Action mechanism of grape seed procyanidine for preventing and treating cataract and medical application of grape seed procyanidine

Abstract

The invention designs a new application of grape seed procyanidine in prevention and treatment of cataract. The method comprises the following steps: constructing a cataract injury model by inducing rabbit crystalline lens oxidative injury with hydrogen peroxide, culturing by adopting a domestic rabbit crystalline lens, detecting the activity of total superoxide dismutase, glutathione peroxidase and catalase and the content of malondialdehyde by observing the opacity and structural form of the crystalline lens, and detecting the expression of apoptosis regulation proteins BCL-2 and BAX in combination with immunohistochemistry. And the protective effect and molecular mechanism of the grape seed procyanidine on crystalline lens injury are researched. Researches show that the grape seed procyanidine has a protective effect on hydrogen peroxide induced rabbit crystalline lens injury, and the mechanism of the grape seed procyanidine is related to an antioxidant effect and adjustment of BCL-2 / BAX apoptotic protein; rabbit eye stimulation experiments prove that the grape seed procyanidine has no stimulation or only slight stimulation to rabbit eyes. Therefore, the grape seed procyanidine can prevent cataract, is a potential cataract treatment medicine, provides a new way for development of related medical products such as eye drops, and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of medical and health technology, specifically involving research on the medical applications of grape seed proanthocyanidins, with a particular focus on the mechanism of action of grape seed proanthocyanidins in preventing and treating cataracts, and the application of grape seed proanthocyanidin eye drops in the prevention and treatment of cataracts. Background Technology

[0002] Cataracts are a common ophthalmic disease, with the highest rate of blindness among all eye diseases, seriously threatening human visual health. Current research confirms that the occurrence and development of cataracts are closely related to various factors such as oxidative stress, ionizing radiation, exposure to toxic substances, and metabolic abnormalities, among which oxidative damage is recognized as the most core pathogenic factor. From the perspective of molecular mechanisms, the essence of cataracts is the abnormal change in the conformation of lens proteins, leading to opacity of the refractive media. This pathological process is directly related to the dysfunction of lens epithelial cells. Existing research (Biochemical and biophysical research communications, 2020, 528(1): 112-119) shows that the physiological dysfunction of lens epithelial cells, as the core of lens metabolic activity, is a key initiating factor for cataracts; and the apoptosis mechanism of lens epithelial cells is initiated in the early stage of cataract development, becoming a common cellular pathological basis for various types of acquired cataracts.

[0003] In clinical treatment, postoperative complications of cataract surgery have become a significant issue affecting patients' postoperative visual function recovery, with posterior capsule opacification being the most common type of complication. Studies have confirmed that abnormal migration, proliferation, and differentiation of lens epithelial cells after surgery are the core mechanism leading to posterior capsule opacification. Based on this pathological mechanism, current research and development of cataract prevention and treatment drugs focuses on enhancing the lens's antioxidant capacity. This involves using antioxidants or antioxidant enzyme activators to clear and neutralize oxidative products within the lens, thereby blocking or reversing abnormal biochemical changes and apoptosis processes within the lens. Among these, glutathione series and related antioxidants play a crucial role in the prevention, control, and treatment of cataracts.

[0004] Proanthocyanidins (PA) are a class of natural polyphenolic compounds widely found in the plant kingdom, with grape seeds being one of the richest natural sources. Originally extracted from pine bark, subsequent studies found grape seeds to be a superior extraction material, with extracts containing up to 95% proanthocyanidins. As one of the most potent free radical scavengers discovered to date, proanthocyanidins exhibit significantly superior antioxidant activity compared to vitamins C and E, with antioxidant efficacy 20 to 50 times greater than the latter two.

[0005] Grape seed proanthocyanidins (GSP), as a high-quality natural antioxidant, meet the modern population's demand for highly effective and low-toxicity functional pharmaceutical ingredients, possessing significant development advantages and broad industrialization prospects. However, this component has an inherent drawback of poor oral absorption, severely limiting its development and application in the pharmaceutical field. Therefore, exploring the application value of grape seed proanthocyanidins in cataract prevention and treatment, and opening up new avenues for its development and utilization, has important research significance and application value.

[0006] Numerous studies have confirmed that grape seed proanthocyanidins possess abundant biological activities, including antioxidant, anti-inflammatory, anti-tumor, anti-aging, and cardiomyocyte protection (Acta Pharmacologica Sinica, 2001, 22(12): 1117-1120; International Immunopharmacology, 2005, 5(7-8): 1247-1257; Canadian Journal of Physiology and Pharmacology, 2005, 83(3): 309-318; Food and Drug, 2020, 22(6): 448-451; Mutation research-fundamental and molecular mechanisms of mutagenesis, 2003, 523-524: 87-97; Acta neurologica Belgica, 2021, 121(2): 357-364; oscience). Letters, 2021, 750: 135793; Life Science Research, 2023, 27(1): 49-55). Given its clear antioxidant activity and good safety, whether grape seed proanthocyanidins can play a protective role in the lens through exogenous supplementation and thus achieve the prevention of cataracts needs to be further studied and verified.

[0007] In terms of experimental model construction, experimental cataract models caused by hydrogen peroxide-induced oxidative damage have been widely validated and applied (New Advances in Ophthalmology, 2020, 40(7): 612-616; New Advances in Ophthalmology, 2025, 45(1): 22-26; Chinese Journal of Ophthalmology Research, 2004, 22(2): 148-151). This study employed an in vitro culture method for rabbit lenses to construct a rabbit lens oxidative damage model induced by hydrogen peroxide. By observing changes in lens opacity and structural morphology, key indicators of the antioxidant defense system (total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity, and malondialdehyde (MDA) content) were detected. Immunohistochemistry was also used to detect the expression differences of lens epithelial cell apoptosis regulatory proteins BCL-2 and BAX. This study systematically explored the protective effect and molecular mechanism of grape seed proanthocyanidins against hydrogen peroxide-induced oxidative damage in rabbit lenses, providing a new direction and experimental basis for the medicinal development of grape seed proanthocyanidins.

[0008] A search revealed no reports in publicly available domestic or international literature regarding the use of grape seed proanthocyanidins for the prevention and treatment of cataracts. Summary of the Invention

[0009] The core objective of this invention is to provide a novel pharmaceutical use for grape seed proanthocyanidins, specifically to clarify the protective effect of grape seed proanthocyanidins against hydrogen peroxide (H2O2)-induced oxidative damage to the rabbit lens and lens epithelial cells (LECs), and to elucidate its potential molecular mechanism. Another objective of this invention is to demonstrate the efficacy of grape seed proanthocyanidins in the prevention and treatment of cataracts, thereby opening up a new path for the research, development, and industrial application of cataract prevention eye drops with grape seed proanthocyanidins as the core component.

[0010] This invention has confirmed through a series of studies that grape seed proanthocyanidins have a significant protective effect against hydrogen peroxide-induced damage to the rabbit lens and oxidative damage to lens epithelial cells. Further mechanistic studies have shown that the protective effect is closely related to the antioxidant activity of grape seed proanthocyanidins, and that they can inhibit apoptosis of the lens and lens epithelial cells by regulating the Bcl-2 / Bax apoptosis-related protein pathway, thereby exerting a cataract prevention and treatment effect.

[0011] Attached Figure Description Figure 1 Photographs showing the lens opacity of rabbits in the normal control group, model control group, and low, medium, and high concentration groups of grape seed proanthocyanidins (25, 50, and 100 μg / mL). Figure 2 Photographs showing the morphological observation of lens epithelial cells (hematoxylin-eosin staining, 400×, scar bars: 50μm). Figure 3 The activity of lens antioxidant enzymes T-SOD, CAT, and GSH-PX, and the content of MDA. Figure 4 Photograph (400×) for immunohistochemical detection of BCL-2 and BAX protein expression in lens epithelial cells. Detailed Implementation

[0012] (I) Lens Preparation

[0013] Rabbits were euthanized via air embolism in the marginal ear vein. The eyeballs were carefully removed, and any attached fibrous tissue was meticulously dissected and cleaned. The treated eyeballs were placed in a saline solution containing penicillin 100 IU / mL + streptomycin 100 mg / mL. A circular incision was made approximately 3 mm at the limbus of the cornea using ophthalmic scissors, and the suspensory ligament of the lens was severed. The lens was rinsed three times with PBS solution containing the penicillin and streptomycin. Fifty clear lenses were randomly divided into five groups (10 lenses per group) and cultured under standard conditions (37°C, 5% CO2, saturated humidity) in serum-free DMEM / F-12 medium.

[0014] (II) Experimental Grouping

[0015] Normal control group: cultured in basal culture medium only, without any additional treatment; Model control group: 200 μmol / L hydrogen peroxide was added to the basal culture medium to induce lens damage and simulate the adverse effects of oxidative stress on the lens; Grape seed proanthocyanidin low, medium and high concentration groups: in addition to hydrogen peroxide damage, 25, 50 and 100 μg / mL of grape seed proanthocyanidins were added for intervention.

[0016] (III) Examination of the degree of lens opacity

[0017] Lenses were cultured under standard conditions for over 24 hours, during which the opacity of each group was recorded in detail. A lens transparency assessment system was established using a white background and 0.5mm black cross lines. Based on clarity, the degree of lens opacity was quantified into four levels: no opacity (-), mild opacity (+), moderate opacity (++), and severe opacity (+++). The frequency of opacity level was used as the primary observation indicator.

[0018] (iv) Observation of the structure of the lens epithelium

[0019] Lens samples were collected from each group after 24 hours of culture and fixed for 24 hours. They were then processed according to routine histopathological techniques, including hematoxylin-eosin staining. The tissue structure of the samples was observed under a microscope, with a focus on the morphological characteristics of the lens epithelial cells.

[0020] (V) Determination of antioxidant enzyme activity and protein concentration, and malondialdehyde content in the lens.

[0021] Lenses cultured for 24 hours were removed using a sterilized stainless steel spatula and rinsed in physiological saline. The samples were then homogenized in PBS buffer (pH 7.2) at a ratio of 1:9 (w / v) for 5 minutes under ice bath conditions. After centrifugation at 4°C and 4000 rpm for 10 minutes, the supernatant was collected. The total protein concentration of each lens was determined using the BCA method according to the kit instructions, and the protein concentration was calculated based on the BSA standard curve. Simultaneously, the malondialdehyde content, total superoxide dismutase, catalase, and glutathione peroxidase activities of each lens were measured.

[0022] (vi) Immunohistochemical detection of the expression of apoptosis-related proteins BCL-2 and BAX in lens epithelial cells

[0023] The experimental procedure was performed according to the kit instructions. (1) Primary antibody concentration adjustment: The dilution ratio of BCL-2 and BAX was 1:200; (2) Tissue fixation was the same as that of sectioning (III); (3) Immunostaining: The sections were dewaxed, hydrated, antigen retrieval, incubated with primary antibody and stained with diaminobenzidine in sequence; (4) Result evaluation: The staining intensity was observed by optical microscope to determine the location of the protein in the cell. PBS was used as a negative control instead of primary antibody. No staining was recorded as (-); the appearance of brownish-yellow or coffee-colored staining in the cytoplasm of the cells was positive. Positive identification indicators: (1) Calculate the percentage of positive cells and assign a score (0-4 points); (2) Score by staining intensity (0-3 points); (3) Calculate the comprehensive score: Total score = percentage score × intensity score; (4) Result grading: 0 points is negative (-), 1-4 points is weakly positive (+), 5-8 points is moderately positive (++), and 9-12 points is strongly positive (+++).

[0024] (vii) Experimental Data Analysis and Statistical Methods

[0025] Organize the experimental data, using the mean ± standard deviation. The statistical analysis and graphing of the data were performed using GraphPad Prism 8 software. One-way ANOVA was used for comparisons of multiple groups, and the Bonferroni method was used for multiple comparisons between groups. Furthermore, the chi-square test was used to analyze the differences in lens opacity between groups using SPSS 26 software. 2 The expression data of BCL-2 and BAX proteins were quantitatively analyzed using the Ridit statistical method, and inter-group comparisons were performed using the independent samples t-test. A p-value < 0.05 was considered statistically significant.

[0026] The instruments and main experimental materials used are as follows:

[0027] 1. Experimental animals: 50 New Zealand white rabbits (weighing 2.0–2.5 kg), male and female, provided by the Medical Animal Experiment Center of Lanzhou University (Production License No.: SCXK(Gan)2023-0003). The experiment followed the animal rights guidelines established by the relevant ethics committee. A total of 100 lenses were tested in two batches for turbidity, antioxidant enzyme assays, and detection of LECs damage and apoptosis-related proteins.

[0028] 2. Experimental Reagents: Grape seed proanthocyanidins were purified by the School of Chemistry and Chemical Engineering, Lanzhou University, with a purity >96%. DMEM / F-12 medium was a product of Hyclone (USA). 30% hydrogen peroxide solution was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. Rabbit two-step detection kit produced by Zhongshan Jinqiao (Beijing) Co., Ltd., Bax rabbit polyclonal antibody (Boson Biotechnology, Beijing) and Bcl-2 polyclonal antibody (ImmunoWay, Texas, USA) were used. MDA, T-SOD, GSH-PX and CAT kits were used by Nanjing Jiancheng Bioengineering Co., Ltd., and penicillin-streptomycin mixture, 4% cell fixation solution and BCA kit were provided by Solarbio (Beijing).

[0029] 3. Experimental instruments: CO2 incubator (SHELLAB, USA), clean bench (Shanghai Boxun, China), Multiskan FC microplate reader (Thermo Fisher Scientific), DM3000 research-grade multi-head microscopy imaging system (Leica, Germany), cell culture plates (ABC, Hong Kong, China).

[0030] result:

[0031] 1. Lens opacity

[0032] Figure 1 Experimental data showed that after 24 hours of culture, each experimental group (n=10) exhibited the following characteristics: all lenses in the model control group showed grayish-white opacity; possibly due to in vitro culture conditions, one lens in the normal control group also showed slight opacity; the low, medium, and high concentration GSP groups had 8, 5, and 3 lenses with slight opacity, respectively. The lens opacity rates in the normal control, model control, and low, medium, and high concentration GSP groups were 10%, 100%, 80%, 50%, and 30%, respectively.

[0033] In the χ2 analysis between groups, compared with the model control group, there were extremely significant differences between the normal control group and the high concentration group of GSP (P<0.01), significant differences in the concentration of GSP (P<0.05), and no significant differences in the low concentration of GSP (P>0.05).

[0035] 2. Morphological observation of LECs under a light microscope

[0036] Figure 2 A shows normal control LECs with normal morphology. The model control shows LEC detachment, disordered arrangement, irregular morphology, and broken lens fibers. Figure 2 B). GSP at 25 μg / mL showed a slight reduction in damage compared to the model control, but abnormal LEC arrangement, occasional degeneration and necrosis, and partial lens fiber breakage were still observed. Figure 2 C).

[0038] 3. Effects of GSP on antioxidant enzymes and malondialdehyde in the lens

[0039] Figure 3 The results showed that the activities of all three antioxidant enzymes in the model control group were significantly lower than those in the normal control group (P<0.01). Compared with the model control group, the activities of total superoxide dismutase, catalase, and glutathione peroxidase were increased in the medium and high concentration groups of grape seed proanthocyanidins (P<0.05); the low concentration group of grape seed proanthocyanidins increased catalase activity (P<0.05), while the activities of total superoxide dismutase and glutathione peroxidase showed no significant change (P>0.05). Compared with the low concentration group of grape seed proanthocyanidins, the high concentration group of grape seed proanthocyanidins significantly increased the activities of all three enzymes (P<0.01). The malondialdehyde content in the model control group was significantly higher than that in the normal control group (P<0.01), while the medium and high concentration groups of grape seed proanthocyanidins were significantly lower than those in the model control group (P<0.05), and there was no statistically significant difference between the low concentration group and the model control group (P>0.05).

[0041] 4. Effects of GSP on the expression of apoptosis-related genes BCL-2 and BAX proteins in lens epithelial cells

[0042] In immunohistochemical analysis of lens epithelial cells, a negative control was replaced with PBS solution, and no specific staining was observed. The expression of BCL-2 and BAX proteins was mainly localized in the cytoplasm; brownish granular staining indicated positive cells. Figure 4 As shown, in normal control LECs, the expression of both proteins was detectable, with BCL-2 expression intensity higher than BAX, and the BCL-2 / BAX ratio at a high level. In the model control, BCL-2 expression level was significantly reduced, while BAX expression level was significantly increased. In contrast, after intervention with GSP (25 μg / mL, 50 μg / mL, and 100 μg / mL), the abnormal expression of both proteins was significantly improved, with BCL-2 level significantly increasing and BAX level significantly decreasing.

[0044] Table 1 shows that, with the model control group as the reference group, the normal control group showed high BCL-2 expression and low BAX expression, with a statistically significant difference (P<0.05). In the medium and high concentration groups of grape seed proanthocyanidins, BCL-2 expression was increased and BAX expression was decreased (P<0.01), which increased the BCL-2 / BAX ratio. However, there was no statistically significant difference in the expression of BCL-2 and BAX in the low concentration group of grape seed proanthocyanidins (P>0.05).

[0045] Table 1. Determination of BCL-2 and BAX protein expression in lens epithelial cells (per cell)

[0046]

[0047] R > 0.50 indicates that the target group expression was higher than the reference group, and R < 0.50 indicates that the target group expression was lower than the reference group. Compared with the model control group, a P<0.01, b P<0.05

[0048] 5. Observational experiment on the irritation of GSP eye drops to rabbit eyes.

[0049] Animal selection: Four healthy New Zealand white rabbits (weighing 2.0-3.0 kg) were selected, and those with eye abnormalities were excluded through pre-screening.

[0050] The testing period is generally 3 days, during which the animal's health status is observed.

[0051] Administration method: Instill 0.1 ml or 2 drops of the test solution into the eyes using physiological saline, or into the low-dose, medium-dose, and high-dose groups of grape seed proanthocyanidins (25 μg / ml, 50 μg / ml, and 100 μg / ml), respectively, and observe immediate and subsequent reactions.

[0052] Observation indicators: Use a magnifying glass to examine corneal transparency, iris texture and conjunctival congestion, and record behavioral indicators such as blinking frequency and secretions.

[0053] Irritation evaluation criteria: Based on the Technical Guidelines for Research on Irritation of Chemical Drugs and other standards, the scoring system includes: (1) Corneal damage: Classified into 0-4 grades according to the degree of turbidity (from no turbidity to corneal opacity).

[0054] (2) Iris reaction: Grade 0 (normal) to Grade 2 (bleeding or no reaction to light).

[0055] (3) Conjunctival congestion / edema: Grade 0 (none) to Grade 3 (severe congestion or edema).

[0056] Stimulation intensity assessment: a total score of ≤3 indicates no stimulation, 4-8 indicates mild stimulation, 9-12 indicates moderate stimulation, and ≥13 indicates severe stimulation.

[0057] Stimulation test results: Low and medium doses of physiological saline and grape seed proanthocyanidins showed no irritation in rabbit eyes; the high-dose grape seed proanthocyanidin group showed mild irritation within 2 minutes of instillation, manifesting only as blinking and mild conjunctival hyperemia. This indicates that grape seed proanthocyanidins have no or only mild irritation to rabbit eyes.

[0058] This study demonstrates that hydrogen peroxide can induce oxidative damage in the rabbit lens, while grape seed proanthocyanidins can alleviate lens damage, thereby increasing the activity of antioxidant enzymes within the lens and reducing MDA content. Furthermore, grape seed proanthocyanidins can regulate the protein expression of BCL-2 and BAX, increasing the BCL-2 / BAX ratio, thus inhibiting oxidative damage and apoptosis of lens epithelial cells. These mechanisms of action may contribute to the prevention of cataracts. Grape seed proanthocyanidins, as a potential cataract treatment, show promising prospects in clinical application.

Claims

1. Use of grape seed proanthocyanidins (GSP) in the preparation of a drug for preventing and treating cataract.

2. Use according to claim 1, characterized in that, The cataract is an oxidative damage-induced cataract.

3. Use according to claim 2, characterized in that, The oxidative damage is hydrogen peroxide-induced oxidative damage of the lens.

4. Use according to claim 1, characterized in that, The drug exerts the effect of preventing and treating cataract by increasing the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT) in the lens and reducing the content of malondialdehyde (MDA).

5. The use according to claim 1, characterized in that, The drug exerts the effect of preventing and treating cataract by regulating the expressions of apoptosis regulatory proteins BCL-2 and BAX, increasing the BCL-2 / BAX ratio, and inhibiting oxidative damage and apoptosis of lens epithelial cells.

6. Use according to claim 1, characterized in that, The drug is an ophthalmic administration preparation.

7. Use according to claim 6, characterized in that, The ophthalmic administration preparation is eye drops.

8. The use according to claim 1, characterized in that, The drug has no irritation or only slight irritation to the eye tissue.

9. A pharmaceutical composition for preventing and treating cataract, characterized by comprising the compound of claim 1. The effective component is grape seed proanthocyanidins, and pharmaceutically acceptable adjuvants are further included.

10. The pharmaceutical composition of claim 9, wherein, The drug composition is an ophthalmic administration preparation, preferably eye drops.

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