Application of apigenin-7-O-beta-D-glucuronide in preparation of medicine for treating fundus diseases

Abstract

The invention discloses an application of apigenin-7-O-beta-D-glucuronide in preparation of a medicine for treating fundus diseases, and establishes a model of ischemic fundus diseases of guinea pigs and a model of eyeball conjunctival microcirculation disturbance of rabbits. The prevention and treatment effects of the apigenin-7-O-beta-D-glucuronide on fundus diseases and fundus lesions are researched, and an experimental basis is provided for clinical treatment of the fundus diseases by the apigenin-7-O-beta-D-glucuronide.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to the use of apigenin-7-O-β-D-glucuronide in the preparation of drugs for treating fundus diseases. Background Art

[0002] Fundus diseases encompass inflammation of the retina, choroid, optic nerve, and vitreous, tumors, various vascular lesions, various degenerative diseases, and multisystem disorders. These diseases are not only numerous but also significantly impair visual function. Consequently, fundus diseases have garnered significant attention from scholars worldwide, with extensive research efforts conducted in diverse fields. Currently, common fundus diseases that severely impact visual function include diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion, and significant progress has been made in clinical research on these diseases.

[0003] 1. Age-related macular degeneration (AMD) Advanced macular degeneration (AMD) is one of the most severe eye diseases that impairs vision in the elderly. Surveys show that the prevalence of AMD ranges from 4.9% to 8.4%, with the incidence increasing significantly with age, reaching 40% in those aged 70 and above. The cause of AMD remains unclear, but studies have shown that smokers have a significantly higher incidence than non-smokers. Environmental, dietary, and genetic factors also warrant attention. The pathogenesis of AMD may be related to chronic light damage, gene mutations, sclerosis and damage of the choroidal capillaries in the macular region, aging of the retinal pigment epithelium, subclinical inflammation, and increased levels of various angiogenic growth factors (such as vascular endothelial growth factor). Currently, AMD is categorized into atrophic and exudative forms based on fundus findings. Epidemiological studies have demonstrated that appropriate use of antioxidants and zinc supplements can delay the onset and progression of AMD. Currently, there is no definitive treatment.

[0004] 2. Retinal vein occlusion (RVO) Retinal vascular occlusion (RVO) is a common fundus vascular disease, including central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO). Vision impairment in patients with RVO is primarily due to macular edema and vitreous hemorrhage caused by retinal neovascularization, leading to neovascular glaucoma.

[0005] In recent years, a number of new treatments for CRVO have emerged, including the use of lasers to anastomose the choroidal vessels and retinal veins in perfused CRVO, improving obstructed venous circulation, reducing the incidence of conversion from perfused CRVO to non-perfused CRVO, alleviating macular edema, and improving vision. Successful treatments for BRVO with arteriovenous sheath incision and optic nerve sclerotomy with perfused CRVOR have also been reported. However, these procedures carry certain complications and require expensive equipment and skilled retinal and vitreous surgery techniques, with the addition of laser retinal photocoagulation when necessary. Therefore, they are currently only performed selectively in larger hospitals. Further randomized controlled clinical trials are needed to confirm their effectiveness.

[0006] 3. Diabetic retinopathy (DR) Dr. Dysregulation (DR) is the leading cause of blindness in diabetes. In 1995, there were approximately 20 million diabetics in my country, and by 2002, the number had reached 30 million. The prevalence of DR increases with the course of diabetes. Inpatient surveys in my country show that the prevalence of DR in diabetic patients has reached 15% to 25%. This indicates that the prevalence of DR in my country is already high and warrants significant attention.

[0007] However, in experimental research, there is still a lack of mature methods to cause fundus diseases in animals. Summary of the Invention

[0008] In response to the problems in the related art, the present invention proposes a use of apigenin-7-O-β-D-glucuronide in the preparation of a drug for treating fundus diseases, so as to overcome the above-mentioned technical problems existing in the existing related technology.

[0009] To this end, the specific technical solutions adopted in the present invention are as follows: Use of apigenin-7-O-β-D-glucuronide in preparing medicine for treating fundus diseases.

[0010] Among them, apigenin-7-O-β-D-glucuronide is a monomeric flavonoid compound extracted from the Asteraceae plant Sophora flavescens.

[0011] Among them, the structure of apigenin-7-O-β-D-glucuronide is as follows:

[0012] Molecular formula: C 21 H 18 O 11 Molecular weight: 456.36.

[0013] The use of the apigenin-7-O-β-D-glucuronide in preparing a drug for treating fundus diseases includes: preparing a drug for treating fundus diseases using apigenin-7-O-β-D-glucuronide as the main active ingredient.

[0014] Optionally, the drugs for treating fundus diseases include: drugs for treating diabetic retinopathy, drugs for treating age-related macular degeneration, and drugs for treating retinal vein occlusion.

[0015] The beneficial effects of the present invention are: The present invention establishes a guinea pig ischemic fundus disease model and a rabbit conjunctival microcirculatory disorder model to study the preventive and therapeutic effects of apigenin-7-O-β-D-glucuronide on fundus diseases and fundus lesions, providing an experimental basis for the clinical treatment of fundus diseases with apigenin-7-O-β-D-glucuronide. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram of the changes in VER in the ipsilateral eye seven days after injection of apigenin-7-O-β-D-glucuronide after ligation of one common carotid artery according to an embodiment of the present invention; Figure 2 Schematic diagram of the dilating effect of apigenin-7-O-β-D-glucuronide on rabbit retinal arteries according to an embodiment of the present invention; Figure 3 Schematic diagram of the effect of apigenin-7-O-β-D-glucuronide on microcirculatory disturbance in rabbits caused by high molecular weight dextran according to an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] Experimental drugs and reagents: (1) Test drug: Apigenin-7-O-β-D-glucuronide (provided by Shenyang Shuangding Pharmaceutical Co., Ltd., purity: more than 90%).

[0020] ⑵High molecular weight dextran: (Dextran produced in Sweden with a weight average molecular weight of 500,000, made into a 10% solution with sterile saline for modeling of animal microcirculatory disorders) (3) Pentobarbital sodium (imported and packaged by Serva, Shanghai Chemical Reagent Purchasing and Supply Station Reagent Factory) Experimental animals: 200 guinea pigs of either sex, weighing 250-300 g, provided by the Institute of Animal Science, Shenyang Medical College. Statistical analysis: Experimental data were expressed as mean ± standard deviation ( ± SD) were expressed, and SPSS13.0 statistical software was used to process the data. One-way ANOVA was used for significance test, and P < 0.05 was considered statistically significant.

[0021] Test method: To measure visual brainstem evoked potentials, guinea pigs were anesthetized with an intraperitoneal injection of 35 mg / kg sodium pentobarbital. Hair was shaved from the mid-parietal region near the frontal end, at the left and right mastoid processes, and connected to the positive, negative, and ground electrodes of a brain disk electrode, respectively. The guinea pigs were then placed in a darkened room. Testing began 20 minutes after anesthesia. Light stimulation was applied to one eye (with the other side shielded from light) using an MS-2PS photostimulator (with a 0.5-second interstimulus interval). The resulting potential changes were fed via the EEG disk electrode into a bioelectrode amplifier, filtered (15 Hz-1000 Hz), and then into a computer. The visual evoked potentials were recorded using an x-y recorder.

[0022] The same method was used to measure conjunctival microcirculatory blood flow velocity. A high-pressure mercury lamp (800W, 200V) was placed in a circular iron lampshade. A small hole 1.5cm in diameter was punched diagonally below the lampshade. A light-guiding glass tube with a 1.5cm diameter opening was inserted into the tube, which gradually tapered downward. The bottom of the tube was blown into a small spherical shape. The tube was filled with distilled water, and the entire tube, except for the bottom of the small sphere, was coated with Chinese ink to block light. The light passed through the water in the tube, turning it into a cold light source. The light was then focused by the small sphere at the bottom of the tube, forming a circular spot approximately 3mm in diameter. This light was projected at a 45-degree angle onto the rabbit conjunctiva. Using a standard microscope with 100x magnification, the conjunctival microvascular blood flow velocity and red blood cell aggregation were observed. The oscilloscope scanning speed was adjusted to synchronize with the blood flow velocity. The oscilloscope scanning speed at this time was the microvascular blood flow velocity.

[0023] Experiments and results: 1. The therapeutic effect of apigenin-7-O-β-D-glucuronide on fundus diseases 1. Measurement of guinea pig visual evoked potential (VER) under normal conditions VER was measured in 40 guinea pigs weighing 200-400 g according to the above method. Except for 8 animals that did not show obvious waveforms, the other 32 animals showed obvious P waves (positive phase waves) and N waves (negative phase waves).

[0024] The amplitudes of the P and N waves vary significantly between animals, ranging from 4 to 34 μV. The latencies of the P and N waves vary relatively little between animals. However, the amplitudes and latencies of the P and N waves in the VER measured at different times within the same animal are similar and relatively stable. Table 1 lists the normal values of the VER measured in 64 eyes of 32 guinea pigs. The table shows that the amplitudes and latencies of the P and N waves in the VER are very similar in both eyes, with the P wave amplitude being greater than the N wave amplitude.

[0025] Table 1. P-wave and N-wave amplitudes (μv) and latency (ms) in normal guinea pig VER

[0026] 2. Replication of guinea pig fundus disease animal model Guinea pigs weighing 250-400g were taken. After measuring their normal visual evoked potentials (VER), they were lightly anesthetized with a small dose of sodium pentobarbital. An incision was made in the neck, and one common carotid artery was dissected. The common carotid artery was then double-ligated and the incisions sutured. VER was measured again one week later. The results are shown in Table 2. Due to the ligation of one common carotid artery, the central retinal artery blood supply to the contralateral eye was insufficient, resulting in ischemia and hypoxia in the fundus, leading to fundus disease in the ipsilateral eye. The results are shown in Tables 2-5.

[0027] Table 2 Changes in visual evoked potential (P wave amplitude) one week after ligation of one common carotid artery in guinea pigs

[0028] Table 3 Changes in visual evoked potential (P wave latency) one week after ligation of one common carotid artery in guinea pigs

[0029] Table 4 Changes in visual evoked potential (N wave amplitude) one week after ligation of one common carotid artery in guinea pigs

[0030] Table 5 Changes in visual evoked potential (N wave latency) one week after ligation of one common carotid artery in guinea pigs

[0031] The results showed that the amplitude of the P wave in the VER of the ipsilateral eye to ligation of the common carotid artery was significantly reduced, from 13.87 to 7.09, with a significant difference (P<0.05). The N wave decreased from 7.14 to 5.60, and the latencies of the P and N waves were also prolonged to a certain extent, indicating that ligation of the common carotid artery on one side can cause fundus disease in the ipsilateral eye, among which the P wave in the VER is the most sensitive.

[0032] 3. Therapeutic effect of apigenin-7-O-β-D-glucuronide on guinea pig fundus disease Thirty male and female guinea pigs weighing 250-400 g were selected for VER measurement. 24 animals with obvious waveforms were selected and one side of the common carotid artery was ligated. The animals were randomly divided into three groups and injected intraperitoneally with normal saline 2 ml / kg and test drug 4 mg / kg daily for seven consecutive days. The VER of the guinea pigs was measured and the effects of the drugs on the amplitude and latency of the P wave in the VER of the eye of the guinea pig with ligated common carotid artery were compared. The results are shown in Tables 6 and 7.

[0033] Table 6 Changes in VER in the ipsilateral eye after injection of normal saline seven days after ligation of one common carotid artery

[0034] Table 7 Changes of VER in the ipsilateral eye after injection of apigenin-7-O-β-D-glucuronide seven days after ligation of one common carotid artery

[0035] Results: The P wave amplitude in the VER of the eye of the guinea pig with ligated common carotid artery in the saline group was significantly decreased compared with that in the saline group (P<0.05), indicating that apigenin-7-O-β-D-glucuronide can significantly treat ischemic fundus disease caused by ligated common carotid artery.

[0036] 2. Effects of apigenin-7-O-β-D-glucuronide on rabbit retinal arteries Nine rabbits, both male and female, weighing 1.85-2.8 kg, were used. Fundus photographs of both eyes were taken before the experiment. The test drug was then injected intravenously at 4 mg / kg. Fifteen minutes after injection, fundus photographs of both eyes were taken under the same conditions. Dosing was continued daily thereafter. Fundus photographs of both eyes were taken again on the seventh day. The film was developed and magnified 2.7 times the diameter. The diameter of the fundus artery was measured 8 mm outward from the mastoid process. Changes in vessel diameter before and after administration were compared. The results are shown in Table 8.

[0037] Table 8 Dilation effect of apigenin-7-O-β-D-glucuronide on rabbit retinal arteries

[0038] The results showed that the diameter of the fundus artery increased 15 minutes and seven days after administration, with an increase of between 8.3% and 3.8% compared with before administration, indicating that apigenin-7-O-β-D-glucuronide has a certain dilating effect on the fundus blood vessels, with an average dilation of 50%.

[0039] 3. Therapeutic effect of apigenin-7-O-β-D-glucuronide on conjunctival microcirculatory disorders in rabbits Twelve rabbits, weighing 1.5-3.0 kg, both male and female, were used. The rabbits were fixed in the ventral position with their heads immobilized. Four to five visual fields were observed in the middle of the right eye, approximately 2 mm from the inferior margin of the bulbar conjunctiva, using the above method. Two to three capillaries of similar size were observed in each field. Blood flow velocity was recorded, and the mean value was taken as the normal value before the experiment. The rabbits were then given an intravenous infusion of 10% high-molecular-weight dextran in saline at 15 ml / kg / day for 1-2 days. This significantly slowed microvascular blood flow and caused severe blood cell aggregation, indicating microcirculatory impairment. Blood flow velocity and blood cell aggregation were recorded. The rabbits were then divided into two groups, each with six rabbits, and each group received an intravenous infusion of 4 mg / kg of apigenin-7-O-β-D-glucuronide and an equal volume of saline two to three times daily for five consecutive days. Microcirculatory status of the bulbar conjunctiva was observed daily at approximately the same location before drug administration. The results are shown in Table 9.

[0040] Table 9. Therapeutic effects of apigenin-7-O-β-D-glucuronide on microcirculatory disturbances in rabbits induced by high molecular weight dextran

[0041] As shown in Table 9, after administration of high-molecular-weight dextran, blood flow velocity in the conjunctival microcirculatory vessels of rabbits significantly slowed, reaching 1 / 4 of the normal value before the experiment. While blood flow velocity in the saline group did not recover within 5 days after injection, it slowly increased in the drug-treated group, returning to approximately 1 / 2 of the normal flow rate before the experiment after 4-6 days of administration. Compared with the saline group, blood flow velocity showed significant recovery (P < 0.05). Following administration of high-molecular-weight dextran, 5-6 or 3-10 red blood cells aggregated into chains. Treatment with the test drug reduced the aggregation, with chains of 2-3 or 3-5 red blood cells forming the majority. However, the aggregation was not eliminated. This demonstrates that apigenin-7-O-β-D-glucuronide has a therapeutic effect on the microcirculatory disturbances caused by high-molecular-weight dextran.

[0042] 4. Experimental Conclusion 1. Establishment of an animal model of fundus disease Clinically, some patients with hypertension, kidney disease, and severe infections can develop fundus diseases due to circulatory disorders such as vasospasm and embolism in the retina. Ligating one common carotid artery can cause insufficient blood flow to the central retinal artery in the ipsilateral eye, leading to ischemia and hypoxia, which can cause fundus diseases. Experimental studies have shown that this method significantly reduces the P-wave amplitude and N-wave amplitude of the retinal retina in guinea pigs with ligated common carotid arteries. Both the P-wave and N-wave latencies are prolonged, demonstrating the development of significant fundus disease. This approach can be used to study the effects of drugs on fundus diseases.

[0043] 2. Apigenin-7-O-β-D-glucuronide has a significant therapeutic effect on fundus diseases After ligating one common carotid artery in guinea pigs and treating them with apigenin-7-O-β-D-glucuronide for one week, the P-wave amplitude of the ligated eye was measured. The saline control group showed a significant decrease in P-wave amplitude and a prolonged latency. However, the apigenin-7-O-β-D-glucuronide group showed no or minimal decrease in P-wave amplitude, and a less prolonged or shortened latency. This suggests that apigenin-7-O-β-D-glucuronide has a significant therapeutic effect on fundus oculopathy.

[0044] 3. Research on the mechanism of therapeutic action Since the cause of guinea pig fundus disease is insufficient blood supply to the central retinal artery, it can be seen that the therapeutic effect of apigenin-7-O-β-D-glucuronide is related to the improvement of fundus circulation; the results of rabbit fundus photography show that apigenin-7-O-β-D-glucuronide can dilate the fundus artery; the rabbit conjunctival microcirculation experiment shows that apigenin-7-O-β-D-glucuronide can significantly improve the systemic microcirculation disorder caused by high molecular weight dextran, and it can be seen that the blood flow velocity of the conjunctival microvessels is significantly increased and the degree of blood cell aggregation is improved. Therefore, it can be considered that the therapeutic effect of apigenin-7-O-β-D-glucuronide on guinea pig fundus disease is related to the drug's effect of dilating blood vessels, increasing blood flow, increasing collateral circulation and improving fundus microcirculation.

[0045] In summary, this experiment confirmed that apigenin-7-O-β-D-glucuronide has a significant preventive and therapeutic effect on fundus diseases in guinea pigs, dilation of retinal arteries in rabbits, and treatment of conjunctival microcirculation disorders in rabbits.

Claims

1. Use of apigenin-7-O-β-D-glucuronide in the preparation of drugs for treating fundus diseases.

2. The use of apigenin-7-O-β-D-glucuronide in the preparation of a drug for treating fundus diseases according to claim 1, characterized in that: Apigenin-7-O-β-D-glucuronide is a monomeric flavonoid compound extracted from the Asteraceae plant Sophora flavescens.

3. The use of apigenin-7-O-β-D-glucuronide in the preparation of a drug for treating fundus diseases according to claim 2, characterized in that: The structure of apigenin-7-O-β-D-glucuronide is as follows: Molecular formula: C 21 H 18 O 11 Molecular weight: 456.

36.

4. The use of apigenin-7-O-β-D-glucuronide in the preparation of a drug for treating fundus diseases according to claim 1, characterized in that: The use of the apigenin-7-O-β-D-glucuronide in preparing a drug for treating fundus diseases includes: using apigenin-7-O-β-D-glucuronide as the main active ingredient to prepare the drug for treating fundus diseases.

5. The use of apigenin-7-O-β-D-glucuronide in the preparation of a drug for treating fundus diseases according to claim 1, characterized in that: The drugs for treating fundus diseases include: drugs for treating diabetic retinopathy, drugs for treating age-related macular degeneration, and drugs for treating retinal vein occlusion.

Images