add secondary antibody, and incubate at room temperature for 2 h. After washing the membrane 3 times with TBST, develop the color using ECL chemiluminescence, analyze the band gray values using ImageJ software, use GAPDH as an internal control, calculate the relative expression level of the target protein, and then create a quantitative analysis statistical graph.
[0054] (2) Experimental results Depend on Figure 3 It can be seen that EELF can reduce the expression levels of FN and α-SMA proteins in the model group in a dose-dependent manner, which further illustrates that EELF has a better effect on improving the fibrotic activity of trabecular meshwork cells.
[0055] Example 4 Effects of EELF on the transcriptional levels of fibronectin gene (FN1), α-smooth muscle actin gene (ACTA2), type I collagen gene (COL1A1), and type IV collagen gene (COL4A1) in a TGF-β1-induced human trabecular meshwork cell fibrosis model.
[0056] (1) Experimental methods Cell samples from each of the above groups were collected, and total RNA was extracted for RT-qPCR detection. Total RNA was extracted from cells using TriQuick Total RNA Extraction Reagent; cDNA was synthesized using the EasyCript one-step gDNA removal and cDNA synthesis kit; RT-qPCR was performed using TB Green Premixed Solution Ex Taq II. The relative mRNA levels were normalized to GAPDH levels, and then a quantitative analysis statistical graph was plotted.
[0057] Table 1 Primer sequence listing
[0058] (2) Experimental results Depend on Figure 4 It can be seen that EELF can inhibit the upregulation of FN1, ACTA2, COL4A1, and COL1A1 transcription levels, further demonstrating that EELF has the activity of improving trabecular meshwork cell fibrosis.
[0059] Example 5 The effects of active ingredients of honeysuckle, including chlorogenic acid (Formula 1), isochlorogenic acid A (Formula 2), Dipsacus saponin B (Formula 3), and Lonicera japonica saponin B (Formula 4), on the activity of human trabecular meshwork cells.
[0060] (1) Experimental methods 1. Passage the trabecular meshwork cells one day in advance. After the cells adhere to the culture plate, take the target cells in the logarithmic growth phase, digest them with trypsin, resuspend them in culture medium and adjust the cell density. Seed 100 μL per well in a 96-well cell culture plate (fill the edge wells with an equal volume of PBS), and incubate them in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere and grow.
[0061] 2. Discard the old culture medium in the 96-well plate. For the experimental group, add 100 μL / well of culture medium containing chlorogenic acid (Formula 1), isochlorogenic acid A (Formula 2), Dipsacus saponin B (Formula 3), and Lonicera japonica saponin B (Formula 4) at concentrations of 0, 1.25, 2.5, 5, 10, 20, and 40 μM respectively to treat trabecular meshwork cells for 24 h. For the control group, add an equal volume of culture medium without the test sample. Set up 3-5 replicates for each group. Continue to incubate under the above culture conditions for 24 h. 3. Add 10 μL of CCK-8 reagent to each well for incubation. After gentle shaking to mix, incubate in the dark for 1–4 h. Then, perform absorbance measurement. Remove the 96-well plate after incubation and measure the absorbance at 450 nm using a microplate reader. Record the experimental data.
[0062] 4. Finally, cell viability was calculated. The absorbance of the blank wells was used as a baseline, and the net absorbance values of the experimental and control groups were subtracted. Quantitative results were obtained using the formulas: "Cell viability (%) = (Net absorbance of experimental group - Net absorbance of blank group) / (Net absorbance of control group - Net absorbance of blank group) × 100%" and "Cell inhibition rate (%) = 100% - Cell viability". The above results, such as Figure 5 As shown.
[0063] (2) Experimental results The result is Figure 5 It can be seen that as the concentration of honeysuckle extract increases, the cell survival rate decreases, while the effect of doses of 20 μM and below on cell viability is relatively small.
[0064] Example 6 The effects of active ingredients from honeysuckle, including chlorogenic acid (Formula 1), isochlorogenic acid A (Formula 2), Dipsacus saponin B (Formula 3), and Lonicera japonica saponin B (Formula 4), on the expression levels of fibrosis markers FN, α-SMA, and COL-1 in a TGF-β1-induced human trabecular meshwork cell fibrosis model.
[0065] (1) Experimental methods 1. Passage trabecular meshwork cells one day in advance. After cell adhesion, add 10 ng / mL TGF-β1 (prepared in 10% complete medium) to construct a trabecular meshwork cell fibrosis model and administer the drug simultaneously. Culture the cells in an incubator (37℃, 5% CO2) according to the following protocols for at least 24 h: ① Blank group (10% complete medium) ② Model group (10 ng / mL TGF-β1) ③ Positive drug group (20 μM Y-27632) ④ Drug-treated group (chlorogenic acid, isochlorogenic acid A, Dipsacus saponin B, Lonicera japonica saponin B 20 μM) for 24 h. 2. Remove cells, add an appropriate amount of RIPA lysis buffer, and lyse on ice for 30 min, shaking every 5 min during lysis. Centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and determine protein concentration using the BCA method. Take an equal volume of protein sample, add 5×SDS loading buffer, and boil for 5 min to denature the protein. Perform SDS-PAGE electrophoresis to transfer the protein to a PVDF membrane. Block the PVDF membrane with 5% skim milk for 2 h, wash the membrane 3 times with TBST, add primary antibody, and incubate overnight at 4℃. The next day, wash the membrane 3 times with TBST for 10 min each time, add secondary antibody, and incubate at room temperature for 2 h. After washing the membrane 3 times with TBST, develop the color using ECL chemiluminescence, analyze the band gray values using ImageJ software, use GAPDH as an internal control, calculate the relative expression level of the target protein, and then create a quantitative analysis statistical graph.
[0066] (2) Experimental results The result is Figure 6 It can be seen that the active ingredients of honeysuckle, including chlorogenic acid (Formula 1), isochlorogenic acid A (Formula 2), Dipsacus saponin B (Formula 3), and Lonicera japonica saponin B (Formula 4), can reduce the expression levels of FN, α-SMA, and COL-1 proteins in the model group, further demonstrating that honeysuckle extract and its active ingredients have the activity of improving trabecular meshwork cell fibrosis.
[0067] Example 7 Effects of Dipsacus asperoides, an active ingredient in honeysuckle, on the expression levels of fibrosis markers FN, α-SMA, and COL-1 in a TGF-β1-induced human trabecular meshwork cell fibrosis model.
[0068] (1) Experimental methods 1. One day in advance, passage trabecular meshwork cells. After cell adhesion, add 10 ng / mL TGF-β1 (prepared in 10% complete medium) to construct a trabecular meshwork cell fibrosis model and administer the drug simultaneously. Culture the cells in an incubator (37℃, 5% CO2) according to the following protocols for at least 24 h: ① Blank group (10% complete medium) ② Model group (10 ng / mL TGF-β1) ③ Positive drug group (20 μM Y-27632) ④ Drug-treated group (Dipsacus asperoides saponin B, dosage (5 μg / mL, 10 μg / mL, 20 μg / mL)) for 24 h. 2. Remove cells, add an appropriate amount of RIPA lysis buffer, and lyse on ice for 30 min, shaking every 5 min during lysis. Centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and determine protein concentration using the BCA method. Take an equal volume of protein sample, add 5×SDS loading buffer, and boil for 5 min to denature the protein. Perform SDS-PAGE electrophoresis to transfer the protein to a PVDF membrane. Block the PVDF membrane with 5% skim milk for 2 h, wash the membrane 3 times with TBST, add primary antibody, and incubate overnight at 4℃. The next day, wash the membrane 3 times with TBST for 10 min each time, add secondary antibody, and incubate at room temperature for 2 h. After washing the membrane 3 times with TBST, develop the color using ECL chemiluminescence, analyze the band gray values using ImageJ software, use GAPDH as an internal control, calculate the relative expression level of the target protein, and then create a quantitative analysis statistical graph.
[0069] (2) Experimental results Depend on Figure 7 It can be seen that the active ingredient of honeysuckle, dipsacin B, can reduce the levels of α-SMA, FN, and COL-1 proteins in the model group in a dose-dependent manner. This further demonstrates that honeysuckle extract and its active ingredients have the activity of improving trabecular meshwork cell fibrosis.
[0070] Example 8 Effect of the active ingredient asperosaponin VI of Lonicerae Japonicae Flos on the transcriptional levels of fibronectin gene (FN1), α - smooth muscle actin gene (ACTA2), type I collagen gene (COL1A1) and type IV collagen gene (COL4A1) in the TGF - β1 - induced fibrosis model of human trabecular meshwork cells
[0071] (2) Experimental method Another aliquot of the cell samples from each group above was taken to extract total RNA for RT - qPCR detection. Total RNA was extracted from the cells using TriQuick total RNA extraction reagent; cDNA was synthesized using the EasyCript one - step gDNA removal and cDNA synthesis kit; RT - qPCR was performed using TB Green Premix Ex Taq II. The relative mRNA levels were normalized to the GAPDH level, and then a quantitative analysis statistical chart was made. The primers are shown in Table 1
[0072] (2) Experimental results It can be seen from Figure 8 that asperosaponin VI can inhibit the up - regulation of the transcriptional levels of FN1, ACTA2, COL4A1, and COL1A1, further indicating that the extract of Lonicerae Japonicae Flos and its active ingredients have the activity of improving trabecular meshwork cell fibrosis
[0073] Example 9 1.1 Experimental materials Experimental animals: SPF - grade SD rats, license number: SCXK (Beijing) 2024 - 0003, 6 - 8 weeks old, body weight 200 - 250 g, half male and half female (a total of 20), in a constant temperature environment of 23 ± 2 °C and a 12 / 12 h light - dark cycle, with free access to food and water
[0074] Reagents and instruments<000,0246>Reagents: Chlorogenic acid monomer of Lonicerae Japonicae Flos (purity ≥ 98%); Modeling drug: Dexamethasone sodium phosphate eye drops (0.1%); Positive drug: Rho kinase inhibitor (Y - 27632)
[0075] Instruments: Applanation tonometer (Reichert VET230750); 1.2 Experimental method<000025,0>1.2.1 Animal grouping and administration The 20 rats were randomly divided into 5 groups (n = 4), with half male and half female in each group Blank control group (Control group): Instilled with normal saline Model group (Model group): Instilled with 0.1% dexamethasone Positive control group: 0.1% dexamethasone eye drops + Y-27632 (40mg / kg) intraperitoneal injection; High-dose chlorogenic acid group: 0.1% dexamethasone eye drops + honeysuckle monomer (40mg / kg) intraperitoneal injection; High-dose chlorogenic acid group: 0.1% dexamethasone eye drops + honeysuckle monomer (1%) eye drops.
[0076] 1.2.2 Glaucoma Model Establishment: Rats were given 0.1% dexamethasone eye drops in their left eye three times a day (9:00, 16:00 and 22:00) for four consecutive weeks to induce a high intraocular pressure model (similar to the pathogenesis of primary open-angle glaucoma); and the intraocular pressure of each group of rats was recorded every Monday.
[0077] 1.2.3 Sample Collection and Testing: HE and Masson staining: Four weeks after modeling, the rats were sacrificed, their eyeballs were removed, and fixed in 4% paraformaldehyde for 24 hours. The fixed rat eyeball tissue was sent to Chengdu Aochuang Technology Co., Ltd. for Masson staining and HE staining.
[0078] 1.3 Experimental Results Depend on Figure 9 Images of paraffin-embedded sections of rat eyes stained with hematoxylin and eosin (HE) and Masson's staining were compared. HE staining showed that the trabecular meshwork structure of rats in the normal group was normal, while the trabecular meshwork in the dexamethasone model group showed significant hyperplasia and reduced cavity area. The degree of trabecular meshwork hyperplasia was significantly reduced in the chlorogenic acid injection and eye drop groups. Masson's trichrome staining confirmed that dexamethasone can induce collagen production, while chlorogenic acid can inhibit collagen production. Compared with the control group, the intraocular pressure of rats in the dexamethasone model group was elevated. However, after treatment with intraperitoneal injection and eye drops of chlorogenic acid, the intraocular pressure of rats was significantly reduced, and the chlorogenic acid eye drop administration method showed better therapeutic effect.
[0079] Statistical analysis of the experimental data from Examples 1-9 showed that chlorogenic acid and bisacodyl saponin B had better anti-fibrotic effects.
[0080] In summary, this invention creatively proposes the application of honeysuckle extract and chlorogenic acid, isochlorogenic acid A, dipsacus saponin B, and honeysuckle saponin B in the preparation of drugs against trabecular meshwork cell fibrosis. It can also be used to prepare drugs that inhibit the upregulation of FN, α-SMA, and COL-1 protein levels, as well as drugs that inhibit the upregulation of FN1, ACTA2, COL1A1, and COL4A1 transcriptional levels. These drugs possess excellent drug development potential and broad value and prospects for development, translation, and clinical application.
[0081] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.
Claims
1. Application of honeysuckle and its extracts in the preparation of drugs for treating glaucoma.
2. The application according to claim 1, characterized in that, The medication mentioned for treating glaucoma is an anti-fibrotic drug for trabecular meshwork cells.
3. The application according to claim 1, characterized in that, The medication mentioned is for treating glaucoma syndrome.
4. The application according to claim 1, characterized in that, The honeysuckle extract includes honeysuckle extract or its target compound.
5. The application according to claim 4, characterized in that, The honeysuckle extract or its target compound includes at least one of the following: the active ingredient of the honeysuckle extract, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, and tautomer of the active ingredient.
6. The application according to claim 5, characterized in that, The active ingredients include at least one of organic acids and triterpenoid saponins.
7. The application according to claim 6, characterized in that, The active ingredients include at least one of the following: chlorogenic acid, isochlorogenic acid A, Dipsacus saponin B, and Lonicera japonica saponin B.
8. The application according to claim 7, characterized in that, The structural formulas of chlorogenic acid, isochlorogenic acid A, dipsacus saponin B, and honeysuckle saponin B are shown in Formulas 1 to 4 respectively: Formula 1: Formula 2: ； Formula 3: ； Formula 4: 。 9. The application according to claim 1, characterized in that, The honeysuckle extract is a honeysuckle alcohol extract.
10. The application according to claim 1, characterized in that, The honeysuckle extract is a water extract of honeysuckle.